JPH08140528A - Transgenic nonhuman animal capable of producing heteroantibody - Google Patents

Abstract

PURPOSE: To provide a transgenic mouse which has heterologous immunoglobulin transgene and is useful to produce human monoclonal antibody to be used for the treatment and diagnosis for human because the mouse causes immune response to heterogenetic antigen to produce a heteroantibody. CONSTITUTION: The animal of the purpose has a homozygote of intrinsic heavy chain allele which is functionally divided (preferably JH region homologous recombination knockout), homozygote of intrinsic light chain allele which is functionally divided (preferably JK region homologous recombination knockout), copy of non-homologous immunoglobulin heavy chain transgene (preferably HC1 human minigene transgene, etc.), and copy of heterologous immunoglobulin heavy chain transgene (preferably KC2 human κ-transgene), and the animal successively causes immunization of antigen and antigen response. For example, the animal is obtd. by producing transgene containing human immunoglobulin heavy chain and light chain genetic locus and microinjecting the transgene to the embryo procaryon of a mouse.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a transgenic non-human animal capable of producing a heterologous antibody, a transgene used for producing such a transgenic animal, and VDJ recombinants. Transgene capable of functionally rearranging a heterologous D gene, immortalized B cells capable of producing a heterologous antibody, a method for producing a heterologous antibody of multiple isotypes, and a transgene therefor, an endogenous immunoglobulin A method for inactivating or suppressing the expression of a locus and a transgene therefor, a method for producing a heterologous antibody wherein the variable region sequence comprises a somatic mutation as compared to the germline rearranged variable region sequence, and To that end, a transgene, which produces an antibody having a human primary sequence and which binds to a human antigen, Scan transgenic non-human animals, hybridomas made from B cells of such transgenic animals, as well as to the monoclonal antibodies expressed by the hybridoma.

[0002]

BACKGROUND OF THE INVENTION One of the major obstacles facing the development of in vivo therapeutic and diagnostic uses for monoclonal antibodies in humans is the intrinsic immunogenicity of non-human immunoglobulin. For example, when a tolerant human patient is administered a therapeutic amount of a rodent monoclonal antibody, the patient produces antibodies against the rodent immunoglobulin sequences, and their human anti-mouse antibody (HAMA) produces the therapeutic antibody. Can neutralize and cause acute toxicity. Therefore, it is desirable to produce human immunoglobulins that are reactive with specific human antibodies that are promising therapeutic and / or diagnostic targets.

Current techniques for producing monoclonal antibodies preexpose or sensitize an animal (usually a rat or mouse) to the antigen, obtain B cells from the animal, and hybridoma clones. It involves creating a library of. By screening the hybridoma population for antigen binding specificity (idiotype) and also for immunoglobulin class (isotype), it is possible to select hybridoma clones secreting the desired antibody.

However, when the current methods for producing monoclonal antibodies are applied for the purpose of producing human antibodies having binding specificity for human antigens, humans typically do not generate an immune response against self-antigens. Thus, obtaining B lymphocytes that produce human immunoglobulin is a serious obstacle. Therefore, the current methods for producing human monoclonal antibodies that specifically react with human antigens are clearly insufficient. B for producing hybridoma
The same limitations apply to making monoclonal antibodies against the true self-antigen when using non-human species as the cell source.

The production of transgenic animals containing a functional heterologous immunoglobulin transgene is a method by which antibodies that react with self-antigens can be produced.
However, in order to express therapeutically useful antibodies, or to obtain hybridoma clones that produce such antibodies, transgenic animals must produce transgenic B cells that can mature through the B lymphocyte development process. I have to. Although such maturation requires the presence of surface IgM on transgenic B cells, isotypes other than IgM are desired for therapeutic applications. Thus, there is a need for transgenes and animals bearing such transgenes that are capable of undergoing functional VDJ rearrangements to produce recombinational diversity and zygosity. Further, such transgenes and transgenic animals preferably have cis-acting sequences that promote isotype conversion from the first isotype required for B cell maturation to the next isotype with superior therapeutic utility. Contains.

Numerous experiments have reported the use of transfected cell lines to determine the specific DNA sequence required for Ig gene rearrangement [Lewis and Geller.
t (1989), Cell , 59 , 585-588]. Such a report identifies putative sequences and concludes that the accessibility of those sequences to the recombinase used for rearrangement is altered by transcription [Yancopoulos and
Alt (1985), Cell , 40 , 271-281]. The sequence for the V (D) J bond was reportedly highly conserved near palindromic heptamers and less conserved high AT separated by spacers of either 12 or 23 bp.
Nanomers (Tonegawa (1983), Nature , 302, 575-5
81; Hesse et al. (1989), Genes in Dev ., 3 , 1053-1061
]. Efficient recombination reportedly occurs only between sites containing recombination signal sequences with spacer regions of different lengths.

Ig gene rearrangements have been studied in tissue culture cells but not in transgenic mice. Only a few reports have been published describing rearrangement test constructs introduced into mice (Buchini et al., Nature , 326: 409-411 (1987).
(Non-rearranged chicken lambda transgene); Go
Odhart et al . , Proc. Natl. Acad. Sci. USA 84 : 4229-4233.
(1987) (unrearranged rabbit kappa gene); and Bruggemann et al . , Proc. Natl. Acad. Sci. U SA 86 : 6.
709-6713 (1989) (hybrid mouse-human heavy chain)]. However, the results of such experiments are variable and in some cases may result in incomplete or minimal rearrangements of the transgene.

In addition, the Fc portion of the molecule allows the antibody molecule to undergo various biological functions, such as interaction with mast cells or basophils via Fcε, and Fcμ or Fcγ.
It is further desirable that the binding of complement due to is exerted and that the change in isotype generate functionally diverse antibodies with specific specificity. Although transgenic animals containing transgenes encoding one or more chains of heterologous antibodies have been generated, there are no reports of heterologous transgenes that undergo successful isotype switching. Transgenic animals unable to switch isotypes are limited to those producing heterologous antibodies of a single isotype, and more particularly to those producing isotypes that are essential for B cell maturation, such as IgM and possibly IgD. These antibodies may have limited therapeutic utility.

Based on the above, it is clear that there is a need for a method of efficiently producing a heterologous antibody encoded by a gene sequence of a first species which is produced in a second species. More specifically, a functional V-containing all or part of a D segment that contributes to recombination diversity
There is a need in the art for heterologous immunoglobulin transgenes and transgenic animals that are capable of undergoing DJ gene rearrangements. Furthermore, transgenes and trans that can support VDJ recombination and isotype switching such that (1) functional B cell development occurs and (2) therapeutically useful heterologous antibodies are produced. There is a need in the art for transgenic animals. There is also a need for a B cell source capable of producing hybridomas producing therapeutic or diagnostic monoclonal antibodies in the particular species for which the antibody is designed.
Heterologous immunoglobulin transgenes capable of undergoing functional V-D-J recombination and / or isotype switching can meet those needs.

According to the above object, a transgenic non-human animal capable of producing a heterologous antibody such as a human antibody is provided. Further provided is a B cell from such a transgenic animal capable of expressing a heterologous antibody, said B cell being immortalized to provide a source of a monoclonal antibody specific for a particular antigen. That is the object of the present invention.

According to this above-mentioned object, it is a further object of the present invention to provide a hybridoma cell capable of producing such a heterologous monoclonal antibody. Furthermore, it is an object of the present invention to provide unrearranged and rearranged heterologous immunoglobulin heavy and light chain transgenes useful in the production of the non-human transgenic animals described above.

Furthermore, it is an object of the present invention to provide a method of disrupting the endogenous immunoglobulin loci in transgenic animals. Furthermore, it is an object of the present invention to provide a method of inducing heterologous antibody production in the transgenic non-human animals described above. It is a further object of the present invention to provide a method of making the immunoglobulin variable region gene segment repertoire used to make one or more transgenes of the invention.

The above references are provided solely for their disclosure prior to the filing date of the present application. It is not to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[0014]

SUMMARY OF THE INVENTION Transgenic non-human animals capable of producing xenoantibodies, eg, human antibodies, are provided. Such heterologous antibodies are IgG1, IgG2, IgG3, IgG4,
It can be of different isotypes including IgM, IgA1, IgA2, IgA _sec , IgD or IgE. In order for such transgenig non-human animals to mount an immune response, B
It is necessary for transgenic B cells and pre-B cells to produce surface-bound immunoglobulins, particularly those of the IgM (possibly IgD) isotype, to effect cell development and antigen-stimulated maturation. Such IgM (or Ig
D) Expression of surface-bound immunoglobulin is required only during the antigen-stimulated maturation phase of B cell development, with only one switched isotype being produced at one time, while mature B cells produce other isotypes. obtain.

Typically, cells of the B cell line will produce only a single isotype at one time, but μ _S (secretory μ) and μ _M (membrane bound μ) forms, as well as μ and δ.
Cis or trans alternative RNA splicing, as occurs naturally in immunoglobulin chains, can result in the homologous expression of multiple isotypes by a single cell. Thus, heterologous antibodies of multiple isotypes, especially Igs that are therapeutically useful
In order to produce heterologous antibodies of the G, IgA and IgM isotypes, isotype switching needs to occur. Such isotype switching may be classical class switching or may result from one or more non-classical isotype switching mechanisms.

The present invention provides heterologous immunoglobulin transgenes and transgenic animals carrying such transgenes, wherein the transgenic animals undergo isotype switching to produce heterologous antibodies of multiple isotypes. be able to. A classical isotype switch has at least one in the transgene
It is caused by a recombination phenomenon involving two switch sequence regions. Non-classical isotype switches are, for example, human σ
It can occur by homologous recombination (δ-related deletion) between the μ sequence and the human Σμ sequence. Another non-classical switch mechanism, such as inter-transgene and / or inter-chromosomal recombination, among others, can perform isotype switching.

Such transgene and transgenic animals produce one or more first immunoglobulin isotypes required for antigen-stimulated B cell maturation and have one or more therapeutic and / or diagnostic utilities. It can be switched to encode and produce a second heterologous isotype. Thus, the transgenic non-human animals of the invention can, in one aspect, produce IgG, IgA and / or IgE antibodies that are encoded by human immunoglobulin gene sequences and that bind with high affinity to specific human antigens.

The present invention provides B from transgenic animals capable of expressing heterologous antibodies of various isotypes.
Regarding cells as well, such B cells are immortalized to provide a source of monoclonal antibodies specific for a particular antigen. Hybridoma cells derived from such B cells can serve as one source of such heterologous monoclonal antibodies.

The present invention is a rearranged, capable of undergoing in vivo isotype switching in the non-human transgenic animals described above or in lymphocytes of the B cell line explanted from such transgenic animals. Non- and rearranged heterologous immunoglobulin heavy and light chain transgenes are provided. Such isotype switching occurs naturally or treats transgenic animals or explanted B cell lineage lymphocytes with an isotype switching enhancer, such as a T cell-derived lymphokine (eg, IL-4 and IFNγ). Can be induced by.

Furthermore, the present invention includes a method of inducing heterologous antibody production in a transgenic non-human animal as described above, wherein such antibody can be of various isotypes. The methods involve raising a stimulatory immune response in transgenic non-human animals for the production of xenoantibodies, particularly xenoantibodies of switched isotypes (IGG. IgA and IgM).

The present invention provides that the transgene is isotype-switched so that the heterologous immunoglobulin produced in the transgenic animal and the monoclonal antibody clones derived from the B cells of said animal can be of various isotypes. Methods are provided that include sequences to fulfill. The invention further provides that transgene isotypes such that the switch between specific isotypes occurs at a much higher or lower frequency or in a different time sequence than typically occurs in germline immunoglobulin loci. Provide a way to facilitate the switch. Switch regions may be allowed to ported from various C _H genes and linked to another of the C _H genes of the transgene constructs in. Such transplantation switch sequences typically function independently of the associated C _H gene, so that the switch in the transgene construct will be the original function of the associated switch region. Alternatively, along with a switch sequence, a delta-binding deletion sequence is linked to the various C _H genes, resulting in two delta-
Non-classical switches can be fulfilled by deletion of sequences between linked deletion sequences. Thus, it is possible to create a transgene in which a particular C _H gene is linked to different switch sequences, thereby switching more frequently than occurs when using the naturally associated switch region.

The present invention also provides a method of determining whether transgene sequence isotype switching has occurred in a transgenic animal containing an immunoglobulin transgene. The present invention provides immunoglobulin transgene constructs, some of which contain a subset of germline immunoglobulin locus sequences (which may include deletions), and methods of making immunoglobulin transgene constructs. To do. The present invention includes a special method for the easy cloning and production of immunoglobulin transgenes, which requires a vector that uses unique XhoI and SalI restriction sites flanked by two unique NotI sites. To do.

The transgene of the present invention comprises at least 1
It includes a heavy chain transgene comprising DNA encoding one variable gene segment, one joining gene segment and one constant region gene segment. The immunoglobulin light chain transgene is a DN encoding at least one variable gene segment, one connecting gene segment and one constant region gene segment.
Comprising A. Gene segments encoding light and heavy chain gene segments are immunoglobulin heavy and light chain genes from species that are heterologous to the transgenic non-human animal from which they are derived or that do not consist of transgenic non-human animals. It corresponds to the DNA encoding the segment.

According to one aspect of the invention, the transgene is such that the individual gene segments are not rearranged, ie not so as to encode a functional immunoglobulin light or heavy chain. Is created. Such non-rearranged transgenes are produced by the recombination (functional rearrangement) of the gene segment in transgenic non-human animals exposed to the antigen, as well as the rearranged immunity produced. Allows expression of globulin heavy and / or light chains.

According to one aspect of the invention, the heterologous heavy and light chain immunoglobulin transgenes comprise relatively large fragments of rearranged heterologous DNA. Such fragments typically contain a substantial portion of the C, J (and D in the case of heavy chains) segments from the heterologous immunoglobulin loci. In addition, such fragments also comprise a substantial portion of the variable gene segment.

In one aspect, such transgene constructs include regulatory sequences such as promoters, enhancers, class switch regions, recombination signals, heterologous DNA.
And the corresponding sequences derived from Alternatively, such regulatory sequences may be incorporated into the transgene from the same or related species as the non-human animal used in the present invention. For example, human immunoglobulin gene segments can be combined in a transgene having a rodent immunoglobulin enhancer sequence for use in transgenic mice.

In the method of the present invention, light chain and heavy chain immunoglobulin transgenes that are not germline rearranged
That is, one that receives VDJ binding during D cell differentiation is contacted with an antigen to induce the production of xenoantibodies in secondary repertoire B cells. Also included in the invention are vectors and methods for disrupting the endogenous immunoglobulin loci in the non-human animals used in the invention. Such vectors and methods use a transgene, preferably a positive-negative selection vector, which is a heavy chain and / or which is endogenous to the non-human animal used in the present invention. Alternatively, it is configured to target functional disruption of a class of gene segments encoding light chain immunoglobulins. Such endogenous gene segments include diversity region, junction region and constant region gene segments.

According to this aspect of the invention, the positive-
After contacting the negative selection vector with at least one non-human animal fetal stem cell, cells in which the positive-negative selection vector has been integrated into the genome of the non-human animal by homologous recombination are selected. After transplantation, the resulting transgenic non-human animals are substantially unable to mount an immunoglobulin-mediated immune response as a result of homologous integration of the vector. Such immunodeficient non-human animals can then be used in the study of immunodeficiency disorders or as receptors for heterologous immunoglobulin heavy and light chain transgenes.

The present invention also provides vectors, methods and compositions useful for suppressing the expression of immunoglobulin chains of one or more species without disrupting the endogenous immunoglobulin loci. Such methods are useful for suppressing expression of one or more immunoglobulin chains encoded by the transgene while allowing expression of one or more immunoglobulin chains encoded by the transgene. Unlike genetic disruption of the endogenous immunoglobulin chain locus, suppression of immunoglobulin chain expression requires time-consuming rearing of transgenic animals that are homozygous for the disrupted endogenous Ig locus. Does not need

An additional advantage of the suppression method as compared to the endogenous Ig gene disruption method is that in some embodiments chain suppression is reversible within the individual animal. For example, Ig chain suppression is
(1) a transgene that encodes and expresses an antisense RNA that specifically hybridizes to an endogenous Ig chain gene sequence, (2) an antisense oligonucleotide that specifically hybridizes to an endogenous Ig chain gene sequence, and ( 3) It can be achieved by using an immunoglobulin that specifically binds to an endogenous Ig chain polypeptide.

The present invention provides a homozygous pair of a functionally disrupted endogenous heavy chain allele, a homozygous pair of a functionally disrupted endogenous light chain allele, and a heterologous immunoglobulin heavy chain transgene. Provided is a non-human transgenic animal comprising at least one copy and at least one copy of a heterologous immunoglobulin heavy chain transgene, which is immunized with an antigen such as a human antigen (eg CD4), followed by an antibody response. To do. The invention also provides that the functionally disrupted endogenous heavy chain allele is a J _H region homologous recombination knockout and the functionally disrupted endogenous light chain allele is a J _k region homologous recombination. A knockout, wherein the heterologous immunoglobulin heavy chain transgene is an HC1 or HC2 human minigene transgene,
Also provided is a transgenic non-human animal, wherein said heterologous light chain transgene is a KC2 or KCle human kappa transgene and said antigen is a human antigen.

The present invention also provides various embodiments for suppressing, excising and / or functionally disrupting the endogenous human immunoglobulin loci. The present invention also provides a transgenic mouse that expresses both a chimeric heavy chain containing a human sequence heavy chain variable region and a mouse sequence heavy chain constant region and a human sequence heavy chain. Such chimeric heavy chains generally include functionally rearranged human transgenes and endogenous mouse heavy chain constant regions (γ1, γ2a, γ).
It is produced by a transswitch between 2b, γ3). Antibodies containing such a chimeric heavy chain, typically combined with a transgene-encoded human sequence light chain or an endogenous mouse light chain, are formed in response to immunization with a predetermined antigen. . The transgenic mice of these embodiments produce (express) human sequence heavy chains at a first time point and at the second (following) time point human variable regions and mouse constant regions (eg, γ1, γ2a, γ2b). , Γ3) can be included in a B cell that transswitches to produce (express) a chimeric heavy chain; such a human sequence and the chimeric heavy chain are incorporated into a functional antibody having a light chain: Antibodies are present in the sera of such transgenic mice.

Thus, in other words, the transgenic mice of these embodiments express human sequence heavy chains and then express human variable regions and alternating constant regions (eg mouse γ1, γ2a, γ2b, γ3: human γ, α, e) can comprise B cells that switch (via trans-switches or cis-switches) to express a heavy chain that has undergone a chimeric or isotype switch; such a human sequence and a chimeric or isotype switched heavy chain is a light chain. Incorporated into a functional antibody (human or mouse) with
Such antibodies are present in the serum of such transgenic mice.

The references cited herein are provided solely for their disclosure prior to the filing date of the present application. It is not to be construed as an admission that the inventors are entitled to antedate such disclosure by virtue of prior invention. (Chemical formula 2) and (Chemical formula 3) show the sequences of the vector pGPe.

(Chemical formula 4) and (Chemical formula 5) show the sequence of the gene V _H 49.8. Table 1 shows the detection of human IgM and IgG in the sera of transgenic mice of the invention. (Chemical formula 6)
And (Chemical formula 7) show the sequence of VDJ junction. Table 2 shows the distribution of J-segments incorporated in the transcript encoded by the pHC1 transgene relative to J-segments found in adult peripheral blood lymphocytes (PBL).

Table 3 shows adult peripheral blood lymphocytes (PBL).
Incorporated into the transcript encoded by the pHC1 transgene for the D segment found in
The distribution of segments is shown. (Chemical formula 8) and (Chemical formula 9) are pH
Figure 3 shows the length of CDR3 peptides from transcripts with in-frame VDJ junctions in C1 transgenic mice or human PBLs.

Table 4 shows the deduced amino acid sequence of the VDJ region of 30 clones analyzed from pHC1 transgenic mice. Table 5 shows the lines used in the indicated experiment
112 shows the transgenic mice; (+) indicates the presence of the transgene, (++) is said animal J _H D
It is shown to be homozygous for the overhanging transgene.

Table A shows the genotypes of several 0011 mice. Table 7 shows the occurrence of somatic mutations within the HC2 heavy chain transgene in transgenic mice.

[0039]

Detailed Description As mentioned above, promising treatments and / or
Alternatively, it may be desirable to produce human immunoglobulins that react with specific human antigens that are diagnostic targets. However, human immunoglobulins that specifically bind to human antigens are problematic. First, the immunized animal, which acts as a source of B cells, must generate an immune response to a given antigen. In order for an animal to mount an immune response, a given antigen must be foreign and the animal must be tolerant to that antigen. Thus, for example, if it is desired to produce a human monoclonal antibody having an idiotype that binds a human protein, self-tolerance will prevent a substantial immune response against the human protein from being generated. This is because the only epitopes of the antigen that can be immunogenic will be those derived from polymorphisms of proteins in the human population (allogeneic epitopes).

Second, if an animal (in one example a human), which acts as a source of B cells for forming hybridomas, develops an immune response against a true autoantigen, a serious autoimmune disease will develop in the animal. obtain. When using humans as the source of B cells for hybridomas, such autoimmunization is considered illogical by modern moral norms.

Since human antibody-secreting B cells capable of inducing an antibody response to a predetermined human antigen are required, a hybridoma secreting a human immunoglobulin chain having specific reactivity with the predetermined human antigen is required. There are many problems in developing. One methodology that can be utilized to obtain human antibodies that specifically react with human antigens is the production of transgenic mice containing the human immunoglobulin transgene constructs of the invention. Briefly, a transgene containing all or part of the human immunoglobulin heavy and light chain loci, or a synthetic "minor locus" comprising essential functional elements of the human heavy and light chain loci (see below) , And co-pending US application No. 07 / 574,748 filed August 29, 1990 and 07 / 575,962 filed August 31, 1990,
And a transgene containing (as described in PCT / US91 / 06185, filed August 28, 1991) are used to make transgenic non-human animals.

Such transgenic non-human animals will be capable of producing immunoglobulin chains encoded by human immunoglobulin genes and, moreover, will be able to generate an immune response against human antigens. Thus, such transgenic non-human animals can serve as a source of immune serum that reacts with specific human antigens, and by fusing B cells from such transgenic animals with myeloma cells, A hybridoma that secretes a monoclonal antibody that specifically reacts can be produced.

The production of transgenic mice containing various forms of immunoglobulin genes has been previously reported. Rearranged mouse immunoglobulin heavy or light chain genes have been used in the production of transgenic mice. In addition, a functionally rearranged human Ig gene containing the μ or γ1 constant region has been expressed in transgenic mice. However, experiments in which the transgene comprises unrearranged (unrearranged VDJ or VJ) immunoglobulin genes are variable and, in some cases, incomplete or minimal transgenes. May cause rearrangement. But,
No example of a rearranged or unrearranged immunoglobulin transgene that undergoes successful isotype switching between C _H genes in the transgene has been reported.

The present invention also provides a method for identifying hybridoma candidates secreting a monoclonal antibody comprising a human immunoglobulin chain consisting essentially of human VDJ sequence in a polypeptide binding to a human constant region sequence. Such hybridoma candidates include (1) a hybridoma clone that expresses an immunoglobulin chain basically consisting of a human VDJ region and a human constant region, and (2)
It is identified from a pool of hybridoma clones containing transswitched hybridomas that express a heterohybrid immunoglobulin chain consisting essentially of the human VDJ region and the murine constant region. The supernatants of individual or pooled hybridoma clones are typically adsorbed onto immobilized substrates (eg microtiter wells) under binding conditions to select for antibodies with a predetermined antigen binding specificity. It is contacted with a predetermined antigen which is the immobilized antigen.

Similarly, for an antigen that specifically binds to a human constant region, on the hybridoma under binding conditions such that the antibody selectively binds to at least one human constant region epitope but not to the mouse constant region epitope. It consists essentially of a hybridoma supernatant (transgenic monoclonal antibody) that has been contacted with a clear antigen and a predetermined antigen, thus bound to the predetermined antigen and an antibody that specifically binds to the human constant region. It forms a complex (and can be labeled with a detectable label or reporter). Detection of the formation of such a complex is indicative of hybridoma clones or pools expressing human immunoglobulin chains).

Definitions As used herein, the term "antibody" refers to a glycoprotein comprising at least two identical light polypeptide chains and two identical heavy polypeptide chains. The heavy and light polypeptide chains each contain a variable region (usually the amino-terminal portion of the polypeptide chain) that contains a binding domain that interacts with an antigen. The heavy and light polypeptide chains are each
It also comprises the constant region (usually the carboxy-terminal portion) of the polypeptide chain, part of the sequence of which is present in various cells of the immune system, certain phagocytes and the first component of the typical complement system (C1q). It mediates the binding of immunoglobulins to host tissues including.

As used herein, "heterologous antibody"
Are defined in relation to transgenic non-human organisms producing such antibodies. It is an antibody that has an amino acid sequence or encodes a DNA sequence that corresponds to that found in an organism that does not consist of transgenic non-human animals. As used herein, "heterohybrid antibody" refers to an antibody that has light and heavy chains of different biological origin. For example, an antibody having a human heavy chain associated with a mouse light chain is a heterohybrid antibody.

As used herein, "isotype"
Refers to the antibody class (eg, IgM or IgG ₁ ) encoded by the heavy chain constant region genes. As used herein, "isotype switching" refers to the phenomenon where the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

As used herein, "non-switched isotype" refers to the class of heavy chains produced when isotype switching did not occur. The C _H gene encoding the non-switch isotype is typically the first C _H gene immediately downstream of the functionally rearranged VDJ gene. As used herein, the term "switch array"
Refers to those DNA sequences that result in switch recombination.
The “switch donor” sequence, typically the μ switch region, will be 5 ′ (ie, upstream) of the constant region that will be deleted during switch recombination. The "switch receptor" region will be between the constant region that will be deleted and the alternative constant region (eg, γ, ε, etc.). The final gene sequence will typically be unpredictable from the construct, since there is no unique site where recombination always occurs.

As used herein, "glycosylation pattern" is defined as the pattern of hydrocarbon units covalently attached to a protein, more particularly an immunoglobulin protein. When a person of ordinary skill in the art recognizes that the glycosylation pattern of a heterologous antibody is more similar to the glycosylation pattern in the species of the non-human transgenic animal than the species from which the transgene C _H gene is derived, the heterologous antibody Can be characterized as being substantially similar to the glycosylation pattern naturally present on antibodies produced by non-human transgenic animal species.

As used herein, "specific binding"
Antibody is (1) at least 1 × against the predetermined antigen.
Binds with an affinity of 10 ⁷ M ⁻¹ and (2) non-specific antigens other than the predetermined antigen (eg BSA, casein)
With an affinity that is at least 2 times greater than the binding affinity for
It refers to the property of preferentially binding to a predetermined antigen. The term "naturally-occurring" as used herein refers to the fact that an object can be found in nature when applied to it. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified by humans in the laboratory is "naturally-occurring."
Things.

As used herein, "rearranged"
The term D-in a structure in which each V segment encodes an essentially complete V _H or V _L domain, respectively.
Refers to the arrangement of the heavy or light chain immunoglobulin loci located immediately adjacent to the J or J segment. Rearranged immunoglobulin loci can be identified by comparison with germline DNA; the rearranged loci will have at least one heptamer / nanomer homology element.

As used herein with respect to V segments, "unrearranged" or "germ cell arrangement"
The term refers to an arrangement in which the V segments are not recombined so that they are in close proximity to the D or J segments. For nucleic acids, "substantial homology" also refers to the two nucleic acids or designated sequences thereof having at least about 80% nucleotides, and usually at least about 90% to 95% when aligned and compared in an optimal fashion. %, More preferably at least about 98-99.5
Indicates identity at% nucleotides with the appropriate nucleotide insertions or deletions. Alternatively, there is substantial homology when the segment hybridizes to the complement of strands under selective hybridization conditions. The nucleic acid may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids, alkaline / SDS treatment, C _S
When other cellular components, such as other cellular nucleic acids or proteins, were purified from other contaminants by standard techniques including Cl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art. "Isolated" or "substantially pure". F. Ausubel, et. A
l., ed. “ Current Protocols in Molecular Biology ”, Gre
See ene, Publishing and Wiley-Interscience, New York (1987).

Nucleic acid compositions of the present invention often contain cD
Although in native sequence (excluding modified restriction sites, etc.) from either NA genomic or a mixture, it can be mutated therefrom to provide the gene sequence according to standard techniques. For coding sequences, these mutations can affect the amino acid sequence as desired. In particular, unmodified D, J. DN substantially homologous to or derived from a coupling switch
A sequences and other such sequences described herein are contemplated (where "derivatized" means that one sequence is the same as or modified from another sequence). It means that).

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid. For example, a promoter or enhancer is "operable when it affects the transcription of a sequence. Regarding transcriptional repression sequence, operably linked refers to linked DNA.
It means that the sequences are contiguous, and if necessary to join the two protein coding regions, they are contiguous and in reading frame. For switch arrays,
Operably linked means that the sequences are capable of switch recombination.

Trans capable of producing heterologous antibody
Design of transgenic non-human animals responsive to foreign antigenic stimulation by the transgenic non-human animal xenoantibody repertoire requires that the heterologous immunoglobulin transgene contained within the transgenic animal function correctly through the B cell development pathway. To do. In a preferred embodiment, the correct function of the heterologous heavy chain transgene comprises an isotype switch. Thus, a transgene of the invention is engineered to produce an isotype switch and result in one or more of the following: (1) high level and cell type specific expression,
(2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) sufficient primary repertoire expression, (5) signal transduction, (6) somatic hypermutation ( hyper-mutation), and (7) dominance of the transgene antibody locus during the immune response.

As will be apparent from the disclosure below, it is not necessary to meet all of the above criteria. For example, in those embodiments in which the endogenous immunoglobulin loci of transgenic animals are functionally disrupted, the transgene need not activate allelic exclusion. Further, in an embodiment wherein the transgene comprises a functionally rearranged heavy and / or light chain immunoglobulin gene, the second reference functional gene rearrangement is at least already rearranged. Not needed for transgenes. For background on molecular immunology, see Fundamental Immunolo - gy , No. 2.
See Edition (1989), edited by Paul William E., Raven Press, NY. This is incorporated herein by reference.

In one aspect of the present invention, the rearranged, unrearranged, or a combination of rearranged and unrearranged in germ cells (germlines) of transgenic animals. Transgenic non-human animals containing heterologous immunoglobulin heavy and light chain transgenes are provided. Each of the heavy chain transgenes comprises at least one C _H gene. In addition, the heavy chain transgene may contain functional isotype switch sequences capable of supporting the isotype switch of a heterologous transgene encoding multiple _CH genes in B cells of transgenic animals. Such a switch sequence can be one that naturally occurs at the germline immunoglobulin locus from the species that serves as the source of the transgene C _H gene, or the species that is to accept the transgene construct. It can also be derived from those present in the (transgenic animal).

For example, the human transgene construct used to produce transgenic mice has a higher frequency if it contains switch sequences similar to those naturally occurring in the mouse heavy chain locus. The isotype switching phenomenon can be brought about. Presumably, the mouse switch sequence would be optimized for the mouse switch recombinase enzyme system to function, whereas the human switch sequence would not. Switch sequences can be isolated and cloned by conventional cloning methods, or can be synthesized initially from overlapping synthetic oligonucleotides designed based on published sequence information for immunoglobulin switch region sequences (Mills et al., Nucl. Acids Res. 18 : 7305-73
16 (1991); Siderras et al . , Intl. Immunol. 1: 631-642 .
(1989), which are incorporated herein by reference).

For each of the above transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes are detected in a significant proportion (at least 10 percent) of B cells of the transgenic animals. . The transgene of the present invention comprises a DNA encoding at least one variable gene segment, one connecting gene segment and one constant region gene segment.
Including a heavy chain transgene comprising. The immunoglobulin light chain transgene comprises at least one variable gene segment, one connecting gene segment and one
It comprises DNA encoding one constant region gene segment. Gene segments encoding light and heavy chain gene segments are immunoglobulin heavy and light chain genes from species that are heterologous to the transgenic non-human animal from which they are derived or that do not consist of transgenic non-human animals. It corresponds to the DNA encoding the segment.

According to one aspect of the invention, the transgene is such that the individual gene segments are not rearranged, ie not to encode a functional immunoglobulin light or heavy chain. Is created. Such unrearranged transgenes support recombination (functional rearrangement) of V, D and J gene segments in transgenic non-human animals upon exposure to antigen, and preferably result. Supporting the incorporation of all or part of the D region gene segment into the rearranged immunoglobulin heavy chain obtained as

According to another aspect of the invention, the transgene comprises a rearranged "minilocus". Such transgenes are typically C, D and J.
It comprises a substantial portion of the segment as well as a subset of V segments. In such transgene constructs, various regulatory sequences, such as promoters, enhancers, class switch regions, splice donors and splice acceptors for RNA processing, recombination signals, etc., have corresponding sequences derived from heterologous DNA. Comprises.

Alternatively, such regulatory sequences may be incorporated into the transgene from the same or related species as the non-human animal used in the present invention. For example, a human immunoglobulin gene segment can be combined with a rodent immunoglobulin enhancer sequence in the transgene for use in transgenic mice.
Alternatively, synthetic regulatory sequences may be incorporated into the transgene, in which case such synthetic regulatory sequences are not homologous to the functional DNA sequences known to occur naturally in the mammalian genome. Synthetic regulatory sequences are designed according to common rules and specify, for example, permissive sequences for splice acceptor sites or promoter / enhancer motifs.

For example, the mini-locus differs from the naturally-occurring germline Ig locus in that it contains at least one internal portion of a non-essential DNA portion (eg, intervening sequence; intron or portion thereof) (ie, at the end of this portion). A) of the genomic immunoglobulin locus having a deletion of The present invention also relates to transgenic animals containing germ cells having heavy and light chain transgenes, wherein one of said transgenes comprises a rearranged gene segment and the other is not rearranged. Contains gene segments. In a preferred embodiment, the rearranged transgene is a light chain immunoglobulin transgene and the unrearranged transgene is a heavy chain immunoglobulin transgene.

Antibody Structure and Production The basic structure of all immunoglobulins is based on a unit consisting of two light chain polypeptides and two heavy chain polypeptides. Each light chain comprises two regions known as the variable light chain region and the constant light chain region. Similarly, immunoglobulin heavy chains comprise two regions called the variable heavy chain region and the constant heavy chain region.

The heavy or light chain constant region is encoded by a genomic sequence called a heavy or light chain constant region gene ( _CH ) segment. The use of specific heavy chain gene segments limits the class of immunoglobulins. For example, in humans, the μ constant region gene segment is the Ig of the antibody.
The M class is limited, while the γ, γ2, γ3 or γ4 constant region gene segment is the IgG class of the antibody as well as the IgG
Subclasses IgG _{1 to} IgG ₄ of Similarly, α ₁
Or the use of α ₂ constant region gene segment
Define the A class and the IgA ₁ and IgA ₂ subclasses. The δ and ε constant region gene segments are Ig
Define D and IgE antibody classes.

The variable regions of the heavy and light chain immunoglobulins together comprise the antigen binding region of the antibody. Due to the need for diversity in this region of the antibody in order to be able to bind a wide range of antigens, the DNA encoding the early or primary repertoire variable region may be a large number of different DNAs derived from a particular family of variable region gene segments. Comprises segments. In the case of the light chain variable region, such a family comprises a variable (V) gene segment and a linked (J) gene segment. Thus, the initial variable region of the light chain is encoded by one V gene segment and one J gene segment, each segment being selected from the family of V and J gene segments contained in the genomic DNA of the organism. In the case of the heavy chain variable region, D encoding the early or primary repertoire variable region of the heavy chain
NA comprises one heavy chain V gene segment, one heavy chain diversity (D) gene segment and one J gene segment, each segment being the V, D of the appropriate immunoglobulin gene segment in the genomic DNA. And J family.

To increase the diversity of sequences that contribute to the formation of antibody binding sites, one can incorporate all or part of the D region in the VDJ gene sequence with the rearranged heavy chain transgene. It is preferred to include cis-acting sequences that support functional VDJ rearrangements. Typically, at least about 1% of the heavy chain (or mRNA) encoded by the expressed transgene contains recognizable D region sequences in the V region. Preferably, at least about 10% of the V region encoded by the transgene,
More preferably at least about 30%, most preferably 50%
Contains a recognizable D region sequence.

The recognizable D region sequence is generally encoded by at least about 8 contiguous nucleotides and / or such a D region nucleotide sequence corresponding to the sequences present in the D region gene segment of the heavy chain transgene. Amino acid sequence. For example, if the transgene contains the D region gene DHQ52, the mRNA encoded by the transgene containing the sequence 5'-TAACTGGG-3 'in the V region between the V gene segment sequence and the J gene segment sequence is: It is recognizable as containing a D region sequence, especially the DHQ52 sequence. Similarly, for example, if the transgene contains the D region gene DHQ52, encoded by a transgene containing the amino acid sequence -DAF- located in the V region between the V gene segment amino acid sequence and the J gene segment amino acid sequence. The heavy chain polypeptides identified are recognizable as containing D region sequences, particularly DHQ52 sequences.

However, since the D area segment can be incorporated into the VDJ joining within various reading frames to various extents, the specific D area
To determine the uptake of the segment, the D region of the heavy chain relative to the D region segment present in the transgene
It is necessary to compare regional areas. Moreover, potential exonuclease digestion during recombination can lead to inaccurate VD and DJ ligations during VDJ recombination.

However, due to somatic mutations or N region additions, some D regions may not exactly match the recognizable but contiguous D region sequence. For example, the nucleotide sequence 5'-CTAAXTGGGG-3 '
(Where X is A, T or G, the sequence being in the heavy chain V region and flanked by the V and J region gene sequences) is the DH52 sequence 5'-CTAACTGGG-3.
It can be recognized that it corresponds to '. Similarly, for example, in the V region and flanked at the amino terminus by the amino acid sequence encoded by the transgene V gene sequence and at the carboxy terminus by the amino acid sequence encoded by the transgene J gene sequence. Polypeptide sequence -DAFDI-, -DYFDY- or -GAFD
I- can be recognized as a D region sequence.

Therefore, since somatic mutation and N region addition can cause a mutation in the sequence derived from the transgene D region, the following definition is given as an index for determining the presence of a recognizable D region sequence. To be An amino acid sequence or nucleotide sequence can be recognized as a D region in the following cases: (1) the sequence exists in the V region, and one side is flanked by the V gene sequence (nucleotide sequence or deduced amino acid sequence) And J on the other side
Flanked by gene sequences (nucleotide sequences or deduced amino acid sequences), and (2) said sequences are substantially the same as or substantially homologous to known D gene sequences (nucleotide sequences or deduced amino acid sequences). If there is.

The term "substantially the same" as used herein means
At least 50% of the polypeptide sequence compared to the reference sequence
Of the polypeptide or nucleotide sequences having a sequence identity of, and the nucleic acid sequence has at least 70% sequence identity compared to the reference sequence. Percentage of sequence matches are calculated excluding small deletions or additions that are less than 35% of the total reference sequence. The reference sequence may be a subset of a larger sequence, eg the complete D gene; however, the reference sequence is at least 8 nucleotides in length for polynucleotides and at least 3 amino acids in length for polypeptides. It is a base. Typically, the reference sequence is at least 8-12 nucleotides or at least 3
~ 4 amino acids, preferably 12-15 nucleotides or more or at least 5 amino acids.

The term “substantial homology” as used herein refers to
Represents a property of a polypeptide sequence in which the polypeptide sequence has at least 80% homology to a reference sequence. Percent sequence homology is calculated by counting identical amino acids or position-conservative amino acid substitutions as homologous.
Position-conservative amino acid substitutions can result from single nucleotide substitutions. The first amino acid is replaced with the second amino acid when the codons of the first and second amino acids can differ by a single nucleotide substitution. For example, the sequence -Lys-Glu-Arg-Val- is the sequence-
It is substantially homologous to Asn-Asp-Ser-Val-. Because the codon sequence -AAA-GAA-AGA-GUU- is introduced by introducing only three substitution mutations (single nucleotide substitutions in three of the two original codons) -AAC-GAC-AGC-. This is because it can be mutated to GUU-. The reference sequence may be a subset of a larger sequence, eg the complete D gene; however, the reference sequence is at least 4 amino acid residues in length. Typically the reference sequence is at least 5
Amino acids, preferably 6 amino acids or more.

DN Encoding Primary Repertoire Heavy and Light Chain Immunoglobulin Genes
The method of making A mainly consists in developing B cells. Prior to binding of various immunoglobulin gene segments, V, D, J and constant (C) gene segments are predominantly V, D in the precursors of primary repertoire B cells.
It is found as a cluster of D, J and C gene segments. Usually, all of the heavy or light chain gene segments are placed in close relative proximity on a single chromosome. Such genomic DNA before recombination of the various immunoglobulin gene segments is referred to herein as "unrearranged" genomic DNA. During B cell differentiation, a heavy chain recombined and functionally rearranged with a gene segment of any one of the appropriate family members of V, D, J (or V and J only for light chain genes) And light chain immunoglobulin genes.

Such a functional rearrangement is a rearrangement of variable region segments forming a DNA encoding a functional variable region. This gene segment rearrangement process appears to be continuous. First, a heavy chain DJ junction is created, followed by a heavy chain V-DJ junction and a light chain VJ junction. The DNA encoding this initial form of the functional variable region in the light and / or heavy chains is called "functionally rearranged DNA" or "rearranged DNA".
In the case of heavy chains, such DNA is "rearranged heavy chain D
Such a DNA in the case of a light chain, called "NA"
Is called "rearranged light chain DNA". Similar terms are used to describe the functional rearrangements of the transgene of the present invention.

Recombination of variable region gene segments to form functional heavy and light chain variable regions is accomplished by recombination signal sequences (RSS) flanking the recombination-responsive V, D and J segments. Mediated. The necessary and sufficient RSS to direct recombination comprises a bimolecularly symmetric heptamer, a high AT nonamer, and an interrupting spacer region of either 12 or 23 base pairs. Their signals are conserved between different loci and species that undergo DJ (or VJ) recombination and are functionally interchangeable. Oettinger et al. (1990), Scie
See nce, 248, 1517-1523 and references cited therein.

There is a heptamer comprising the sequence CACAGTG or an analogue thereof, followed by a spacer of non-conserved sequence, then a nonamer having the sequence ACAAAAACC or an analogue thereof. The sequences are found on the J, or downstream, side of each V and D gene segment. Immediately before the germline and J gene segments are again two recombination signal sequences, first a nonamer and then a heptamer separated by a non-conserved sequence. V _L, heptamer and nonamer sequences behind the V _H or D segment are complementary to their set switched J _L, and that of the front of the D or J _H segments. The spacer between the heptamer and nonamer sequences is 12 base pairs long or 22-24
It is either the length of a base pair.

In addition to the rearrangement of the V, D and J segments, variable recombination between the V and J segments of the light chain and the D and J segments of the heavy chain results in immunoglobulin heavy chain And additional diversity is created in the primary repertoire of light chains. Such various recombination is generated by the displacement (shift) of the exact location where such segments join. Such shifts in the light chain typically occur within the last codon of the V gene segment and within the first codon of the J segment. Similar misalignment occurs on the heavy chain chromosome between the D and _JH segments and can extend to as many as 10 nucleotides. Furthermore, between the D segment and the J _H segment and between the V _H segment and D
Some nucleotides may be inserted between the segments that are not encoded by genomic DNA. The addition of those nucleotides is known as N-region diversity.

After VJ and / or VDJ rearrangement, transcription of the rearranged variable region gene segment and one or more constant region gene segments located downstream of the rearranged variable region is determined by primary RNA transcripts. Produces
When it is subjected to proper RNA splicing, it yields a full length heavy or light chain immunoglobulin encoding mRNA. Such heavy and light chains include a leader sequence that directs the insertion and / or secretion of immunoglobulins into the transmembrane region of B cells. The DNA encoding this signal sequence is contained within the first exon of the V segment used to form the variable region of the immunoglobulin heavy or light chain. Appropriate regulatory sequences are also present in the mRNA to regulate translation of the mRNA that produces the encoded immunoglobulin heavy and light chain polypeptides, which form the antibody molecule upon proper association with each other.

The end result of such rearrangements in the variable region gene segments and variable recombination that may occur during such ligation is the production of a primary antibody repertoire.
Generally, each B cell differentiated to this stage produces a single primary repertoire antibody. During this differentiation process,
The cellular phenomenon occurs that suppresses the functional rearrangement of gene segments other than those contained within the functionally rearranged Ig gene. The process by which diploid B cells maintain such monospecificity is called allelic exclusion.

Secondary Repertoire B cell clones expressing immunoglobulins from within the set of sequences comprising the primary repertoire are readily available to respond to foreign antigens. Simple VJ and VD
Due to the limited diversity produced by J-binding, the antibodies produced by the so-called primary response are of relatively low affinity. Two different types of B cells create this initial response: a precursor of primary antibody-forming cells and a precursor of secondary repopulation B cells [Linton et al., Cell , 59 : 1049-1059.
(1989)]. The first type of B cell matures into an IgM-secreting plasma cell in response to certain antigens. The other B cell is T
It responds to initial exposure to antigen by entering the cell-dependent maturation process.

T of B cell clones sensitized with antigen
During cell-dependent maturation, the structure of antibody molecules on the cell surface changes in two ways. First, the constant region can switch to a non-IgM subtype, and the sequence of the variable region can be altered by multiple single amino acid substitutions to produce higher affinity antibody molecules. As I pointed out earlier, Ig
Each variable region of the heavy or light chain contains an antigen binding region.
Somatic mutations during the secondary response are due to amino acid and nucleic acid sequencing of the V region containing three complementarity determining regions (CDR1, CDR2 and CDR3; which are also called hypervariable regions 1, 2 or 3). It has been determined to occur everywhere [Kabat et al., Sequences of Proteins of Immunolo
gical Interest (1991) US Department of Health an
d Human Services, Washington, DC; which is incorporated herein by reference]. CDR1 and CDR2 are located within the variable gene segment, while CDR3 is largely the result of recombination between the V and J gene segments or between the V, D and J gene segments.

The portions of the variable region that do not constitute CDR1, 2 or 3 are commonly referred to as the framework regions designated FR1, FR2, FR3 and FR4. See FIG. During hypermutation, the rearranged DNA is mutated to yield new clones with different Ig molecules. Those clones with higher affinity for the foreign antigen are selectively expanded by helper T cells, causing affinity maturation of the expressed antibody. Clonal selection typically results in the expression of clones containing novel mutations in the CDR1, CDR2 and / or CDR3 regions. However, mutations outside of the antigen binding regions that affect the specificity and affinity of those regions also occur.

Trans capable of producing heterologous antibody
Transgenic Non-Human Animal In one aspect of the invention, a transgenic non-human animal is produced by introducing at least one immunoglobulin transgene of the invention (described below) into a zygote or early embryo of the non-human animal. . Non-human animals useful in the present invention generally include any mammal that is capable of rearranging immunoglobulin gene segments to produce a primary antibody response. Such non-human transgenic animals include, for example, transgenic pigs, transgenic rats, transgenic rabbits, transgenic cows, and other transgenic animal species known in the art, particularly mammalian species. . Particularly preferred non-human animals are mice or other members of rodents.

However, the invention is not limited to the use of mice. Rather, any non-human mammal capable of mounting a primary and secondary antibody response can be used. Such animals include non-human primates such as chimpanzees, cows, sheep and pig species, other members of rodent dentistry such as rats, and rabbits and guinea pigs. Particularly preferred animals are mice, rats, rabbits and guinea pigs, most preferably mice.

In one aspect of the invention, various gene segments from the human genome are used in unrearranged heavy and light chain transgenes. In this aspect, such a transgene is introduced into mice. The unrearranged gene segment of the light and / or heavy chain transgene has a human species-specific DNA sequence that is distinguishable from the endogenous immunoglobulin gene segment in the mouse genome. They can be easily detected in unrearranged form in somatic cells that do not consist of germ cells and B cells and in rearranged form in B cells.

In another aspect of the invention, the transgene comprises a rearranged heavy and / or light chain immunoglobulin transgene. Specific segments corresponding to functionally rearranged VDJ or VJ segments of such transgenes include immunoglobulin DNA sequences that are clearly distinguishable from endogenous immunoglobulin gene segments in mice.

Such a difference in DNA sequence is caused by the mouse B
The amino acid sequence encoded by such a human immunoglobulin transgene is also reflected when compared to the amino acid sequence encoded by the cell. Thus, antibodies specific for immunoglobulin epitopes encoded by human immunoglobulin gene segments can be used to detect human immunoglobulin amino acid sequences in the transgenic non-human animals of the invention.

Transgenic B cells containing unrearranged transgenes from humans or other species are functionally rearranged light and heavy chains upon functional recombination of the appropriate gene segments. Form a variable region. Antibodies encoded by such rearranged transgenes include DNA and / or heterologous to those normally encountered in the non-human animals used to practice the invention.
Or it will be readily apparent to have the amino acid sequence.

Unrearranged Transgene As used herein, an "unrearranged immunoglobulin heavy chain transgene" means at least one variable gene segment, one diversity gene segment,
It comprises DNA encoding one joining gene segment and one constant region gene segment. Each gene segment of the heavy chain transgene has a sequence that is derived from or corresponds to the DNA encoding the immunoglobulin heavy chain gene segment of a species that does not consist of a non-human animal into which the transgene is introduced.

Similarly, as used herein, "unrearranged immunoglobulin light chain transgene"
Comprises DNA encoding at least one variable gene segment, one connecting gene segment and at least one constant region gene segment. Here, each gene segment of the light chain transgene is
It has a sequence derived from or corresponding to the DNA encoding an immunoglobulin light chain gene segment of a species not consisting of a non-human animal into which said light chain transgene is introduced.

Such heavy and light chain transgenes in this aspect of the invention contain the gene segments described above in unrearranged form. Thus, a suitable recombination signal sequence (RSS) is placed between the V, D and J segments in the heavy chain transgene and between the V and J segments in the light chain transgene. In addition, such transgenes also contain suitable RNA splicing signals for joining constant region gene segments with VJ or VDJ rearranged variable regions.

Upstream of each constant region gene segment and downstream of the variable region gene segment to facilitate isotype switching within the heavy chain transgene containing multiple C region gene segments, eg, Cμ and Cγ1 from the human genome. In order to allow recombination between the constant regions taking into account immunoglobulin class switches, eg IgM to IgG class switches, a "switch" region as described below is incorporated. Such heavy and light chain immunoglobulin transgenes also contain transcriptional regulatory sequences (typically containing a TATA motif) containing a promoter region located upstream of the variable region gene segment.

A promoter region can be roughly defined as a DNA sequence capable of directing the transcription of a downstream sequence when operably linked to the downstream sequence. The promoter may require the presence of additional cis-acting sequences linked to it to direct efficient transcription. More preferably, other sequences involved in transcription of the non-fertility transcript are included. Examples of sequences involved in the expression of non-fertile transcripts are described in Rothman et al . , Intl. Immunol. 2: 621-627 (19
90); Reid et al., Proc . Natl. Acad. Sci. USA86 : 840-844
(1989); Stavnezer et al., Proc . Natl. Acad. Sci. USA
85 : 7704-7708 (1988) and Mills et al . , Nucl. Acids.
Res. 18 : 7305-7316 (1991), each of which is incorporated herein by reference, and can be found in the published literature.

The sequences are typically at least about 50 bp immediately upstream of the switch region, preferably at least about 200 bp upstream of the switch region, more preferably at least about 200-1000 bp or more of the switch region. including. Suitable sequences are human Sγ1, Sγ2, Sγ3, Sγ4, S
Although present immediately upstream of the α1, Sα2 and Sε switch regions, sequences immediately upstream of the human Sγ1 and Sγ3 switch regions are preferred. Alternatively, or in combination with it, a murine Ig switch array can be used. It is often advantageous to use the same species of Ig switch sequence as the transgenic non-human animal. In particular, interferon (IFN) -inducible transcriptional regulatory elements such as IF
It is preferred that the N-inducible enhancer be included immediately upstream of the transgene switch sequence.

In addition to the promoter, other regulatory sequences that function primarily in B-lineage cells are used. For example, a light chain enhancer sequence, preferably placed between the J segment and the constant region gene segment on the light chain transgene, is used to enhance expression of the transgene, thereby promoting allelic exclusion. . In the case of heavy chain transgenes, regulatory enhancers are also used. Such regulatory sequences are used to maximize transcription and translation of the transgene, thus inducing allelic exclusion and providing relatively high levels of transgene expression.

Although the promoter and enhancer regulatory sequences described above have been comprehensively described, such regulatory sequences can be heterologous to the non-human animal,
The heterologous transgene immunoglobulin gene segment can be derived from the resulting genomic DNA. Alternatively, such regulatory gene segments are derived from corresponding regulatory sequences in the genome of non-human animals or closely related species, including heavy and light chain transgenes.

In a preferred embodiment, the gene segment is derived from human. Transgenic non-human animals containing such heavy and light chain transgenes can mount an Ig-mediated immune response against specific antigens administered to such animals. B cells capable of producing xenogeneic human antibodies are produced in such animals. After immortalization, and with the appropriate monoclonal antibody (Mab
), Eg, after selection of hybridomas, a source of therapeutic human monoclonal antibodies is provided. Such a human Mab
Has substantially reduced immunogenicity when administered therapeutically to humans.

Although the preferred embodiment discloses the production of heavy and light chain transgenes comprising human gene segments, the present invention is not so limited. In this regard, it should be understood that the teachings described herein can be readily modified for use with immunoglobulin gene segments from species other than human. For example, in addition to therapeutic treatment of humans with the antibodies of the invention, therapeutic antibodies encoded by appropriate gene segments can be used to generate monoclonal antibodies for use in veterinary science.

Rearranged Transgene In another embodiment, the transgenic non-human animal functionally contains at least one rearranged heterologous heavy chain immunoglobulin transgene in the germ cells of the transgenic animal. Such animals contain primary repertoire B cells expressing the rearranged heavy chain transgene as described above. Such B cells are preferably
Upon contact with an antigen, it can undergo somatic mutations to form xenoantibodies with high affinity and specificity for that antigen. The rearranged transgene will contain at least two C _H genes and related sequences required for isotype switching.

The invention also includes germ cells having heavy and light chain transgenes, one of the transgenes comprising a rearranged gene segment and the other comprising an unrearranged gene segment. , Also to transgenic animals. In such animals, the heavy chain transgene will have an associated sequences required for at least two of the C _H gene and isotype switching.

The present invention further relates to methods of making synthetic variable region gene segment repertoires that can be used in the transgenes of the present invention. The method comprises generating a population of immunoglobulin V segment DNA. Here, each of the V segment DNAs encodes an immunoglobulin V segment and contains at each end a cleavage recognition site for a restriction endonuclease. The population of immunoglobulin V segment DNA is then
Concatenated to form a synthetic immunoglobulin V segment repertoire. Such synthetic variable region heavy chain transgenes will have an associated sequences required for at least two of the C _H gene and isotype switching.

During the development of isotype switched B lymphocytes, the cells first produce IgM with a binding specificity determined by the productively rearranged V _H and V _L regions. Subsequently, each B cell and its progeny cells synthesize antibodies with the same L and H chain V regions, which are capable of switching the H chain isotype.

The use of the μ or δ constant regions is for the most part
Determined by alternating splicing, which allows IgM and IgD to be co-expressed in a single cell. The other heavy chain isotypes (γ, α and ε) are expressed only essentially after the gene rearrangement event deletes the Cμ and Cδ exons. This gene rearrangement process is called isotype switching, and typically each heavy chain gene (except δ)
It occurs by recombination between so-called switch segments placed immediately upstream of.

Each switch segment has a length of 2-10.
kb, consisting mainly of short repeats. The exact location of recombination varies for each individual class-switch phenomenon.
Experiments using Southern blotting or solution hybridization kinetics with C _H probes derived from cDNA
Switch has confirmed that may relate to loss of the C _H sequences from the cell.

The switch (S) region of the μ gene, Sμ,
Located about 1-2 kb 5'to the coding sequence, and (GAGC
It consists of multiple tandem repeats of an array of the form T) _n (GGGGT). Here n is usually 2 to 5, but can range up to 17 or so. (T. Nikaido et al., Nature , 292: 845-848 (1
See 981). ] A similar internal repetitive switch sequence extending over several kilobase pairs has been found in the 5'of other C _H genes. The Sα region has been sequenced and found to consist of tandemly repeated 80 bp of homology units. On the other hand, S _2a , S _2b and S
₃ all contain homology units of 49 bp was repeated very similar to each other [P. Szurek et al, J. Immunol, 135: 6
20-626 (1985) and T. Nikaido et al., J. Biol. Chem.
257: 7322-7329 (1982); these are incorporated herein by reference]. All sequenced S regions contain multiple occurrences of the pentameric GAGCT and GGGGT, which are the basic repeats of the Sμ gene [T. Nikai
do et al., J. Biol. Chem. 257: 7322-7329 (1982); which is incorporated herein by reference]; in other S regions, their pentamers are exactly tandem as Sμ. Not repeated but instead buried in a large repeating unit. The Sγ ₁ region has an additional conformation, ie
Two direct repeats flank each of two clusters of 49 bp tandem repeats [MR Mowatt et al., J. Immunol . 13
6: 2674-2683 (1986); which is incorporated herein by reference].

The switch region of the human heavy chain gene has been found to be very similar to their mouse homologs. In fact, the similarity between the human and mouse clones 5'to the C _H gene was found to be limited to the S region, a fact confirming the biological importance of those regions. . Switch recombination between the μ and α genes results in a complex Sμ-Sα sequence. Typically, there are no specific sites in the Sμ region or in other S regions where recombination always occurs.

In general, unlike the enzymatic machinery of VJ recombination, the switch machinery is clearly able to accommodate different alignments of the repetitive homology regions of germline S precursors, which in turn position the sequences at different positions. Can be connected in a straight line. (TH Rabbitts et al., Nucleic Acids Res. 9: 450
9-4524 (1981) and J. Ravetch et al . , Proc. Natl. Aca.
d. Sci. USA , 77 : 6734-6738 (1980); these are incorporated herein by reference. ] The details of the mechanism that selectively activates the switch to a specific isotype are unknown. Although exogenous influencers such as lymphokines and cytokines may upregulate isotype-specific recombinases, this enzyme mechanism catalyzes a switch to any isotype, and specificity targets this enzyme mechanism to a specific switch. It may also be in the area.

Lymphokines IL-4 and IFN from T cells
γ has been demonstrated to specifically promote the expression of several isotypes: IL-4 decreases IgM, IgG2a, IgG2b and IgG3 expression and increases IgE and IgG1 expression; IFNγ expresses IgG2a. Selectively induces and antagonizes the expression of IgE and IgG1 [Coffman
Et al., J. Immunol . 136: 949-954 (1986) and Snapper.
Et al., Science 236: 944-947 (1987); these are incorporated herein by reference].

Most of the experiments involving T cell effects on switches suggest that the increase observed in cells with specific switch recombination may reflect the selection of preswitched or pretilted cells. I couldn't ignore it. However, the most likely explanation is that lymphokines actually promote switch recombination.

Induction of class switching is initiated by the stelile transcrip that begins upstream of the switch segment.
t)) (Lutzeker et al. Mol. Cell Biol .
8: 1849 (1988); Stavnezer et al., Proc . Natl . Acad. Sc
i. USA 85 : 7704 (1988); Esser and Radbruch, EMBO
J. 8: 483 (1989); Berton et al . , Proc. Natl. Acad. Sc
i. USA 86 : 2829 (1989); Rothman et al . , Int. Immunol .
2: 621 (1990); each of which is incorporated herein by reference]. For example, the observed induction of γ1 non-reproducible transcripts by IL-4 and inhibition by IFNγ promotes the class switch to γ1 in B cells by IF-4
Is consistent with the observation that inhibits γ1 expression. Thus, the inclusion of regulatory sequences that affect the transcription of non-fertile transcripts may also affect the rate of isotype switching. For example, increasing transcription of a particular non-fertile transcript can be expected to increase the frequency of isotype switch recombination, which typically involves flanking switch sequences.

For those reasons, approximately 1-2 k of each switch region that the transgene will use for the isotype.
b It is desirable to include a transcriptional regulatory sequence within the upstream region. Those transcriptional regulatory sequences preferably include promoters and enhancer elements, and more preferably include 5'flanking (ie, upstream) regions naturally associated with the switch region (ie, present in the germline configuration). The 5'flanking region is typically at least 50 nucleotides in length, preferably at least about 200 nucleotides in length, more preferably at least 500-1000 nucleotides.

A 5'flanking sequence from _one switch region can be operably linked to another switch region for a transgene construct (eg, a 5'flanking sequence from a human Sγ1 switch can be linked to the Sα ₁ switch). Can be fused immediately upstream; the murine Sγ1 flanking region can be grafted next to the human γ1 switch sequence; or the murine Sγ1 switch can be grafted to the human γ1 coding region), but in some embodiments , Each switch region contained in the transgene construct is immediately upstream of the naturally occurring germline arrangement 5 '
It is preferred to have flanking sequences.

Monoclonal Antibodies Monoclonal antibodies can be obtained by various techniques well known to those skilled in the art. Briefly, splenocytes from animals immunized with the desired antigen are immortalized, generally by fusion with myeloma cells. (Kohler
And Milstein, Eur. J. Immunol. , 6 ; 511-519 (197).
See 6)). Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes or retroviruses, or other methods well known in the art.

Colonies generated from a single immortalized cell are screened for the production of antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibody produced by such cells is determined in the peritoneal cavity of a vertebrate host. It can be enhanced by a variety of techniques, including infusion. Effective in these fields, including immunization of animals to produce immunoglobulins, production of monoclonal antibodies; labeling of immunoglobulins for use as probes; immunoaffinity purification; and immunoassays Various techniques, such as Harlow and
Lane, Antibodies: The Lab Manual , Cold Spring Harb
Or, New York (1988).

Transgenic Primary Repertoire A. An important prerequisite for human immunoglobulin locus transgene function is the production of a primary antibody repertoire that is sufficiently diverse to mount a secondary immune response against a wide range of antigens. The rearranged heavy chain gene contains signal peptide exons,
It consists of variable region exons and tandemly aligned regions of multidomain constant regions, each encoded by several exons. Each of the constant region genes encodes the constant portion of a different immunoglobulin class. During B cell development, the V region near the constant region is deleted resulting in the expression of a novel heavy chain class. For each heavy chain class, distinct patterns of RNA splicing result in both transmembrane and secretory immunoglobulins.

The human heavy chain locus spans approximately 200 Mb and is approximately 200 Mb.
V gene segment (This data is about 50-100
Supports the presence of the V gene segment. ), About 40 k
It is expected to consist of approximately 30 D gene segments spanning b, 6 J segments clustered within 3 kb, and 9 constant region gene segments spanning approximately 300 kb. The entire locus is about the distal part of the longer arm of chromosome 14.
It reaches 2.5 Mb (Fig. 4).

B. Gene fragment transgene 1. Heavy Chain Transgene In a preferred embodiment, the immunoglobulin heavy and light chain transgenes are human derived unrearranged genomic D
Comprising NA. For heavy chains, the preferred transgene comprises a NotI fragment having a length of 670-830 kb. The length of this fragment is ambiguous because the 3'restriction site has not actually been mapped. However, α
It is known to be between 1 and the ψα gene segment. This fragment contains all members of the 6 known V _H families, the D and J gene segments, and μ,
Includes δ, γ3, γ1 and α1 constant regions. Berman et al.
EMBOJ. 7: 727-738 (1988). A transgenic mouse line containing this transgene correctly expresses the heavy chain class (IgM) required for B cell development and is sufficient to initiate a secondary response to most antigens. It expresses at least one switched heavy chain class (IgG ₁ ) with multiple repertoires.

2. Light chain transgene A genomic fragment containing all the necessary gene segments and regulatory sequences from the human light chain locus can be similarly made. Such a construct is made as described in this example.

C. Made intracellularly by in vivo recombination
It is not necessary to isolate all or part of the heavy chain locus on the transgene single DNA fragment. For example, the 670-830 kp NotI fragment from the human immunoglobulin heavy chain locus can be formed in vivo in non-human animals during transgenesis. Such in vivo transgene production is carried out by introducing two or more overlapping DNA fragments into the embryonic nucleus of a non-human animal. Overlapping portions of DNA fragments have DNA sequences that are substantially homologous. Upon exposure to the recombinase contained within the fetal nucleus, overlapping DNA fragments recombine in the proper orientation to form a 670-830 kb NotI heavy chain fragment.

It is understood that in vivo transgene production can be used to generate large numbers of immunoglobulin transgenes whose size is difficult or impossible to produce or manipulate by existing techniques. Should be. For example, in vivo transgene production is performed by YAC
Vectors [Murray and Szostak, Nature 305: 189-19
3 (1983)], and is useful for producing an immunoglobulin transgene larger than a DNA fragment that can be engineered by Such in vivo transgene production is
It can also be used to introduce into a non-human animal a substantially complete immunoglobulin locus of a species that does not consist of a transgenic non-human animal.

In addition to forming genomic immunoglobulin transgenes, in vivo homologous recombination can also be used to form "minilocus" transgenes as described in the Examples. In a preferred embodiment utilizing in vivo transgene production, each overlapping DNA fragment preferably comprises a terminal portion of the first DNA fragment and a second D fragment.
Substantially homologous DNs overlapping between the terminal parts of the NA fragment
It has an A sequence. Such overlapping portions of DNA fragments are
Preferably about 500 bp to about 2000 bp, most preferably 1.0
Includes kb to 2.0 kb. Homologous recombination of overlapping DNA fragments to form a transgene in vivo was performed on March 19, 1992.
Published in Japan, "Homologous Recombination in Mammalian Cells (Ho
mologous Recombination in Mammalian Cells)
Further details are provided in PCT Publication WO 92/03917.

D. Minilocus transgene As used herein, the term "immunoglobulin minilocus" usually refers to less than about 150 kb, typically about 25 kb.
~ 100 kb of DNA sequence, wherein said DNA sequence is at least one substantial discontinuity (eg homologous genomic DN
With respect to the A sequence, it refers to a DNA sequence, each of which has at least one of the following, usually having at least about 2-5 kb, preferably 10-25 kb or more deletions: functional variable (V B.) A gene segment, a functional junction (J) region segment, at least one functional constant (C) region gene segment, and in the case of a heavy chain minilocus, a functional diversity (D) region segment. The light chain minilocus transgene is at least 25 kb in length,
It will typically be 50-80 kb. The heavy chain transgene will have two constant regions operably linked to the switch region, typically about 70-80 kb, preferably at least about 60 kb in length. In addition, the individual elements of the minilocus are preferably in the germline (germline) arrangement, and may express functional antibody molecules with diverse antigenic specificities fully encoded by the elements of the minilocus. In particular, it can undergo gene rearrangement in precursor B cells of transgenic animals. In addition, the heavy chain minilocus, which comprises at least two C _H genes and the necessary switch sequences, typically causes functional antibody molecules of different immunoglobulin classes to be generated,
Can receive an isotype switch. Such isotype switching can occur in vivo in B cells present within transgenic non-human animals, or in cultured cells of the B cell line explanted from transgenic non-human animals.

[0125] In another preferred embodiment, it comprises an immunoglobulin heavy and light chain transgenes V _H, one each of D and J _H gene segments or more and a C _H gene of two or more. At least one of the appropriate type of gene segment is integrated into the minilocus transgene. With regard to the C _H segment of the heavy chain transgene, the transgene comprises at least one μ gene segment and at least one other constant region gene segment, more preferably the γ gene segment, most preferably the γ3 or γ1 gene segment. It is preferable. This preference is due to the Ig of the encoded immunoglobulin.
It takes into account the class switch between M and IgG forms and the production of secreted forms of high affinity non-IgM immunoglobulins. Other constant region gene segments, eg IgD, IgA
And those encoding the production of IgE can also be used.

Those skilled in the art will also produce transgenes in which the order of development of the heavy chain C _H genes will be different from the naturally occurring spatial order found in the germ cells of the species that act as donors for the C _H gene. right. Further, those skilled in the art
A C _H gene can be selected from multiple individuals of one species (eg, an allogeneic C _H gene) and integrates the gene into the transgene as an excess C _H gene that can undergo isotype switching. be able to. The resulting transgene non-human animal can then, in one embodiment, produce various classes of antibodies that include all of the allotypes symbolized by the species from which the transgene C _H gene was obtained.

Furthermore, the person skilled in the art is able to select _CH genes from different species and integrate them in the transgene. A functional switch sequence is included with each C _H gene, but the switch sequence used does not necessarily have to be naturally occurring next to the C _H gene. Interspecies C _H gene combinations will result in transgenic non-human animals capable of producing diverse classes of antibodies corresponding to C _H genes from different species. Transgenic non-human animals containing interspecific C _H transgenes include
It can serve as a source of B cells to produce hybridomas that produce veterinary monoclonals.

The human heavy chain J region segment is a 3 kb D region.
It comprises 6 functional J segments and 3 pseudogenes clustered in NA length. Its relatively small size and their segments
Can be isolated as a single 23 kb SFiI / SpeI fragment with the 5'portion of the gene [Sado et al . , Biochem. Biophys. Re
S. Commun . 154: 246-271 (1988); which is incorporated herein by reference], it is preferred to use the entire J region gene segment in the minilocus construct. Furthermore, since this fragment spans the region between the μ and δ genes, it is appropriate to include all of the cis-linked 3 ′ regulatory elements required for μ expression.

In addition, since this fragment contains the complete J region, it contains the heavy chain enhancer and the μ switch region [Mills
Et al., Nature 306: 809 (1983); Yancopoulos and Al.
t, Ann. Rev. Immunol. 4: 339-368 (1986); these are incorporated herein by reference]. That is VDJ
It also contains a transcription initiation site that initiates binding to form primary repertoire B cells [Yancopoulos and Alt, Cell.
40 : 271-281 (1985); which is incorporated herein by reference]. Alternatively, instead of the 23 kb SfiI / SpeI fragment, a 36 kb BssHII / SpeI fragment containing part of the D region can be used. The use of such fragments is efficient for D-
Increase the amount of 5'flanking sequences that promote J-joining.

The human D region has 4 or 5 tandemly linked regions.
Consisting of 9 homologous 9 kb subregions [Siebenlist et al., Natu
re 294: 631-635 (1981)]. Each subregion contains up to 10 individual D segments. Some of these segments have been mapped and are shown in FIG. The minilocus D region is created using two different strategies. The first strategy is to use only those D segments placed in a continuous strand of short DNA containing one or two repeating D subregions. The candidate is a single 15 kb fragment containing 12 individual D segments.

This fragment of DNA contains two consecutive EcoRI
It consists of fragments and is fully sequenced [Ichi
Hara et al., EMBO J. 7: 4141-4150 (1988)]. Twelve D segments would be sufficient for the primary repertoire. However, given the dispersive nature of the D region, another strategy is to ligate several discontinuous D fragment containing fragments together to create a smaller DNA fragment with a larger number of segments. is there. For example, additional D segment genes can be identified by the presence of characteristic flanking nonamer or heptamer sequences (anterior) and by reference to the literature.

At least one, and preferably multiple V gene segments are used to create the heavy chain minilocus transgene. With or without flanking sequences,
The rearranged or unrearranged V segment is a "transgenic non-human animal C capable of producing a heterologous antibody".
apable of Producing Heterologous Artibodies). PCT Publication WO 92/0391 published on March 19, 1992
It can be isolated as described in 8.

The rearranged or unrearranged V segment, D segment, J segment and C gene with or without flanking sequences are described in PCT Publication WO 92/03918, published Mar. 19, 1992. Can be isolated as described in. The minilocus light chain transgene is
It can be similarly prepared from the human λ or κ immunoglobulin loci. That is, for example, a plurality of DNs
From the A fragment, at least 2, 3 or 4 DNA fragments, coding for V, D, J and constant region sequences, each sequence being substantially homologous to a human gene sequence, eg about 75 kb The globulin heavy chain minilocus transgene construct can be formed. Preferably, the sequences are operably linked to transcriptional regulatory sequences and are capable of undergoing rearrangement. Switch recombination also occurs at two or more appropriately placed constant region sequences (eg, μ and γ) and the switch region. A typical light chain transgene construct is described in co-pending application USS filed August 29, 1990.
Multiple DNAs that are substantially homologous to human DNA and are capable of undergoing rearrangement, as described in N 07 / 574,748.
The fragments can be similarly formed.

E. Can receive an isotype switch
Transgene constructs Ideally, the transgene construct that is to undergo the class switch should contain all of the cis-acting sequences necessary to regulate the non-fertile transcript. Naturally occurring switch regions and upstream promoter and regulatory sequences (eg IFN-inducible elements) are preferred cis-acting sequences contained in transgene constructs capable of undergoing isotype switching. A sequence of at least about 50 base pairs, preferably at least about 200 base pairs, more preferably at least 500-1000 base pairs or more immediately upstream of the switch region, preferably the human γ1 switch region, is present in the switch sequence, preferably human γ1.
It will be operably linked to the switch array. In addition, the switch region can be linked next to the particular switch region upstream (and in the vicinity) of the non-naturally occurring _CH gene. For example, without limitation, human γ
The 1 switch region may be linked upstream of the human α2C _H gene or the mouse γ1 switch region may be linked to the human C _H gene.

An alternative method for obtaining non-classical isotype switches (eg δ-related deletions) in transgenic mice is a 400 bp direct repeat sequence (σμ and εμ) flanking the human μ gene [Yasui et al. Eur. J. Immunol.
19 : 1399 (1989)]. Homologous recombination between those two sequences results in the deletion of the μ gene in IgD-only B cells. The heavy chain transgene can be represented by the formula: (V _H ) _x- (D) _y- (J _H ) _z- (S _D ) _m- (C ₁ ) _n -[(T)-
(S _A ) _p − (C ₂ )] _{q In the} above formula, V _H is a heavy chain variable region gene segment,
D is a heavy chain D (diversity) region gene segment, J
_H is a heavy chain J (connecting) region gene segment,

S _D is _a donor region segment that can participate in recombination events with the S _a acceptor region segment such that isotype switching occurs, and C ₁ is an isotype used in B cell development (eg μ Or δ)
A heavy chain constant region gene segment encoding
Is a cis-acting transcriptional regulatory region segment containing at least a promoter, and S _A is an acceptor region segment capable of participating in recombination with a selected S _D donor region segment such that isotype switching occurs. And

C ₂ is an isotype other than μ (eg, γ ₁ ,
γ ₂ , γ ₃ , γ ₄ , α ₁ , α ₂ , ε) is a closed constant region gene segment encoding x, y, z, m,
n, p and q are integers, x is 1 to 100 and n is 0.
10, y is 1-50, p is 1-10, z is 1-50, q is 0-5
0 and m are 0-10. Typically, when the transgene is capable of undergoing isotype switching, q is at least 1, m is at least 1, n is at least 1, and m is greater than or equal to n. There must be.

The V _H , D, J _H , S _D , C ₁ , T, S _A and C ₂ segments may be selected from various species, preferably mammalian species, more preferably human and mouse germline DNA. it can. V _H segments, V _H segment which is naturally present in the human germ cells, e.g. V _H251
You can choose from. Typically about 2 V _H
Segments are included, preferably about 4 and more preferably at least about 10 _VH segments.

Typically at least one D segment is included, but preferably at least 10 D segments are included, and in some embodiments 10 or more D segments are included. Some preferred embodiments include human D segments. Although typically at least one J _H segment is included in the transgene, it is preferred that it contains about 6 J _H segments, and some preferred embodiments will contain about 6 or more J _H segments. Does not contain any human J _H segment.

The S _D segment is the S _{A of the} transgene
It is a donor region that can participate in the recombination phenomenon with the segment. For classic isotype switches, S _D
And S _A region is Sμ, Sγ ₁ , Sγ ₂ , Sγ ₃ , Sγ
Switch regions such as ₄ , Sα, Sα ₂ and Sε. Preferably, the switch region is mouse or human, more preferably S _D is human or mouse Sμ and S _A is human or mouse Sγ ₁ . In nonclassical isotype switching (δ-related deletion), S _D and S _A
The region is preferably a 400 base pair direct repeat sequence flanking the human mu gene.

The C ₁ segment is typically μ or δ
It is a gene, preferably a μ gene, and more preferably a human or mouse μ gene. The T segment typically contains 5'flanking sequences which flank the naturally occurring (ie germline) switch region. The T segment is typically at least about 50 nucleotides in length, preferably at least about 200 nucleotides in length, and more preferably at least 500-1000 nucleotides in length.
Preferably the T segment is a 5'flanking sequence which is located immediately upstream of the human or mouse switch region in the germline configuration. The T segment comprises a cis-acting transcriptional regulatory sequence not naturally present in animal germ cells (eg, viral enhancers and promoters such as those found in SV40, adenovirus, and other viruses that infect eukaryotic cells). However, it is obvious to those skilled in the art.

The C ₂ segment is typically γ ₁ ,
It is a γ ₂ , γ ₃ , γ ₄ , α ₁ , α ₂ or εC _H gene, preferably an isotype of the human C _H gene, and more preferably a human γ ₁ or γ ₃ gene. Mouse γ _2a and γ _2b may be used as downstream (switched) isotype genes from various species. If the heavy chain transgene comprises an immunoglobulin heavy chain minilocus, the total length of the transgene will typically be less than 150 kilobase pairs.

In general, the transgene will be other than the native heavy chain Ig locus. For example, there may be deletions of unwanted regions or substitutions with corresponding regions from other species.

F. Functional isota in the Ig transgene
Methods of Determining Ipswitches The occurrence of isotype switches in transgenic non-human animals can be identified by any method known in the art. Preferred embodiments include the following, which are used alone or in combination. 1. At least one transgene downstream C _H other than δ
Sequence and the transgene is homologous to a gene V _H -D _H -
Detection of mRNA transcripts containing flanking sequences homologous to the J _H rearranged gene; such detection includes Northern hybridization, S ₁ nuclease protection assays,
It can be by PCR amplification, cDNA cloning or other methods.

2. In the serum of transgenic animals,
Alternatively, detection of an immunoglobulin protein encoded by a downstream C _H gene in the culture supernatant of hybridoma cells prepared from B cells of the transgenic animal. It is also possible here to prove by immunochemical methods that such proteins comprise functional variable regions.

3. Detection of DNA rearrangements in DNA from B cells of transgenic animals or in genomic DNA from hybridoma cells, consistent with the occurrence of an isotype switch in the transgene. Such detection can be accomplished by Southern blot hybridization, PCR amplification, genomic cloning or other methods.

4. Other signs of isotype switching, such as production of non-fertile transcripts, production of characteristic enzymes involved in the switch (eg "switch recombinase"), or detection, measurement or observation by current techniques. Identification of other indications that can be made. Each transgenic line represents a different integration site of the transgene and a potentially different tandem alignment of transgene inserts, and different placements of each transgene and flanking DNA sequences can affect gene expression. It is preferred to identify and use mouse strains that express high levels of human immunoglobulins, particularly those of the IgG isotype, and that contain the lowest copy number of the transgene. Single copy transgenic animals minimize the problem of possible defective allele expression.

The transgene is typically host chromosome D
In NA, most commonly incorporated into germ cell DNA,
It is then bred by the subsequent breeding of germline transgenic breeding animals. However, the practitioner can, if appropriate and desired, substitute other vectors and transgenic methods known in the art or later developed.

Transswitches to endogenous non-human heavy chain constant region genes may occur to produce chimeric heavy chains and antibodies containing such chimeric human / mouse heavy chains. Such chimeric antibodies may be desirable for some of the uses described herein, but may not be desirable in some cases.

G. Functions of endogenous immunoglobulin gene chains
Disruption successful in rearranged immunoglobulin heavy and expression of light chain transgenes is expected to have a dominant effect by suppressing the rearrangement of the endogenous immunoglobulin genes in the transgenic nonhuman animal . However, another way to generate non-human animals lacking endogenous antibodies is by mutating the endogenous immunoglobulin loci. The fetal stem cell technology and homologous recombination can be used to easily eliminate the endogenous immunoglobulin repertoire. The following describes the functional disruption of the mouse immunoglobulin loci. However,
The disclosed vectors and methods can be readily modified for use in other non-human animals.

Briefly, this technique involves the inactivation of genes by homologous recombination in pluripotent cell lines capable of differentiating into germ cell tissues. A DNA construct containing altered copies of mouse immunoglobulin is introduced into the nucleus of fetal stem cells. In some of the cells, it recombines with the endogenous copy of the introduced DNA mouse gene and replaces it with the altered copy. Cells containing the newly engineered genetic lesion are injected into host mouse embryos and reimplanted into recipient females. Some of these embryos develop into chimeric mice with germ cells that are entirely derived from the mutant cell line. Therefore, by mating chimeric mice,
It is possible to obtain a new strain of mice containing the introduced genetic lesion [Capecchi (1989) Science, 244,
1288-1292].

Since the mouse λ locus contributes only 5% of immunoglobulins, inactivation of the heavy and / or kappa light chain loci is sufficient. There are three ways to disrupt each of these loci, deletion of the J region, J
-Deletion of the C intron enhancer and disruption of the constant region coding sequence by the introduction of a stop codon. DNA
From a component design standpoint, the last option is the easiest to choose. Exclusion of the μ gene disrupts B cell maturation, thereby preventing class switching to any functional heavy chain segment. The strategy for knocking out those loci is outlined below.

To disrupt the mu and kappa genes in mice, Jaenisch and coworkers [Zijlstra et al. (1989), Na
ture , 342, 435-438] for the successful disruption of the mouse β2 microglobulin gene. The neomycin resistance gene (neo) from plasmid pMCIneo is inserted into the coding region of the target gene. The pMCIneo insert uses a hybrid viral promoter / enhancer sequence to direct neo expression. This promoter is active in fetal stem cells. Thus, neo can be used as a selectable marker for the incorporation of knockout constructs.
HSV as a negative selection marker for the random insertion phenomenon
A thymidine kinase (tk) gene is added to the end of the construct (Zijlstra et al., Supra).

The preferred strategy for disrupting the heavy chain locus is the deletion of the J region. This region is quite small in mice, extending to only 1.3 kb. To create a gene targeting vector, a 15 kb KpnI fragment containing all of the secreted A constant region exons is isolated from a mouse genomic library. The 1.3 kb J region is replaced by the 1.1 kb insert from pMCIneo. The HSV tk gene is then added to the 5'end of the KpnI fragment. Correct integration of this construct by homologous recombination will result in replacement of the mouse _JH region with the neo gene. Recombinants are screened by PCR using a primer based on the neo gene and a primer homologous to the 5'mouse sequence of the KpnI site in the D region.

Alternatively, the heavy chain locus is destroyed (knocked out) by destroying the coding region of the μ gene. This approach requires the same 15 kb Kpn I fragment used in the above approach. The 1.1 kb insert from pMCIneo is inserted at exon II at the unique BamHI site and the HSK tk gene is added to the 3'Kpn I terminus. Select the double crossover on one side of the neo insert, which deletes the tk gene. They are,
Detected by PCR amplification from a pool of selected clones. One of the PCR primers is derived from the neo sequence,
And the other comes from the mouse sequence outside the targeting vector. Functional disruption of the mouse immunoglobulin locus is described in the examples.

G. Expression of endogenous immunoglobulin loci
In addition to functional disruption of the endogenous Ig locus,
Another way to prevent expression of a locus is suppression. Endogenous I
Suppression of the g gene can be achieved by antisense RNA produced by one or more integrated transgenes, by antisense oligonucleotides, and / or by administration of antisera specific for one or more endogenous Ig chains. Can be achieved. Antisense Polynucleotides Antisense RNA transgenes can be used to partially or totally disrupt the expression of specific genes [Pepin et al. (1991) Nature 355: 725; Helene, C.
And Toulme, J. (1990) Biochi - mica Biophys. Acta
1049 : 99; Stout, J. and Caskey, T. (1990) Somat.
Cell Mol. Genet. 16 : 369; Munir et al. (1990) Soma
t. Cell Mol. Genet. 16 : 383; each of these is incorporated herein by reference].

“Antisense polynucleotide” refers to
(1) Reference sequence, for example C of endogenous Ig _HOr C_Lregion
Complementary to all or part of the sequence, and (2) complementary
Target sequences such as chromosomal loci or Ig mRNA
A polynucleotide that specifically hybridizes.
Such complementary antisense polynucleotides have
Specific hybridization to the contiguous target sequence is
As long as it is retained as a functional property of the polynucleotide,
It may include nucleotide substitutions, additions, deletions or rearrangements.
Yes.

Complementary antisense polynucleotides are those that hybridize specifically to an individual mRNA species and prevent transcription and / or RNA processing of the mRNA species and / or translation of the encoded polypeptide, Soluble antisense RNA or DNA oligonucleotides may be mentioned [Ching et al . , Proc. Natl.
Acad. Sci. USA 86 : 10006-10010 (1989); Broder et al.,
Ann. Int. Med. 113: 604-613 (1990); Loreau et al., FE
BS Letters 274: 53-56 (1990); Holcenberg et al., W09
1/11535; USSN 07 / 530,165 ("Novel human CRIPTO gene"); WO91 / 09865; WO91 / 04753; WO90 / 13641 and EP 386563; each of which is incorporated herein by reference].

Antisense sequences have a length of at least about.
A polynucleotide sequence that is complementary to at least one immunoglobulin gene sequence of 15 consecutive nucleotides, more preferably about 30 or more consecutive nucleotides in length. However, in some embodiments, the antisense sequence can have substitutions, additions or deletions relative to the complementary immunoglobulin gene sequence, so long as specific hybridization is retained as a property of the antisense polynucleotide. Generally, the antisense sequence is complementary to an endogenous immunoglobulin gene sequence that encodes an immunoglobulin chain or, after DNA rearrangement, has the potential to encode an immunoglobulin chain. In some cases, sense sequences that correspond to immunoglobulin gene sequences may act to suppress expression, especially by interfering with transcription.

Accordingly, antisense polynucleotides are
Inhibits production of the encoded polypeptide. In this regard, antisense polynucleotides that inhibit the transcription and / or translation of one or more endogenous Ig loci are used in non-human animals capable of producing immunoglobulin chains encoded by the endogenous Ig loci and / or Or the specificity can be changed.

Antisense polynucleotides can be expressed in transfectant cells or transgenic cells,
For example, it can be produced from a heterologous expression cassette in transgenic pluripotent hematopoietic stem cells used to reconstitute all or part of the hematopoietic stem cell population of an individual, or in transgenic non-human animals. Alternatively, the antisense polynucleotides may comprise soluble oligonucleotides that are administered to the external environment, either in culture in vitro or in vivo in the circulatory system or interstitial fluid. Soluble antisense oligonucleotides present in the external milieu approach the cytoplasm and reach specific mR
It has been proven to inhibit translation of NA species.

In some embodiments, the antisense polynucleotide comprises a methylphosphonate component, or phosphorothionate or O-methylribonucleotides may be used, and chimeric oligonucleotides may be used. Dagle et al. (1990) Nucleic Acids Re
s. 18 : 4751]. For some applications, antisense oligonucleotides may include polyamide nucleic acids [Nielsen
(1991) Science 254: 1497]. For general information about antisense polynucleotides, see Antisense RNA
and DNA (1988), DA Melton, Cold Spring Harbor
See Labo-ratory, Cold Spring Harbor, NY.

Antisense polynucleotides complementary to one or more sequences are used to inhibit transcription, RNA processing, and / or translation of a cognate mRNA species, thereby determining the amount of each polypeptide encoded thereby. Reduce. Such antisense polynucleotides can provide a therapeutic function by inhibiting the formation of one or more endogenous Ig loci in vivo.

The antisense polynucleotides of the invention, either as soluble antisense polynucleotides or as antisense RNA transcribed from the antisense transgene, are preferential to endogenous Ig sequences in physiological conditions in vivo. Selected to hybridize to Most typically, the selected antisense polynucleotides will not hybridize to the heterologous Ig sequences encoded by the heavy or light chain transgenes of the invention so much (ie, the antisense oligonucleotide will contain about 25-35% or more Transgene I
Will not inhibit gf expression). Antiserum Suppression Partial or complete suppression of endogenous Ig chain expression can be achieved by injecting mice with antiserum against one or more endogenous Ig chains [Weiss et al. (1984) P
roc. Natl. Acad. Sci. USA 81 : 211; which is incorporated herein by reference]. The antisera react specifically with one or more endogenous Ig chains, but the Ig of the invention
It is selected to have minimal or no reactivity with the heterologous Ig chains encoded by the transgene. Thus, administration of selected antisera according to a schedule, such as that typified by Weiss et al. (Supra), suppresses endogenous Ig chain expression but encodes one or more heterologous Ig encoded by the transgene of the invention. Allow expression of chains.

Suitable antibody sources of antibodies include the following: (1) those which bind specifically to mouse μ, γ, κ or the incoming chain but are encoded by the human Ig transgene of the invention. A monoclonal antibody, such as a monoclonal antibody, that does not react with human immunoglobulin chains; Or a mixture of such monoclonal antibodies which bind to multiple epitopes and multiple chains or endogenous immunoglobulins; (3) a polyclonal antiserum or a mixture thereof. Typically, such an antiserum (single chain) will bind to a single species of endogenous Ig chain (eg mouse μ, mouse γ, mouse κ, mouse λ) or multiple species of endogenous Ig chain). Most preferably, such antisera have negligible avidity for human immunoglobulin chains encoded by the transgenes of the invention: and / or

(4) A polyclonal which binds to a single or multiple species of endogenous Ig chain and most preferably has negligible binding strength to the human immunoglobulin chain encoded by the transgene of the present invention. A mixture of antisera and monoclonal antibodies. Polyclonal antibodies are generally preferred and such substantially monospecific polyclonal antibodies are advantageously of a non-human animal species (eg mouse).
Affinity adsorption of the antiserum or a purified fraction thereof on an affinity resin containing, for example, immobilized human Ig and preadsorption with an antibody derived from Enriched for the desired anti-human Ig, this bound fraction can be produced from antisera raised against human immunoglobulin by standard elution at low pH or chaotropic salt solution).

Cell division and / or complement fixation can be used to enhance cell depletion induced by antibody to lymphocytes expressing endogenous (eg, murine) immunoglobulin chains. For example, in one embodiment, the antibody is utilized for ex vivo depletion of explanted hematopoietic cells expressing mouse Ig and / or B-lineage lymphocytes obtained from transgenic mice harboring the human Ig transgene. To be done. Thus, hematopoietic cells and / or B lineage lymphocytes are explanted and explanted from a non-human transgenic animal that harbors a human Ig transgene (preferably harboring both a human heavy chain transgene and a human light chain transgene). The transplanted cells bind (1) to endogenous immunoglobulins (eg mouse mu and / or kappa),
(2) Incubated with antibody (s) that lack substantial binding to the human immunoglobulin chain encoded by the transgene.

Such antibodies are referred to as "suppressor antibodies" for clarity. The explanted cell population is selectively depleted of cells that bind inhibitory antibody (s); such depletion is (1) physical separation to remove inhibitory antibody-bound cells from unbound cells. (For example, the suppressor antibody can be bound to a solid support or magnetic beads to immobilize and remove cells that bind to the suppressor antibody),
(2) Antibody-dependent cell killing of cells bound by inhibitory antibodies (eg, by ADCC, complement fixation, or toxins linked to inhibitory antibodies), and (3) clonal anergy induced by inhibitory antibodies, etc. Can be achieved by various methods.

Frequently, the antibodies used to suppress antibody production of endogenous Ig chains can fix complement. Semi-vivo / in such as rabbit or guinea pig
It is often desirable to be able to select such antibodies so that they react well with a convenient source of complement for vitro depletion. I
For in vivo depletion, it is generally preferred that the inhibitory antibody have effector function in transgenic animal species other than human. Thus, for use in transgenic mice, inhibitory antibodies containing mouse effector functions (eg, ADCC and complement fixation) are generally preferred.

In one variation, inhibitory antibodies that specifically bind to a predetermined endogenous immunoglobulin chain are used for ex vivo / in vitro depletion of lymphocytes that express endogenous immunoglobulin. Cell explants (eg, lymphocyte samples) from nonhuman transgenic animals that harbor the human immunoglobulin transgene are contacted with a suppressor antibody and cells that specifically bind to the suppressor antibody are depleted (eg, immobile). Cationization, complement fixation, etc.), thus producing a depleted cell subpopulation within cells expressing endogenous (non-human) immunoglobulin (eg, lymphocytes expressing mouse Ig).

The resulting depleted lymphocyte population (T cells, human Ig-positive B cells, etc.) is immunocompatible (ie, MHC matched) of the same species that is substantially incapable of producing endogenous antibodies. Non-human animals (eg SC)
ID mouse, RAG-1 or RAG-2 knockout mouse) can be transferred into the body. The reconstituted animal (mouse) is then immunized with the antigen (or reimmunized with the antigen used to immunize the donor animal that was donated with the explant), high affinity (affinity) Antibodies (matured) and B cells that produce such antibodies can be obtained. Such B cells can be used to generate hybridomas by conventional cell fusion and screened.
Antibody suppression can also be used in combination with other methods of inactivating / suppressing endogenous Ig (eg, J _H knockout, C _H knockout, D-region excision, antisense suppression, compensated frameshift inactivation). .

Complete Endogenous Ig Locus Inactivation In certain embodiments, hybrid immunoglobulin chains containing human variable regions and non-human (eg, murine) constant regions cannot be formed (eg, trans. It is desirable to effect a complete inactivation of the endogenous Ig locus) (by trans-switching between the gene and the endogenous Ig sequences). Knockout mice bearing an endogenous heavy chain allele that is functionally disrupted only in the J _H region (error with with which) has a relocated human variable region normally encoded by the transgene. (VDJ)
Frequently show transswitches expressed as fusion proteins linked to the endogenous mouse constant region, although other transswitched ligations are possible.

To overcome this potential problem, it is generally desirable to completely inactivate the endogenous heavy chain locus by any of a variety of methods including, without limitation, the following: : (1) Functionally disrupting and / or deleting at least one, and preferably all of the endogenous heavy chain constant region genes by homologous recombination, (2)
Mutating at least one and preferably all endogenous heavy chain constant region genes to encode a stop codon (or frameshift) and produce a truncated or frameshifted product (if transswitched) , And other methods and strategies apparent to those of ordinary skill in the art. Deletion of a substantial portion or all of the heavy chain constant region genes and / or D-region genes is accomplished by a variety of methods, including sequential deletions with homologous recombination targeting vectors, especially of the "hit end run" type. it can. Similarly, functional disruption and / or deletion of at least one endogenous light chain gene (eg, kappa) to excise an endogenous light chain constant region gene is often preferred.

Frequently, the heterologous transgene will be within the early region (ie, amino-terminal coding portion) of one or more (preferably all) of the frameshift and transgene constant region genes within the J segment (s). It is preferred to utilize a frame-shifted transgene such as that which includes a compensating frame-shift of (e.g., to reproduce the original open reading frame). Endogenous IgH
Transswitches to locus constitutive genes (those that do not include compensatory frameshifts) result in truncated or missense products, resulting in the transswitched B cells being deleted or not selected. Thus, the transswitched phenotype will be suppressed.

Perform substantially complete functional inactivation of endogenous Ig gene product expression (eg, mouse heavy and light chain sequences) and / or transswitched antibody (eg, human variable / mouse constant chimeric antibody). To do so, antisense suppression and antibody suppression can be used as well. Various combinations of inactivation and suppression strategies can be used to achieve essentially complete suppression of endogenous (eg, murine) Ig chain expression.

Trans-Switches In some variations, it may be desirable to produce trans-switched immunoglobulins. For example, such transswitched heavy chains can be chimeric (ie, non-mouse (human) variable regions and mouse constant regions). Antibodies containing such transswitched chimeric immunoglobulins are nonhuman (eg, mouse)
A variety of applications where it is desired to have a constant region (eg, for the retention of effector functions in the host for the presence of mouse immunological determinants, such as for the binding of secondary antibodies that do not bind the human constant region). Can be used for. As an example, for certain antigens, the human variable region potency may be advantageous over the mouse variable region potency (repertoire).

Presumably, the human V _H , D, J _H , V _L and J _L genes were selected during evolution due to their ability to encode immunoglobulins that bind certain evolutionarily important antigens. It may have been: The antigens that provided evolutionarily selective pressure for the mouse competence range could be quite different from the antigens that provided evolutionary pressure to shape the human competence range. is there. There may be other range of benefit advantages that, when combined with the mouse constant region (eg, of the transswitched mouse) isotype, favor human variable region coverage. The presence of the mouse constant region may provide advantages over the human constant region.

For example, a mouse γ constant region linked to a human variable region by a transswitch is a chimeric antibody (preferably monoclonal) reactive with a predetermined antigen (eg, human IL-2 receptor) T
Providing an antibody with mouse effector functions (eg ADCC, mouse complement fixation) so that lymphocytes can be tested in a mouse disease model, such as a mouse model of transplanted cells versus host disease expressing a functional human IL-2 receptor. You can. Following this, a human variable region coding sequence (eg, cDNA from a source (hybridoma clone)
A human sequence antibody which is isolated (by NA cloning or PCR amplification) and spliced into a sequence encoding the desired human constant region, which is more suitable for human therapeutic use, where immunogenicity is preferably minimized. It is possible to

The resulting polynucleotide (s) containing the fully human coding sequence (s) can be expressed in a host (eg, from an expression vector in mammalian cells) and purified for pharmaceutical purposes. is there. In some applications, chimeric antibodies can be used directly without replacing the mouse constant region with the human constant region. Other variations and uses of the transswitched chimeric antibody will be apparent to the party.

The present invention generally provides a non-human transgenic animal containing B lymphocytes expressing a chimeric antibody resulting from a transswitch between a human heavy chain transgene and an endogenous mouse heavy chain constant region gene. provide. Such chimeric antibodies generally contain mouse constant regions and human sequence variable regions, which are mouse switched (ie, non-μ, non-δ) isotypes. Non-human transgenic animals that are capable of producing chimeric antibodies against a predetermined antigen will typically have both human heavy and light chain transgenes encoding human variable and human constant region genes incorporated, It also has the ability to produce antibodies of fully human sequence.

Most typically, one or more endogenous heavy chain constant region genes (eg γ) that are homozygous for a functionally interrupted heavy and / or light chain locus, but which are transswitchable. , Α, ε), and frequently a cis-chain enhancer. Such mice are usually immunized with a predetermined antigen in combination with Ashbant, and a detectable amount of the chimeric chimera comprising a heavy chain consisting of a human sequence variable region linked to a mouse constant region sequence. An immune response including antibodies is generated. Typically, the sera of such immunized animals will be at least about 1 μg / ml, often more than about 10 μg / ml, frequently 30 μg.
Such chimeric antibodies may be included at concentrations of g / ml or higher, and up to 50-100 μg / ml or higher.

Antisera containing antibodies containing chimeric human variable / mouse constant region heavy chains typically also include antibodies containing human variable / human constant region (fully human sequence) heavy chains. A transswitched chimeric antibody usually comprises (1) a chimeric heavy chain consisting of a human variable region and a mouse constant region (normally mouse gamma) and (2) a human transgene-encoded light chain (typically B. kappa) or mouse light chain (typically lambda in the kappa knockout packground).

Such a transswitched chimeric antibody is generally about 1 × 10 ⁷ M ⁻¹ or more, preferably 5 × 10 ⁷
M ⁻¹ or more, more preferably 1 × 10 ⁸ M ⁻¹ to 1 × 10 ⁹ M ⁻¹
It binds to a predetermined antigen (eg, immunogen) with the above affinity. Frequently, the predetermined antigen is a human protein, such as a human cell surface antigen (eg CD4, CD8,
IL-2 receptor, EGF receptor, PDGF receptor) and other human biological macromolecules (eg thrombomodulin, protein C, carbohydrate antigen, sialyl-
Lewis antigen, L-selectin) or non-human disease-related macromolecules (eg bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.

The present invention provides a non-human transgenic animal which comprises a genome which includes: (1) (eg cis-link to a functional switch recombination sequence, typically a functional enhancer). In)
A homozygous functionally disrupted endogenous heavy chain gene containing at least one mouse constant region gene capable of transswitching; (2) rearranging to encode and express a functional human heavy chain variable region And transswitchable (eg cis-chained R
A human heavy chain transgene (with SS); and optionally (3) a human light chain (eg kappa) trans that can be rearranged to encode a functional human light chain variable region and express a human sequence light chain. Gene; and optionally,
(4) a homozygous functionally disrupted endogenous light chain locus (κ, preferably κ and λ); and optionally (5) a human sequence variable region encoded by a human transgene and an endogenous mouse. A serum containing an antibody containing a chimeric heavy chain consisting of a mouse constant region sequence encoded by a heavy chain constant region gene (eg, γ1, γ2a, γ2b, γ3).

Such a transgenic mouse is further provided with about 1 × 10 ⁴ M ⁻¹ or more, preferably about 5 × 10 ⁴ M ⁻¹ or more, more preferably 1 × 10 ⁷ M ⁻¹ to 1 × 10 ⁹ M ^−1. With the above affinity, a predetermined human antigen (eg, CD4, CD
8, CEA) can be included in the serum containing the chimeric antibody. Frequently, the monoclonal antibodies so produced can produce hybridomas with an affinity of at least 8 x 10 ⁷ M ^-1 . Chimeric antibodies containing heavy chains consisting of mouse constant regions and human variable regions, which are often capable of binding non-human antigens, can also be present in serum or as antibodies secreted from hybridomas.

In some variations, the gene has an inactivated endogenous mouse heavy chain locus carrying an intact heavy chain constant region gene and has a human heavy chain transgene capable of transswitching, optionally It is desirable to generate transgenic mice that also carry the human light chain transgene, with one or more inactivated endogenous mouse light chain loci. Such mice can advantageously produce, by transswitching, B cells capable of alternately expressing an antibody containing a fully human heavy chain and an antibody containing a chimeric (human variable / mouse constant) heavy chain. . This mouse serum includes antibodies containing fully human heavy chains and preferably antibodies containing chimeric (human variable / mouse constant) heavy chains in combination with fully human light chains. Be done. Hybridomas can be generated from the B cells of this mouse.

In general, such chimeric antibodies can be produced by transswitch, in which the human variable region and human constant region encoded by the in vivo productive VDJ rearrangement, typically human μ, are The encoding human transgene undergoes switch recombination with a non-transgene immunoglobulin homeostatic gene switch sequence (RSS) and thus typically encodes on the endogenous mouse immunoglobulin heavy chain homeostatic region or a second transgene. A non-homologous (eg, human) heavy chain constant region, which is a non-transgene-encoded human heavy chain constant region and a transgene-encoded human variable region. A cis-switch is an isotype switch that results from recombination of RSS elements in the transgene, and transswitch is often associated with transgene RSS on a chromosome different from the chromosome that harbors the transgene. Recombination between RSS elements outside the transgene is involved.

Transswitches generally involve the expression of the expressed transgene heavy chain constant region gene and the endogenous murine constant region gene (of the non-μ isotype, typically γ) or often on separate chromosomes. Occurring between either one of the RSS of the human constant region genes contained on the integrated second transgene. If the transswitch occurs between the RSS of the first, expressed transgene heavy chain constant region gene (eg, μ) and the RSS of the human heavy chain constant region gene contained on the second transgene, then A non-chimeric antibody having a fully human sequence is produced. As a non-limiting example, human heavy chain constant region (eg, γ1) and operably linked RS
Introducing a polynucleotide encoding S (eg, γ1 RSS) into a population of hybridoma cells generated from B cells (or B cell population) of transgenic mice expressing an antibody containing a human μ chain that has undergone transgene coding. (Eg, transfection).

The resulting hybridoma cells were transformed for the presence of the introduced polynucleotide and / or with the introduced polynucleotide sequence having the variable region (idiotype / antigen reactivity) of human μ chain ( For example, for expression of a transswitched antibody containing a heavy chain with a constant region encoded by human γ1),
You can choose. Transgene-encoded human μ chain RSS and downstream isotype (eg γ1)
Transswitch recombination between the RSS's of the introduced polynucleotides encoding the thus can produce transswitched antibodies.

The present invention also provides (1) a homozygous, functionally disrupted, endogenous endogenous gene containing at least one mouse constant region gene (eg, γ2a, γ2b, γ1, γ3) capable of transswitching. Heavy chain locus,
(2) A human heavy chain transgene capable of rearranging to encode a functional human heavy chain variable region, expressing a human sequence heavy chain, and capable of undergoing an isotype switch (and / or transswitch); Optionally (3) a human light chain (eg kappa) transgene capable of rearranging to encode a functional human light (eg kappa) chain variable region and expressing a human sequence light chain, and optionally ( 4) a human sequence encoded by a homozygous functionally disrupted endogenous light chain locus (typically kappa, preferably both kappa and lambda), and optionally (5) a human transgene. Variable region and endogenous mouse heavy chain constant region genes (eg, γ1, γ2a, γ2b,
γ3) such a transswitch comprising the step of immunizing a transgenic mouse containing a genome containing a serum containing an antibody containing a chimeric heavy chain consisting of a mouse constant region sequence, with a predetermined antigen. Also provided are methods for producing chimeric antibodies.

Affinity labeling: switched isotype
Selection Advantageously for the process of somatic mutation is associated with trans-switching (and cis switch). Somatic mutations extend the range of antibody affinities encoded by the clonal progeny of B cells. For example, antibodies produced by hybridoma cells that have undergone a switch (trans- or cis-) exhibit a broader range of antigen binding affinity than is present in hybridoma cells that have not undergone a switch. Therefore, the first human heavy chain constant region (eg, μ)
To a second heavy chain constant region (typically human γ) that expresses a hybridoma cell population (typically clonal) expressing a first antibody comprising a heavy chain comprising a first human heavy chain variable region in a polypeptide binding to , Α or ε constant region) can be screened for a hybridoma cell clone mutant expressing an antibody comprising a heavy chain comprising the first human heavy chain variable region in a polypeptide binding.

Such clonal mutants may be lymphokines (eg, IL-4 and IF) induced by natural clonal mutations that produce cisswitches in vitro or by T cells.
A heterologous (eg human) heavy chain constant region gene to serve as a substrate for a class switch (trans- or cis-), such as through administration of an agent that promotes an isotype switch such as Nγ), or as a substrate for a transswitch. And a functional RSS-containing polynucleotide, or a combination of the above, and the like. Polynucleotides containing human downstream isotype constant regions (eg, γ1, γ3, etc.) with operably linked RSS are often also introduced into hybridoma cells to switch isotype switches via the trans-switch mechanism. Facilitate.

Class switching and affinity maturation occur within the same population of B cells derived from the transgenic animals of the invention. Therefore, identification of class-switched B cells (or hybridomas derived therefrom) can be used as one screening step to obtain high affinity monoclonal antibodies. Various approaches can be utilized to facilitate class switch events such as cis-switch (transgene switch), transswitch or both. For example, a single contiguous human genomic fragment containing both mu and gamma constant region genes with associated RSS elements and switch regulatory elements (eg, a sterile promoter) can be used as a transgene.

However, a portion of the desired single contiguous human genomic fragment may be difficult to clone effectively, for example due to instability problems when replicated in cloning hosts and the like. : In particular, the region between δ and γ3 is the μ gene, γ3
It may prove difficult to clone as efficiently as flanking fragments containing genes, V genes, D gene segments and J gene segments.

Similarly, as an example, human μ gene, human γ3
Gene, human V gene (s), human D gene segment and human J gene segment, intervening (intron) or otherwise non-essential sequences (eg, single or multiple V, D, and / or J There are discontinuous human transgenes (minigenes), with single or multiple deletions of segments and / or single or multiple non-μ constant region genes). Such minigenes have several advantages over separating a single flanking segment of genomic DNA that spans all of the essential elements for efficient immunoglobulin expression and switching.

For example, such a minigene eliminates the need to separate large fragments of DNA that may contain sequences that are difficult to clone (eg, unstable sequences, toxic sequences, etc.). In addition, mini loci containing elements necessary for isotype switching for cis- or trans-switches (eg, the human γ-sterile promoter) are advantageously subject to somatic mutations and class switching in vivo. be able to. Since many eukaryotic DNA sequences have proven difficult to clone, it has proved advantageous to delete the non-essential sequences.

In a variant, an antibody with a high binding affinity (eg 1 × 10 ⁷ M ⁻¹ or more, preferably 1 × 10 ⁸ M ⁻¹ or more, more preferably 1 × 10 ⁹ M ⁻¹ or more) is used. The hybridoma clone that produces is productive (framed)
From B cells of a transgenic mouse harboring a human heavy chain transgene capable of isotype switching that is substantially lacking an endogenous mouse heavy chain locus capable of undergoing VDJ rearrangement (see above) From the pool of induced hybridoma cells, one obtains by selecting hybridomas that express an antibody containing a heavy chain containing a human sequence heavy chain variable region in polypeptide binding to a human (or mouse) non-μ heavy chain constant region. These antibodies are termed "switched antibodies" because they include "switched heavy chains" produced as a result of cis- and / or trans-switching in vivo or in cell culture.

Hybridomas that produce switched antibodies have generally undergone a somatic mutation process and this pool of hybridomas will generally have a broader range of antigen binding affinities, from among which high affinity antibodies. Hybridoma clones that secrete Normally, a human sequence antibody having a substantial binding affinity (1 × 10 ⁷ M ⁻¹ to 1 × 10 ⁸ M ⁻¹ or more) for a predetermined antigen is secreted, and this human sequence antibody is secreted by human immunity. Hybridomas containing globulin variable region (s) can be selected by methods involving a two-step process. One step is to bind the hybridoma cells to an immobilized immunoglobulin that does not substantially bind to (eg, specifically binds to a switched heavy chain and does not substantially bind to an unswitched isotype, eg, μ). ), To identify and isolate hybridoma cells that secrete immunoglobulins containing switched heavy chains.

Another step is (eg ELISA of hybridoma clone supernatant, using labeled antigen).
The purpose is to identify hybridoma cells that bind a predetermined antigen with a substantial binding affinity (such as by FACS analysis). Typically, the selection of hybridomas secreting the switched antibody is done prior to identifying hybridoma cells that bind the predetermined antigen. Hybridoma cells expressing a switched antibody with a substantial binding affinity for a predetermined antigen are isolated and cultured under suitable growth conditions known in the art, typically as individual selected clones. To be done.
Optionally, the method comprises culturing the selected clone under conditions suitable for expression of the monoclonal antibody. This monoclonal antibody can be collected and administered for therapeutic, prophylactic and / or diagnostic purposes.

Frequently, selected hybridoma clones will be used to isolate immunoglobulin sequences encoding immunoglobulins (eg, variable regions) that bind (or confer on the binding capacity) of a predetermined antigen. It can serve as a source of DNA or RNA. Following this, the human variable region coding sequences are isolated (eg, PC
C from R amplification or source (hybridoma clone)
(By DNA cloning), preferably splicing to a sequence encoding the desired human constant region to encode a human sequence antibody more suitable for therapeutic use in humans where immunogenicity is minimized. The resulting polynucleotide (s) with fully human coding sequence (s) can be expressed in host cells (eg from expression vectors in mammalian cells) and purified for pharmaceutical purposes. is there.

Xeno (heterologous) enhancers Heterologous transgenes capable of encoding human immunoglobulins (eg heavy chains) are advantageously derived from the mouse genome and / or with exons of the heterologous transgene. It contains a cis-linked enhancer that is not naturally associated with cis. For example, a human κ transgene (e.g., κ minilocus) can advantageously, may contain human V _K gene, the human J _K gene, the human C _K gene and xenon Bruno enhancer, Typically, this The xeno-enhancer comprises a human heavy chain intron enhancer and / or a mouse heavy chain intron enhancer, which is typically between the J _K gene and the C _K gene or located downstream of the C _K gene. For example, the mouse heavy chain J-μ intron enhancer (Banerji
et al. (1983) Cell 33 : 729) is a plasmid pKVe2.
Can be isolated on a 0.9 kb Xba I fragment of
See ).

The human heavy chain J-μ intron enhancer (Hayday et al. (1984) Nature 307 : 334) was 1.4 k
It can be isolated as a Mlu I / Hind III fragment of b (see below ). Addition of a transcriptionally active xeno enhancer to the transgene, such as a combined xeno enhancer consisting essentially of a human J-μ intron enhancer linked in cis to the mouse J-μ intron enhancer, is particularly If the transgene encodes a light chain such as human kappa, it can confer high expression levels of the transgene. Similarly,
The rat 3'enhancer can advantageously be included within a minilocus construct that can encode a human heavy chain.

Nucleic acid The term “substantially homologous” refers to at least about 80% nucleotides, usually at least about 90% when two nucleic acids or designed sequences thereof are optimally aligned and compared.
It is pointed out that% -95%, more preferably at least about 98% -99.5% nucleotides are identical. Alternatively,
Substantial homology exists when a segment hybridizes to the complement of its strands under selective hybridization conditions. The nucleic acid can be present in whole cells, in cell lysates, or in partially purified or substantially pure form.

Nucleic acids may be labeled with other cellular components or other contaminants, such as by alkaline / SDS treatment, CsCl band precipitation, column chromatography, agarose gel electrophoresis and other methods known in the art by standard techniques, eg, "Isolated" or "substantially pure" when separated and purified from other cellular nucleic acids or proteins. F.
Ausubel et al., Current Protocols in Molecular Biolog
y , Greene Publishingand Wiley-Interscience, New Yo
See rk (1987).

The nucleic acid compositions of the present invention often mutate the native sequence (excluding modified restriction sites, etc.), either from cDNA, genomic DNA or a mixture, according to standard techniques for providing gene sequences. be able to. For coding sequences, these mutations may affect the amino acid sequence if desired. Especially the native V,
DNA sequences that are substantially homologous to or derived from D, J, constants, switches and other such sequences described herein are contemplated (where "derivative" is meant). , Point out that one sequence is identical or has been modified with another sequence).

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer acts on the transcription of a coding sequence, they are operably linked to the coding sequence. For transcriptional regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, if necessary to concatenate the two protein coding regions, contiguous and read. It means within the frame. For switch sequences, operably linked means that the sequences are capable of switch recombination.

[0207]Certain preferred embodiments Preferred embodiments of the invention are described in Examples 5, 6, 8 or 14.
Animal containing a single copy of the described light chain transgene
And the transgene described in Example 12 (eg,
at least one copy of pHC1 or pHC2), typically
Includes 2 to 10 copies, sometimes 25 to 50 or more.
Animal having, and J described in Example 10 _HFrom the deletion animal
It is a descendant that was bred. Animals have these three characteristics
And are mated to homozygous.

Such animals have the following genotypes:
Single copy (per haploid pair of chromosomes) of the unrearranged human heavy chain minilocus (described in Example 12), rearranged human kappa light chain construct (described in Example 14). A single copy (per haploid pair of chromosomes) and functional J _H
Deletions at each endogenous mouse heavy chain locus that removes all of the segments (described in Example 10). When such an animal is bred to a mouse that is homozygous for the J _H segment deletion (Example 10), it is homozygous for the J _H deletion and hemizygous for the human heavy and light chain constructs. Produce offspring that are. The resulting animals are injected with antigens and used to produce human monoclonal antibodies against those antigens.

B cells isolated from such animals are
It is monospecific with respect to human heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monospecific for human or mouse heavy chains.
Because both endogenous mouse heavy chain gene copies are non-functional due to the deletion spanning the J _H region introduced as described in Examples 9 and 12. Furthermore, a substantial portion of B cells will be monospecific for human or mouse light chains. Because rearranged human kappa
This is because expression of a single copy of the light chain gene will allelically and isotopically eliminate rearrangements of the endogenous mouse kappa and lambda chain genes in a substantial portion of B cells.

The transgenic mice of the preferred embodiment will exhibit immunoglobulin production with a significant repertoire, ideally substantially the same as that of the native mouse. For example, in an embodiment where the endogenous Ig gene is inactivated, the total immunoglobulin level is about 0.1-10 mg.
/ Ml serum, preferably 0.5-5 mg / ml, ideally at least about 1.0 mg / ml. When a transgene capable of switching from IgM to IgG is introduced into transgenic mice, the ratio of mature serum IgG to IgM mice is preferably about 10: 1. Of course, IgG vs. Ig
The M ratio will be much lower in immature mice. In general,
Greater than about 10% of spleen and lymph node B cells, preferably 40-80%, express exclusively human IgG protein.

The repertoire will ideally approximate that shown in non-transgenic mice and will usually be at least about 10% higher, preferably 25-50% or higher. Usually at least about 1000 different immunoglobulins (ideally, depending on the number of different V, J and D regions introduced into the mouse genome (ideally
IgG), preferably 10 ⁴ to 10 ⁶ or more immunoglobulins will be produced. The immunoglobulins will typically recognize about half or more of the highly antigenic proteins.

Examples of the antigenic protein include pigeon cytochrome C, chicken lysozyme, pokeweed mitogen (PWM), bovine serum albumin, mussel hemocyanin, influenza hemagglutinin, staphylococcus protein A, sperm whale myoglobin, influenza neuraminidase and Examples include, but are not limited to, the lambda repressor protein. Some of the above immunoglobulins, against preselected antigens,
At least about 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 to 10 ⁹ M ^-1
Or it will show greater affinity.

In some embodiments, it may be preferable to generate mice with a predetermined potency range in order to limit the selection of V genes represented in the antibody response to the predetermined antigen type. There is a nature. Heavy chain transgenes with a predetermined potency range can include, for example, the human _VH gene, which is preferably used in antibody response to a predetermined antigen type in humans. Alternatively, varying extents (eg, low probability of encoding a high affinity V region for a predetermined antigen; less prone to somatic mutations and affinity exaggeration; or certain human immunogens). Some _VH genes can be excluded from the defined potency range.

Prior to rearrangement of the transgene containing various heavy or light chain gene segments, those gene segments may be labeled, for example, by hybridization or DN.
By A sequencing, it can be easily identified as originating from a species other than the transgenic animal. Although preferred embodiments of the transgenic animals of the invention have been described above, other embodiments are defined by the disclosure herein and more specifically by the transgenes described in the Examples. Four categories of transgenic animals can be defined:

I. A transgenic animal containing an unrearranged heavy chain immunoglobulin transgene and a rearranged light chain immunoglobulin transgene. II. A transgenic animal containing an unrearranged heavy chain immunoglobulin transgene and an unrearranged light chain immunoglobulin transgene. III. A transgenic animal containing a rearranged heavy chain immunoglobulin transgene and an unrearranged light chain immunoglobulin transgene.

IV. A transgenic animal containing a rearranged heavy chain immunoglobulin transgene and a rearranged light chain immunoglobulin transgene. A preferred preference for the above categories of transgenic animals is II>I>III> IV if the endogenous light chain gene (or at least the kappa gene) is disrupted by homologous recombination (or otherwise). , And I>II>III> IV if the endogenous light chain gene is not disrupted and must be dominant by allelic exclusion.

[0219]

EXAMPLES Methods and Materials Transgenic masses are described by Hogan et al., "Manipulatin.
g the Mouse Embryo: A Laboratory Manual ", Cold Spr
ing Harbor Laboratory, which is incorporated herein by reference.

Embryonic stem cells are engineered according to published methods [Terato-carcinomas and embryonic stems.
cells: a practical approach, EJ Robertson, IR
L Press, Washington, DC, 1987; Zjilstra et al., Natu
re 342: 435-438 (1989) and Schwartzberg, P. et al., Sc.
ience 246: 799-803 (1989); each of which is incorporated herein by reference].

The DNA cloning method is described in J. Sambrook.
Et al., Molecular Cloning: A Laboratory Manual, 2nd
Edition, 1989, Cold Spring Harbor Labo-ratory Press, Co
ld Spring Harbor, NY, which is incorporated herein by reference. Oligonucleotides are labeled with Appl according to the specifications given by the manufacturer.
Synthesized on an oligonucleotide synthesizer from ied Biosystems.

Hybridoma cells and antibodies are labeled as "Antib
odies: A Laboratory Manual ", Harlow and David La
ed., Cold Spring Harbor Labo-ratory (1988), which is incorporated herein by reference.

Example 1 Genomic Heavy Chain Human Ig Transgene This example describes cloning and microinjection of human genomic heavy chain immunoglobulin transgene microinjected into mouse zygotes.

Marzluff, WF et al., "Transcription and
Translation: A Pra-ctical Approach ", BD Hammes
And SJ Higgins, 89-129, IRL Press, Oxford
Nuclei are isolated from fresh human placental tissue as described by (1985). The isolated nuclei (or human spermatocytes washed with PBS) were embedded in a low-melting point agarose matrix, dissolved in EDTA and proteinase K to expose high molecular weight DNA, and this DNA was then analyzed by M. Finney. Protocols in Molecular Biology (F. Ausu
edited by bel et al., John Wiley & Sons, 4th Supplement, 1988, No. 2.5.1
Restriction enzyme Not in agarose as described in
Digest with I.

The Not I digested DNA was then digested with Anand,
Fractionation by pulsed field gel electrophoresis as described by R. et al . , Nucl. Acids Res. , 17 : 3425-3433 (1989). Fractions rich in Not I fragment are assayed by Southern blot hybridization to detect one or more sequences encoded by this fragment. Such sequences include the heavy chain D segment, J segment, μ and γ1 constant regions together with a representative of all six V _H families [this fragment was derived from HeLa cells by Berman et al. (1988) supra]. Although it has been identified as a 670 kb fragment, the present inventors have discovered that it is as an 830 kb fragment from human placenta and sperm DNA].

Fractions containing this NotI fragment (see FIG. 4) were pooled and NotI of the vector pYACNN in yeast cells.
Clone into the site. The plasmid pYACNN is pYAC-4
Neo 〔Cook, H. et al . , Nucl. Acids Res. 16 : 11817 (198
8)] was digested with EcoRI and the oligonucleotide 5 '
-AAT TGC GGC Prepared by ligation in the presence of CGC-3 '.

Brownstein et al. (1989), Science, 244, 134.
8-1351 and Green, E. et al . , Proc. Natl. Acad. Sci. US
YAC clones containing the heavy chain NotI fragment are isolated as described by A 87 : 1213-1217 (1990), which are incorporated herein by reference. M. Finne
by cloning pulsed-field gel electrophoresis from high molecular weight yeast DNA as described by Y. (supra).
The insert is isolated. This DNA is condensed by the addition of 1 mM spermine and microinjected directly into the nuclei of the single cell embryos described above.

Example 2 Genomic kappa light chain human Ig formed by in vivo homologous recombination
A map of the transgene human kappa light chain is given by Lorenz. W. et al . , Nucl. Acids Res.
15 : 9667-9677 (1987), which is incorporated herein by reference. A 450 kb XhoI-NotI fragment containing all CK, 3'enhancers, all J segments and at least 5 different V segments was isolated as described in Example 1 and isolated in the nucleus of single cell embryos. Microinjected into.

Example 3 Genomic kappa light chain human Ig formed by in vivo homologous recombination
Transgene A 750 kp MluI-NotI fragment containing all of the above components and at least 20 more V segments is isolated as described in Example 1 and digested with BssHII to generate an approximately 400 kb fragment.

450 kb XhoI-Not I fragment and approximately 400 kb Mlu
The I-BssHII fragment has a sequence overlap defined by the BssHII and XhoI restriction sites. Homologous recombination of these two fragments by microinjection of mouse zygotes results in a transgene containing at least 15-20 additional V segments than found in the 450 kb XhoI / Not I fragment (Example 2). .

Example 4 Construction of Heavy Chain Minilocus A. Construction of pGP1 and pGP2 pBR322 is digested with EcoRI and StyI and ligated with the following oligonucleotides to construct pGP1 containing a 147 base pair insert having a restriction site shown in FIG.
The general overlap of those oligos is also shown in FIG.

The oligonucleotides are: Oligo-1 5'-CTT GAG CCC GCC TAA TGA GCG GGC
TTT TTT TTG CAT ACTGCG GCC -3 'oligo-2 5'-GCA ATG GCC TGG ATC CAT GGC GCG
CTA GCA TCG ATA TCTAGA GCT CGA GCA -3 '

Oligo-3 5'-TGC AGA TCT GAA TTC
CCG GGT ACC AAG CTT ACG CGT ACTAGT GCG GCC GCT −
3'oligo-4 5'-AAT TAG CGG CCG CAC TAG TAC GCG
TAA GCT TGG TAC CCGGGA ATT -3 'oligo-5 5'-CAG ATC TGC ATG CTC GAG CTC TAG
ATA TCG ATG CTA GCGCGC CAT GGA TCC -3 'oligo-6 5'-AGG CCA TTG CGG CCG CAG TAT GCA
AAA AAA AGC CCG CTCATT AGG CGG GCT -3 '

This plasmid contains a large polylinker flanked by rare cleavable Not I sites to construct large inserts that can be isolated from vector sequences for microinjection. This plasmid has a relatively low copy number compared to pUC-based plasmids
Derived from pBR322 (pGP1 retains the pBR322 copy number regulatory region near the origin of replication). Low copy number reduces the potential toxicity of the insert. In addition, pGP1 is a strong transcription terminator sequence from trpA [Christ inserted between the ampicillin resistance gene and the polylinker.
ie, GE et al . , Proc. Natl. Acad. Sci. USA 78 : 4180 (1
981)] is also included. This reduces the toxicity associated with certain inserts by preventing readthrough transcription that occurs from the ampicillin promoter.

Plasmid pPG2 is derived from pGP1 to introduce an additional restriction site (Sfi I) in the polylinker. pGP1 is digested with MluI and SpeI to excise the recognition sequence in the polylinker part of the plasmid. The thus-digested pGP1 is ligated with the following adapter oligonucleotide to prepare pGP2.

5′CGC GTG GCC GCA ATG GCC A 3 ′ 5′CTA GTG GCC ATT GCG GCC A 3 ′ pGP2 is an additional S located between the MluI and SpeI sites.
It is the same as pGP1 except that it contains a fiI site. This allows the insert to be completely excised with SfiI as well as NotI. B. Construction of pRE3 (rat enhancer 3 ') An enhancer sequence placed downstream of the rat constant region is introduced into the heavy chain construct.

Pettersson et al., Nature 344: 165-168 (199
The heavy chain region 3'enhancer described by (0) is isolated and cloned. The following oligonucleotides: 5'CAG GAT CCA GAT ATC AGT ACC TGA AAC AGG GCT TGC
3 ′ 5 ′ GAG CAT GCA CAG GAC CTG GAG CAC ACA CAG CCT TCC
The 3'is used to PCR amplify the rat IGH 3'enhancer sequence.

The thus formed double-stranded DNA encoding the 3'enhancer was cleaved with BamHI and SphI to obtain BamHI /
Cloning into pGP2 cut with SphI gives pRE3 (rat enhancer 3 '). C. Cloning of the human J-μ region A substantial portion of this region is cloned by combining two or more fragments isolated from the λ phage insert. See FIG.

Oligonucleotide GGA CTG TGT CCC TGT
A 6.3 kb BamHI / HindIII fragment containing the entire human J segment using the GTG ATG CTT TTG ATGTCT GGG GCCAAG [Matsuda et al., EMBO J. 7: 1047-1051 (1988); Ravete
ch. et al., Cell 27 : 583-591 (1981), which is incorporated herein by reference], from a human genomic DNA library.

Oligonucleotides CAC CAA GTT GAC CTG
CCT GGT CAC AGA CCTGAC CAC CTATGA was used to flank adjacent 10 kb HindIII / BamHI fragments containing enhancers, switches and constant region coding exons [Yasui et al. , Eur.
J. Immunol. 19 : 1399-1403 (1989)] is similarly isolated. The clone pMUM insert (pMUM
Is a μ-membrane exon 1 oligonucleotide: CCT GTG GAC
4 kb E isolated from human genomic DNA library using CAC CGC CTC CAC CTT CAT CGT CCT CTT CCT CCT
flanking 3'1.5 kb using coRI / HindIII fragment)
The BamHI fragment is likewise isolated and cloned into pUC19.

PGP1 is digested with BamHI and BglII and then treated with calf intestinal alkaline phosphatase. Fragments (a) and (b) from Figure 9 are cloned into the digested pGP1. A clone is then isolated in which the 5'BamHI site has been engineered to be disrupted by the BamHI / BglII fusion. Name it pMU (see Figure 10). pMU is digested with BamHI and the fragment (c) from Figure 9 is inserted. Confirm the direction by HindIII digestion. The resulting plasmid pHIG1 (Fig. 1
0) contains an 18 kb insert encoding J and Cμ segments. D. Cloning of the Cμ region pGP1 is digested with BamHI and HindIII and then treated with calf intestinal alkaline phosphatase (Figure 14). The fragment (b) of FIG. 14 and the fragment (c) of FIG.
Clone into pGP1 cut with HI / HindIII. Hi
Confirm the correct orientation of fragment (c) by digesting ndIII
We obtain pCON1 containing a 12 kb insert encoding the μ region.

PHIG1 contains a J segment, a switch and a μ in the 18 kb insert with SfiI 3'and SpeI 5'sites in the polylinker flanked by Not I sites.
It will be used for the rearranged VDJ segment because it contains the sequence. pCON1 is similar to pHIG1 except that it lacks the J region and contains only a 12 kb insert. Rearranged V
The use of pCON1 in the generation of fragments containing the DJ segment is described below. E. FIG. Cloning of the γ-1 constant region (pREG2) Cloning of the human γ-1 region is depicted in Figure 16.

Yamamura et al . , Proc. Natl. Acad. Sci. USA
86 : 2152-2156 (1986) report expression of membrane bound human γ-1 from transgene constructs that were partially deleted during integration. Their result was that the 3'BamHI site was
It is pointed out that it outlines a sequence containing a transmembrane rearranged and switched gamma gene copy with a V-C intron of less than 5 kb. Thus, in a non-rearranged, unswitched gene, the entire switch region is contained in the sequence starting less than 5 kb from the 5'end of the first γ-1 constant exon.

It is therefore contained in the 5'5.3 kb Hind III fragment [Ellison, JW et al., Nucleic. Acids Res.
10 : 4071-4079 (1982); which is incorporated herein by reference]. Takahashi et al., Cell 29 : 671-679 (19
82), which is hereby incorporated by reference, also reported that this fragment contained a switch sequence, which when combined with the 7.7 kb Hind III-BamHI fragment resulted in the transgene. It must contain all of the sequences required for the construct. An intron sequence is a nucleotide sequence of at least 15 contiguous nucleotides present in an intron of a particular gene.

The following oligonucleotides, which are specific for the third exon of γ-1 (C _H 3) are used to identify and isolate phage clones containing the γ-1 region. 5 ′ TGA GCC ACG AAG ACC CTG AGG TCA AGT TCA ACT GG
T ACG TGG 3 ′ 7.7 kb Hind III-Bgl II fragment (fragment (a) in FIG. 11)
Is cloned into HindIII / BglII digested pRE3 to create pREG1. The upstream 5.3 kb HindIII fragment (fragment (b) in FIG. 11) is cloned into HindIII-digested pREG1 to generate pREG2. BamHI / Spe I
Confirm the right direction by digestion. F. Binding of Cγ to Cμ The above-mentioned plasmid pHIG1 contains the human J segment and the Cμ constant region exon. PHIG1 was added to Sfi to provide a transgene containing the Cμ constant region gene segment.
Digested with I (Fig. 10). Plasmid pREG2 was also digested with SfiI to obtain a 13.5 kb insert containing human Cγ exon and rat 3'enhancer sequence. The sequences were ligated together, and the human J segment and human Cμ were inserted on the 31.5 kb insert.
A plasmid pHIG3 'containing the constant region, human Cγ1 constant region and rat 3'enhancer sequence was prepared (Fig. 1).
2).

PCON1 was digested with SfiI and pREG2 was digested.
By ligating to the Sfi I fragment containing the human Cγ region obtained by digestion with Sfi I and the rat 3 ′ enhancer, a second plasmid encoding human Cμ and human Cγ1 but not containing the J segment is prepared. . The resulting plasmid pCON (FIG. 12) contains a 26 kb NotI / containing human Cμ, human γ1 and rat 3 ′ enhancer sequences.
Contains the Spe I insert. G. Cloning of the D segment The strategy for cloning the human D segment is shown in Figure 13.
Pictured in. A phage clone from a human genomic library containing a D segment was cloned into a diversity region sequence [Y.
chihara et al., EMBO J. 7: 4141-4150 (1988)] for identification and isolation. The following oligonucleotides are used.

DXP1: 5'-TGG TAT TAC TAT GGT TCG
GGG AGT TAT TAT AAC CAC AGT GTC-3 'DXP4: 5'-GCC TGA AAT GGA GCC TCA GGG CAC AGT
GGG CAC GGA CAC TGT-3 'DN4: 5'-GCA GGG AGG ACA TGT TTA GGA TCT GAG
From the phage clones identified using GCC GCA CCT GAC ACC-3 'oligo DXP1, 5.2 kb XhoI containing DLR1, DXP1, DXP'1 and DA1
The fragment (fragment (b) in Figure 13) is isolated.

From phage clones identified using oligo DXP4, 3.2 kb Xba containing DXR4, DA4 and DK4.
The I fragment (fragment (c) in Figure 13) is isolated. Fragments (b), (c) and (d) in FIG. 13 were ligated to each other, and Xba I of pGP1 was ligated.
/ Clone into the XhoI site to form pHIG2 containing the 10.6 kb insert. This cloning is done continuously. First, the 5.2 kb fragment (b) in Figure 13 and the
The 2.2 kb fragment (d) is treated with calf intestinal alkaline phosphatase and cloned into XhoI and XbaI digested pGP1. The resulting clones were cloned into the 5.2 kb and 2.2
Screen with the kb insert. Half of those clones that tested positive with the 5.2 kb and 2.2 kb inserts were in the correct orientation as confirmed by BamHI digestion.
It has a 5.2 kb insert. The 3.2 kb XbaI fragment of Figure 13 is then cloned into this intermediate plasmid containing fragments (b) and (d) to form pHIG2. This plasmid contains a unique 5'Sfi I site and a unique 3'Spe I site.
It contains a diversity segment cloned into a polylinker with sites. The complete polylinker is flanked by NotI sites. H. Production of Heavy Chain Minilocus The following describes the production of a human heavy chain minilocus containing one or more V segment.

Newkirk et al., J. Clin. Invest. 81 : 1511-
An unrearranged V segment corresponding to that identified as the V segment contained in the hybridoma of 1518 (1988), which is incorporated herein by reference, was prepared with the following oligonucleotides: 5'- GAT CCT GGT TTA GTT AAA GAG GAT TTT ATT CAC C
Isolate using CC TGT GTC-3 '.

Examination of the restriction map of the unrearranged V segment provides a DNA fragment having a length of approximately 2 kb which comprises the unrearranged V segment together with the 3'and 5'flanking sequences upon digestion. Identify unique restriction sites that The 5'starting sequence will contain the promoter and other regulatory sequences, while the 3'flanking sequence will provide the recombination sequences necessary for V-DJ binding. This approximately 3.0 kb V segment insert was cloned into the polylinker of pGB2 to generate pVH1
To form.

PVH1 was digested with SfiI and the resulting fragment was adjusted to pH
It is cloned into the SfiI site of IG2 to make pHIG5 '. Since pHIG2 contains only the D segment, the generated p
The HIG5 'plasmid contains a single V segment along with a D segment. The size of the insert contained in pHIG5 is the size of the 10.6 kb + V segment insert.
The insert is excised from pHIG5 by digestion with Not I and Spe I. PHIG containing J, Cμ and Cγ1 segments
The 3'is digested with SpeI and NotI and the 3 kb fragment containing the above sequence and the rat 3'enhancer is isolated. The two fragments were combined and ligated into NotI-digested pGP1 to form a V segment, 9 D segments, 6 functional J segments, Cμ, Cγ and rat 3 '.
A pHIG containing an enhancer is made. The size of this insert is approximately 43 kb plus the size of the V segment insert. I. Generation of Heavy Chain Minilocus by Homologous Recombination As pointed out in the previous section, the pHIG insert is approximately 43-45 kb using a single V segment. This insert size is at or near the limit at which it can be easily cloned into a plasmid vector. To prepare for the use of a larger number of V segments, a zygote or E
In vivo homologous assembly of overlapping DNA fragments forming a transgene containing a rat 3'enhancer sequence, human Cμ, human Cγ1, human J segment, human D segment and multiple human V segments by homologous recombination in S cells. The replacement is described below.

6.3 kb BamHI / Hi containing human J segment
The ndIII fragment (see fragment (a) in Figure 9) was transformed with the following adapters: 5'GAT CCA AGC AGT 3'5'CTA GAC TGC TTG 3'5'CGC GTC GAA CTA 3 '5'AGC TTA. GTT CGA 3'is used to clone in Mlu I / Spe I digested pHIG 5 '.

The plasmid generated is named pHIG5'O (overlap). The insert contained in this plasmid contains human V, D and J segments. The size of this insert is approximately 17 k when a single V segment from pVH1 is used.
b + 2 kb. This insert is isolated and combined with the insert from pHIG3 'containing human J, Cμ, γ1 and rat 3'enhancer sequences. Both inserts contain the human J sequence with an approximately 6.3 kb overlap between the two DNA fragments. When they are co-injected into mouse zygotes, in vivo homologous recombination occurs, producing a transgene equivalent to the insert contained in pHIG.

This approach provides for the addition of multiple V segments into the transgene formed in vivo.
For example, instead of incorporating a single V segment into pHIG5 ', (1) isolated genomic DNA, (2) ligated DNA derived from genomic DNA, or (3) synthetic V
The DNA encoding the segment repertoire, multiple V segments contained above, is cloned into the SfiI site of pHIG2 to create pHIG5'V _N. The J segment fragment (a) of Figure 9 is then cloned into pHIG5'V _N and the insert is isolated. This insert is pHIG
It will contain a number of V segments and J segments that overlap with the J segment contained on the insert isolated from 3 '. When this is co-injected into the nucleus of a mouse zygote,
Homologous recombination occurs, resulting in multiple V and multiple J segments, multiple D segments, Cμ region, Cγ1
A transgene encoding the region (all from human) as well as the rat 3'enhancer sequence is generated.

Example 5 Construction of Light Chain Minilocus Generation of pEμ1 The generation of pEμ1 is depicted in FIG. Oligo: 5′GAA TGG GAG TGA GGC TCT CTC ATA CCC TAT TCA GAA
678 bp XbaI-EcoRI from the phage clone using CTG ACT 3 '
The mouse heavy chain enhancer is isolated in a fragment [J. Banerji et al., Cell 33 : 729-740 (1983)].

This Eμ fragment is cloned into EcoRV / Xba I digested pGP1 by blunt-end fill-in at the EcoRI site. The resulting plasmid is named pEμ1. B. Generation of the kappa light chain minilocus The kappa construct comprises at least one human Vk segment, 5
It contains all four human Jκ segments, the human J-Cκ enhancer, the human kappa constant region exon, and ideally the human 3′κ enhancer [K. Meyer et al., EMBO J. 8: 19].
59-1964 (1989)]. The mouse kappa enhancer is from Ck
9 kb downstream. However, it has not been identified in humans. In addition, the construct also contains a copy of the mouse heavy chain J-Cμ enhancer.

This minilocus is made up of the following four component fragments: (a) A 16 kb Sma I fragment containing the human CK exon and the 3'human enhancer by analogy with the mouse locus; (b) 5 5'adjacent 5 kb Sma containing all one J segment
I fragment; (c) mouse heavy chain intron enhancer isolated from pEμ1 (this sequence is included to induce expression of the light chain construct as early as possible in B cell development. The heavy chain gene is the light chain gene). This heavy chain enhancer is probably more active than the intron kappa enhancer because it is transcribed earlier); and (d) a fragment containing one or more V segments.

The preparation of this construct is as follows. Human placental DNA is digested with SmaI and fractionated on agarose by electrophoresis. Similarly, human placental DNA is digested with BamHI and fractionated by electrophoresis. From a gel digested with SmaI
The 16 kb fraction is isolated and the 11 kb region is isolated from a gel containing DNA also digested with BamHI. The 16 kb Sma I fraction was digested with Xho I and treated with Klenow fragment DNA polymerase to fill in the Xho I restriction digestion product Lambda FIX II (Stratagene, La Jolla, Califo
rnia). Ligation of the 16 kb Sma I fraction destroys the Sma I site but leaves the Xho I site intact.

The 11 kb BamHI fraction was cloned into B
Lambda FIX II digested with amHI (Stratagene, La Jolla,
California). Clones from each library were cloned into Cκ specific oligos: 5′GAA CTG TGG CTG CAC CAT CTG TCT TCA TCT TCC CGC.
Exploration using CAT CTG 3 '.

16 kb Xh so that Cκ is flanked by SmaI
The I insert is subcloned into Xho I cut pEμ1. The resulting plasmid is named pKap1.
Λ EMBL3 / B using the above Cκ-specific oligonucleotide
Explore the amHI library and identify an 11 kb clone. The 5 kb SmaI fragment (fragment (b) of Figure 25) is subcloned and then inserted into SmaI digested pKap1. Those plasmids containing the J segment, Cκ and Eμ enhancers in the correct orientation are designated pKap2.

Then, one or more Vκ segments are
Plasmid pKapH subcloned into MluI of pKap2 and encoding human Vκ segment, human Jκ segment, human Cκ segment and human Eμ enhancer
Give rise to. The insert is excised by digesting pKapH with NotI and purified by agarose gel electrophoresis. The insert thus purified is microinjected into the pronucleus of mouse zygotes as described above. C. Generation of the kappa light chain minilocus by in vivo homologous recombination The 11 kb BamHI fragment is cloned into BamHI digested pGP1 with its 3'end facing towards the SfiI site. The resulting plasmid is named pKAPint. pKAPin
One or more Vκ segments are inserted in the polylinker between the BamHI and SpeI sites in t to create pKapHV. The pKapHV insert is excised by digestion with NotI and purified. NotI insert from pKap2
It is cut out by digestion with and purified. Each of these two fragments is homologous in that the fragment from pKapHV contains a 5 kb DNA sequence containing a Jκ segment that is substantially homologous to the 5 kb SmaI fragment contained in the insert obtained from pKap2. Contains sex areas. Therefore, those inserts can homologously recombine when microinjected into the mouse zygote to form transgenes encoding Vκ, Jκ and Cκ.

Example 6 Rearranged and expressed codons of immunoglobulin kappa light chain gene
Isolation of a Genomic Clone Corresponding to a Pea This Example describes the cloning of the immunoglobulin kappa light chain gene from cultured cells expressing the immunoglobulin of interest. Such cells may contain multiple alleles of a given immunoglobulin gene. For example, a hybridoma contains 4 copies of the kappa light chain gene, 2 copies from the cell line of the fusion partner, and 2 copies from the original B cells expressing the immunoglobulin of interest. Of the four copies, only one encodes the immunoglobulin of interest, despite the fact that some can rearrange. The procedure described in this example allows for selective cloning of the expression copy of the kappa light chain. A. Cells from double-stranded cDNA human hybridomas or lymphomas, or cell surface and / or secreted forms of kappa
Other cell lines that synthesize light chain-containing IgM are used for isolation of poly A ⁺ RNA. The RNA is then used for the synthesis of oligo dT-initiated cDNA using reverse transcriptase. Then single-stranded cDNA
Is isolated and a G residue is added to the 3'end using a polynucleotide terminal transferase enzyme. The G-terminated single stranded cDNA is then purified and used as a primer for the following oligonucleotides: 5'-GAG GTA CAC TGA CAT ACT GGC ATG CCC CCC CCC C
Used as template for second-strand synthesis (catalyzed by DNA polymerase enzyme) using CC-3 '.

Double-stranded cDNA is isolated and used to determine the nucleotide sequence at the 5'end of the mRNA encoding the heavy and light chains of the expressed immunoglobulin molecule. Genomic clones of those expressed genes are then isolated. The method of cloning the expressed light chain gene is summarized in Part B below. B. The double- stranded cDNA described in the A part of the light chain was denatured and the following oligonucleotide primer: 5'-GTA CGC CAT ATC AGC TGG ATG AAG TCA TCA GAT G
GC GGG AAG ATG AAG ACA GAT GGT GCA-3 'used as a template for the third round of DNA synthesis.

This primer was used for the sequence (TCA TCA GAT GGCGGG AAG ATG AAG ACA) specific to the constant part of the kappa light chain information.
GAT GGT GCA) and P of newly synthesized DNA chain
It contains a unique sequence (GTA CGC CAT ATC AGC TGG ATG AAG) that can be used as a primer for CR amplification. This sequence is amplified by PCR using the following two oligonucleotide primers: 5'-GAG GTA CAC TGA CAT ACT GGC ATG -3 '5'-GTA CGC CAT ATC AGC TGG ATG AAG -3'.

The PCR amplified sequence was purified by gel electrophoresis and used as a template for a dideoxy sequencing reaction using the following oligonucleotides as primers: 5'-GAG GTA CAC TGA CAT ACT GGC ATG-3 '. To do. The first 42 nucleotides of the sequence are then used to synthesize a unique probe to isolate the gene into which the immunoglobulin information has been transcribed. Synthesis of this DNA
The 42 nucleotide segment will be referred to hereinafter as o-kappa.

Southern blots of DNA isolated from Ig-expressing cell lines and digested individually and in combination with several different restriction endonucleases, including SmaI,
Probing with ³² P-labeled unique oligonucleotide o-kappa. Unique restriction endonuclease sites are identified upstream of the rearranged V segment. The DNA from the Ig-expressing cell line was then digested with SmaI and a second enzyme (Ba if the SmaI site was inside the V segment).
Cut with mHI or KpnI). Any generated non-blunt ends are treated with T4 DNA polymerase enzyme to give blunt ended DNA molecules. Then a linker that encodes a restriction site (BamHI, depending on which site is not present in the fragment,
EcoRI or XhoI) is added and cleaved with the corresponding linker enzyme to give a DNA fragment with BamHI, EcoRI or XhoI ends. The DNA is then size fractionated by agarose gel electrophoresis and the fraction containing the DNA fragment containing the V segment to be expressed is cloned into lambda EMBL3 or lambda FIX (Stratagene, La Jolla, Calif.). V segment containing clones are isolated using the unique probe o-kappa. DNA is isolated from positive clones and subcloned into the polylinker of pKap1. The resulting clone is named pRKL.

Example 7 Immunoglobulin Heavy Chain μ Gene Rearranged and Expressed Co
Isolation of a Genomic Clone Corresponding to a Pea This Example describes the cloning of the immunoglobulin heavy chain μ gene from cultured cells expressing the immunoglobulin of interest. The procedure described in this example allows for selective cloning of expression copies of the mu heavy chain gene.

Double-stranded cDNA is prepared and isolated as described above. This double-stranded cDNA was denatured and the following oligonucleotide primers were: 5'-GTA CGC CAT ATC AGC TGG ATG AAG ACA GGA GAC G
AG GGG GAA AAG GGT TGG GGC Used as a template for the third round of DNA synthesis using GGA TGC-3 '.

This primer was used for the sequence (ACA GGA GAC GAGGGG GAA AAG GGT TGG) specific to the constant part of the μ heavy chain information.
GGC GGA TGC) and P of newly synthesized DNA chain
It contains a unique sequence (GTA CGC CAT ATC AGC TGG ATG AAG) that can be used as a primer for CR amplification. This sequence is amplified by PCR using the following two oligonucleotide primers: 5'-GAG GTA CAC TGA CAT ACT GGC ATG -3 '5'-GTA CTC CAT ATC AGC TGG ATG AAG -3'.

The PCR amplified sequence was purified by gel electrophoresis and used as a template for a dideoxy sequencing reaction using the following oligonucleotides as primers: 5'-GAG GTA CAC TGA CAT ACT GGC ATG-3 '. use. The first 42 nucleotides of the sequence are then used to synthesize a unique probe to isolate the gene into which the immunoglobulin information has been transcribed. Synthesis of this DNA
The 42 nucleotide segment will be referred to hereinafter as o-mu.

Isolated from Ig-expressing cell lines and individually and paired with several different restriction endonucleases, including MluI (MluI is a rare-cutting enzyme that cleaves between the J segment and μCH1). Southern blots of digested DNA are probed with ³² P-labeled unique oligonucleotide o-mu. Unique restriction endonuclease sites are identified upstream of the rearranged V segment.

The DNA from the Ig expressing cell line was then digested with MluI.
And cut with the second enzyme. A MluI or SpeI adapter linker is then ligated to the ends and cleaved to convert the upstream site to MluI or SpeI. The DNA is then size fractionated by agarose gel electrophoresis and the fraction containing the DNA fragment containing the expressed V segment is cloned directly into plasmid pGP1. A V segment containing clone is isolated using the unique probe o-mu and the insert is subcloned into plasmid pCON2 cut with MluI or MluI / SpeI.
The resulting clone is named pRMGH.

Example 8 Preparation of the Human Kappa Minilocus Transgene A light chain minilocus kappa light chain specific oligonucleotide probe was used to screen a human genomic DNA phage library to isolate clones spanning the Jκ-C region. did. A 5,7 kb ClaI / Xho I fragment containing Jκ1 contains Jκ2-5 and Cκ
Cloned into pGP1d with the 13 kb XhoI fragment,
It was then used to create the plasmid pKcor. This plasmid contains the Jκ1-5, κ inroton enhancer and Cκ with 4.5 kb of 5 ′ and 9 kb of 3 ′ flanking sequences. It also has a unique 5'Xho I site for cloning V segments and a unique 3'Sal I site for insertion of additional cis-acting regulatory sequences. A human genomic DNA phage library was screened with a Vκ gene Vκ light chain specific oligonucleotide probe to isolate clones containing the human Vκ segment. D
Functional V segments were identified by NA sequence analysis. These clones include a TATA box, an open reading frame encoding a leader and variable peptide (containing two cysteine residues), a splice sequence, and a recombinant heptamer-
It contains the 12 b spacer-nonamer sequence. Three of the clones were mapped and sequenced. Two of the clones, 65.5 and 65.8, were functional and
It appears to contain a TATA box, a leader and open reading frame encoding a variable peptide (containing two cysteine residues), a splice sequence, and a recombinant heptamer-12b spacer-nonamer sequence. 3rd clone 6
Since 5.4 contains a non-canonical recombinant heptamer, Vκ
It seems to encode the I pseudogene.

One of the functional clones encoding the VkIII family of genes, Vκ 65-8, was used to generate a light chain minilocus construct. The κ light chain minilocus pKC1 (FIG. 38) was generated by inserting a 7.5 kb XhoI / Sal I fragment containing pKC1 Vκ65-8 into the 5′XhoI site of pKcor. The transgene insert was isolated by digestion with NotI prior to injection.

The purified insert was fertilized (C57BL / 6x
CBA) F ₂ mouse embryos were microinjected into the pronucleus,
Hogan et al. (Methods of Manipulating the Mouse
Embryo, 1986, Cold Spring Harbor Laboratory, New Y
Surviving embryos were transferred to pseudopregnant females as described in (ork). Mice developed from injected embryos were analyzed for the presence of transgene sequences by Southern blot analysis of tail DNA. The transgene copy number was evaluated by band intensity compared to a control containing a known amount of cloned DNA. Isolation of serum from those animals,
And Harlow and Lane (Antibodies: A Laboratory M
anual, 1988, Cold Spring Harbor Laboratory, New Yo
rk) and analyzed by ELISA for the presence of the human Igκ protein encoded by the transgene.

Microtiter plate wells with human Ig
κ (Clone 6E1, # 0173, AMAC Inc., Westbrook, ME
), Human IgM (clone AF6, # 0285, AMAC Inc., Wes
tbrook, ME) and human IgG1 (clone JL512, # 0280,
AMAC Inc., Westbrook, ME) was coated with a specific mouse monoclonal antibody. Serum samples were serially diluted in the wells and affinity-purified alkaline phosphatase-conjugated goat anti-human Ig pre-adsorbed to minimize cross-reactivity with mouse immunoglobulin.
(Polyvalent) was used to detect the presence of specific immunoglobulins.

FIG. 41 shows eight mice (ID # 676, 674,
EL of sera from 673, 670, 666, 665, 664 and 496)
The results of the ISA assay are shown. The first 7 of those mice
The animals develop from embryos injected with the pKC1 transgene insert, and the eighth mouse is from a mouse born by microinjection of the pHC1 transgene (supra). 2 out of 7 mice from pKC1 injected embryos
The animals (ID # 666 and 664) did not contain the transgene insert as analyzed by DNA Southern blot analysis and 5 (ID # 676, 674, 673, 670 and 665) did contain the transgene. .

All but one of the pKC1 transgene-positive animals expressed detectable levels of human Igκ protein and the other non-expressing animal appeared to be a genetic mosaic based on DNA Southern blot analysis. . pHC1 positive transgenic mice express human IgM and IgG1 but not Igκ, demonstrating the specificity of the reagents used in the assay. An 8 kb XhoI / Sal I fragment containing pKC2 Vκ 65.5 was added to the 5'of pKC1.
By inserting at the Xho I site, the κ light chain minilocus pK
C2 was produced. The resulting transgene insert containing the two Vκ segments was not
Isolated by digestion with I. pKVe2 This construct is similar to pKC1 except that it contains an additional 1.2 kb sequence 5 ′ of Jκ and lacks the 4.5 kb sequence 3 ′ of Vκ65.8. The construct further comprises the mouse heavy chain Jm intron enhancer [Banerji et al., Cell 3
3 : 729-740 (1982)] and a human heavy chain Jm intron enhancer [Hayday et al., Nature 307: 334-340 (1984)] inserted downstream 1.4.
It contains the kb MluI-HindIII fragment. This construct is tested for the potential to initiate an initial rearrangement of the light chain minilocus that performs allelic exclusion and isotype exclusion. Different enhancers, namely the mouse or rat 3'κ or heavy chain enhancers [Meyer and Neuberger, EMBO J.
8: 1959-1964 (1989); Petterson et al., Nature 344: 16
5-168 (1990); these are incorporated herein by reference]. A kappa light chain expression cassette was designed to reconstitute a functionally rearranged light chain gene amplified by PCR from rearranged light chain transgene human B cell DNA. The strategy is shown in Figure 39. The PCR amplified light chain gene was isolated from the kappa light chain gene 65.5 3.
Clone into vector pK5nx containing the 7 kb 5'flanking sequence. Then a vector pK31s containing the Jκ2-4, Jκ intron enhancer, Cκ and 9 kb of downstream sequence.
The VJ segment fused to a 5'transcriptional regulatory sequence is cloned into the unique XhoI site of. The resulting plasmid contains the rearranged, functionally rearranged kappa light chain transgene, which can be excised with NotI for microinjection into the embryo. The plasmid also contains a unique SalI site at the 3'end for insertion of additional cis-acting regulatory sequences.

Two synthetic oligonucleotides (o-130, o-131) were used to amplify the rearranged kappa light chain gene from human splenic genomic DNA. Oligonucleotide o-130 (gga ccc aga (g, c) gg aac cat gga a (g, a)
(g, a, t, c)) is 5'of VκIII family light chain gene
First ATC complementary to region and in leader sequence
Overlaps with. Oligonucleotide o-131 (gtg caa
tca att ctc gag ttt gac tac aga c) is about Jκ1
It is complementary to the sequence from 150 bp 3'and contains an Xho I site. These two oligonucleotides amplify a 0.7 kb DNA fragment from human spleen DNA that corresponds to the rearranged VκIII gene linked to the Jκ1 segment.

PCR-amplified DNA was digested with NcoI and XhoI
Digested with and individual PCR products cloned into plasmid pNN03. DNA sequencing of 5 clones identified 2 clones with a functional VJ junction (open reading frame). Additional functionally rearranged light chain clones are also collected. Functionally rearranged clones can be individually cloned into the light chain expression cassette (Figure 39) above. By crossing transgenic mice produced with rearranged light chain constructs with heavy chain minilocus transgenic mice, a fully human antibody in which all of the diversity of the primary repertoire is conferred by the heavy chain It is possible to produce a mouse strain expressing the spectrum of.

One source of light chain diversity can be from somatic mutations. Since not all light chains are equal in their ability to bind a variety of different heavy chains, different mouse strains, each containing a different light chain construct, can be generated and tested. The advantage of this strategy is that the light chain resulting from having a light chain that readily pairs with the heavy chain and immediately undergoes heavy chain VDJ ligation, as opposed to the use of unrearranged light chain minilocus. Increased allele and isotype exclusion. This combination results in an increased frequency of B cells that express fully human antibodies, thus facilitating the isolation of human Ig expressing hybridomas.

The NotI insert of the plasmids pIGM1, pHC1, pIGH1, pKC1 and pKC2 was isolated from the vector sequence by agarose gel electrophoresis. The purified inserts were microinjected into the pronucleus of fertilized (C57BL / 6 x CBA) F2 mice and as described by Hogan et al. (Hogan et al., Methods of Manipulating the
Mouse Embryo , Cold Spring Harbor Laboratory, New Y
ork (1986)], surviving embryos were transferred into pseudopregnant females.

Example 9 Inactivation of the Mouse Kappa Light Chain Gene by Homologous Recombination This example illustrates the inactivation of the mouse endogenous kappa locus by homologous recombination in embryonic stem (ES) cells. Describes the introduction of mutant genes into the mouse germline by injecting target ES cells with an inactivated κ allele into early mouse embryos (blastocysts).

The strategy was to perform homologous recombination using a vector containing a DNA sequence homologous to the mouse κ locus in which the 4.5 kb segment of the locus spanning the Jκ gene and Cκ segment has been deleted and replaced by the selectable marker neo. To delete the Jκ gene and the Cκ gene. Construction of κ targeting vector Plasmid pGEM7 (KJ1) (MA Rudnicki, Whitehead Ins
titute) is the mouse phosphoglycerate kinase (pgk) promoter (Xba) in the cloning vector 7Zf (+).
I / TaqI fragment; Adra, CN et al., Gene 60 : 65-74 (198
7)] under the transcriptional regulation, it contains neomycin resistance gene (neo) used for drug selection of transfected ES cells. This plasmid is derived from the 3'region of the mouse pgk gene and is a heterologous polyadenylation site for the neo gene [Pvu II / HindIII fragment; Boer, PH et al., Biochemica.
l Genetics 28 : 299-308 (1990)]. This plasmid was used as a starting point for the construction of the kappa targeting vector. The first step was to insert a homologous sequence into the 3'kappa locus of the neo expression cassette.

Oligonucleotide probe specific for Cκ locus: 5′-GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC
ACC ATC CAG-3 ′ and Jκ gene segment-specific oligonucleotide probe: 5′-CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG
The mouse kappa chain sequence (Figure 25a) was isolated from a genomic phage library derived from liver DNA using AAA CGT AAG-3 '.

In two fragments from the positive phage clone, namely as a 1.2 kb BglII / SacI fragment and a 6.8 kb SacI fragment, 8 kb BglII spanning 3'of the mouse Cκ segment.
/ SacI fragment was isolated and it was subcloned into BglII / SacI digested pGEM (KJ1) to obtain plasmid p
NEO-K3 'was prepared (Fig. 25b). Spans 5'of the Jκ region
A 1.2 kb EcoRI / SphI fragment was also isolated from a positive phage clone. SphI / Xba I / Bg at the SphI site of this fragment
l II / EcoRI adapter was ligated, and the resulting EcoRI fragment was the same as the neo gene and the downstream 3'κ sequence 5 '→
In the 3'direction, it was ligated to EcoRI-digested pNEO-K3 'to generate pNEO-K5'3' (Fig. 25c).

Mansour et al., Nature 336: 348-3.
52 (1988), which is incorporated herein by reference, to prepare for the enrichment of ES clones with homologous recombinants, the herpes simplex virus ( The HSV) thymidine kinase (TK) gene was included. The HSV TK cassette was obtained from the plasmid pGEM7 (TK). This cassette contains the structural sequence of the HSV TK gene flanked by the mouse pgk promoter and a polyadenylation sequence, as described above for pGEM7 (KJ1). pG
Change EcoRI site of EM7 (TK) to BamHI site, and TK
The cassette was cut out as a BamHI / HindIII fragment and pGP1
Subcloned into b to create pGP1b-TK. This plasmid was linearized at the Xho I site and contains the neo gene flanked by the genomic sequences from 5'of Jκ and 3'of Cκ, derived from pNEO-K5'3 '.
The XhoI fragment was inserted into pGP1b-TK and the targeting vector J / C KI
(Fig. 25d) was prepared. The putative structure of the genomic kappa locus after homologous recombination with J / C K1 is shown in Figure 25e. Generation of ES cells by targeted inactivation of κ allele and
The ES cells used for analysis were essentially as described (Robe
rtson, EJ, Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, edited by EJ Robertson (Oxfo
rd: IRL Press) 71-112 (1987)], a mitotically inactive SN.
L76 / 7 feeder layer [McMahon and Bradley, A. Cell
62 : 1073-1085 (1990)], which was the AB-1 line grown on the above. Other suitable ES systems include the E14 system [Hooper et al., Natu
re 326: 292-295 (1987)], D3 series (Doetschman et al., J. E.
mbryol. Exp. Morph. 87 : 27-45 (1985)], and CCE
Systems [Robertson et al., Nature 323: 445-448 (1986)], but are not limited thereto. The successful generation of mouse lines from ES cells with specific target mutations is that pluripotency of ES cells (ie, when injected into a host blastocyst, allows them to participate in embryogenesis and Their ability to contribute to the germ cells of animals).

The pluripotency of any given ES cell line may depend on the culture time and the attention given to it. The only definitive assay for pluripotency determines whether the particular population of ES cells to be used can generate chimeras that are capable of germline transmission of the ES genome That is. For this reason, prior to gene targeting, a portion of the parental population of AB-1 cells was injected into C57B1 / 6J blastocysts to determine if the cells were able to generate chimeric mice and the large size of those chimeras. See if the part can transfer the ES genome to offspring.

The kappa chain inactivation vector J / C K1 was digested with NotI and the method described [Hasty, PR et al., Nature.
350: 243-246 (1991)] and electroporated into AB-1 cells. 10 electroporated cells
The cells were plated on 0 mm dishes at a density of 2-5 × 10 ⁶ cells / dish. twenty four
After hours, G418 (200 μg / ml active ingredient) and FIAU (0.
5 μM) was added to the medium to develop drug resistant clones for 10 to 11 days. Clones were picked, trypsinized, split into two parts and expanded further. Half of the cells from each clone were then frozen and the other half analyzed for homologous recombination between the vector and the target sequence.

DNA analysis was performed by Southern blot hybridization. As stated [Laird, PW
Et al . , Nucl. Acids Res. 19 : 4293 (1991)] clone, DNA was isolated by digestion with XbaI and probed with the 800 bp EcoRI / XbaI fragment indicated in Figure 25e as a characteristic probe. This probe is 3.7k in the wild type locus
b The XbaI fragment and a characteristic 1.8 kb band in the locus homologously recombined with the targeting vector were detected (Fig. 25a).
And e)). Of the 901 G418 and FIAU resistant clones screened by Southern blot analysis, 7 clones displayed the 1.8 kb XbaI band indicating homologous recombination into one of the kappa genes. The four clones were further digested with BglII, SacI and PstI enzymes to confirm that the vector was homologously integrated into one of the kappa genes. Typical 800 bp EcoRI /
When probed with the Xba I fragment (probe A), BglII, Sac I and Pst I digests of wild type DNA were obtained, respectively.
The 4.1, 5.4, and 7 kb fragments were generated, while the presence of the targeted kappa allele was indicated by the 2.4, 7.5 and 5.7 kb fragments, respectively (see Figures 25a and e). All seven positive clones detected by the XbaI digest showed the expected BglII, SacI and PstI restriction fragments that are characteristic of homologous recombination at the kappa light chain. Five of the targeted ES clones described in the previous section Preparation of mice with inactivated κ chains, as described [Bradley, A., in Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, EJ R
Edited by Obertson (Oxford: IRL Press), pp. 113-151 (198
7)] Injected into C57B1 / 6J blastocysts and transferred to the uterus of pseudopregnant females to generate chimeric mice derived from a mixture of cells derived from injected ES cells and host blastocysts. Black C5
On the 7B1 / 6J background, the extent of ES cell line-derived agouti coat coloring visually identifies the extent of ES cell contribution to the chimera. Almost half of the progeny obtained from blastocyst injections of target clones were chimeric (ie, showed agouti and black staining), and most of them contributed excess ES cells to capsular staining (> 70%). )showed that. AB1 ES cells
The XY cell line, the majority of those high rate chimeras were male due to sex reversal of female embryos by colonized male ES cells.

Male chimeras are crossed with C57BL / 6J and offspring are observed for the presence of a dominant agouti coat color, an indicator of germline transmission of the ES genome. Chimeras from two of those clones consistently produced agouti progeny. Since only one copy of the kappa locus was targeted to the infused ES clone, each agouti progeny had a 50% chance of inheriting the mutant locus. BglII digested D from tail biopsy sample
By Southern blot analysis of NA, using the probe used to identify the target ES clone (Probe A, Figure 25e),
The target gene was screened. As expected, approximately 50% of agouti progeny had 2.4 kb in addition to the 4.1 kb wild-type band.
The kb hybridizing BglII band is shown. This result demonstrates germline transmission of the targeted kappa locus.

To generate mice homozygous for the mutation, heterozygotes were mated and the progeny kappa genotype determined as described above. As I expected,
Three genotypes were derived from the heterozygous mating: wild-type mice with two copies of the normal kappa locus, one copy of the target kappa gene and a heterozygote with one NTkappa gene, and for the kappa mutation. A mouse that is homozygous. Deletion of the kappa sequence from these latter mice was confirmed by Southern blot hybridization using a probe specific for Jkappa (Probe C, Figure 25a). The Jκ
Hybridization of the probe was observed in DNA samples from the heterozygous and wild-type progeny, whereas no hybridization signal was detected in the homozygotes. This demonstrates the development of a novel mouse strain in which both copies of the kappa locus are inactivated by deletion as a result of targeted mutations.

Example 10 Inactivation of the mouse heavy chain gene by homologous recombination This example describes activation of the endogenous mouse immunoglobulin heavy chain gene by homologous recombination in embryonic stem (ES) cells. To do. The strategy is to delete the endogenous heavy chain J segment by homologous recombination with a vector containing the heavy chain sequence in which the _JH region has been deleted and replaced by the selectable marker gene neo. Preparation of heavy chain targeting vector J _H 4 specific oligonucleotide probe: 5′-ACT ATG CTA TGG ACT ACT GGG GTC AAG GAA CCT C
Using the AG TCA CCG-3 ′, the D3 ES cell line [Gossler et al . , Proc. Natl. Ac
ad. Sci. USA 83 : 9065-9069 (1986)], a mouse heavy chain sequence containing the J _H region (Fig. 26a) was isolated from a genomic phage library.

3.5 kb genomic Sac I / St spanning the J _H region
The uI fragment was isolated from a positive phage clone and the
Subcloned into pUC18 digested with cI / SmaI. The resulting plasmid was named puc18 J _H. The neomycin resistance gene (neo) used for drug selection of transfected ES cells was derived from the repair variant of plasmid pGEM7 (KJ1). Report in literature [Yenofski et al., Pr
oc. Natl. Acad. Sci. USA 87 : 3435-3439 (1990))
Is worked pMC1neo [Thomas and Cappechi as the source of the neo gene used in pGEM7 (KJ1), Cell 51: 50
3-512 (1987)].
Demonstrate point mutations in the eo coding sequence.

This mutation reduces the activity of the neo gene product, but was repaired by replacing the restriction fragment containing the mutation with the corresponding sequence from the wild-type neo clone. pGEM
The HindIII site in 7 (KJ1) was changed to a SalI site by the addition of synthetic adapters and the neo expression cassette was excised by digestion with XbaI / SalI. Then both ends of the neo fragment were blunted by treatment with Klenow form of DNA pol I, and subcloned into the NaeI site of pUC18 J _H, to generate the plasmid pUC18 J _H -neo (Fig 26b).

Further construction of the targeting vector is carried out by plasmid
It was performed on a derivative of pGP1b. Restriction enzyme Not for pGP1b
Digested with I and the following oligonucleotide as an adapter: 5'-GGC CGC TCG ACG ATA GCC TCG AGG CTA TAA ATC T
AG AAG AAT TCC AGC AAA GCT TTG GC-3 '

The resulting plasmid (designated pGET) was used to construct a mouse immunoglobulin heavy chain targeting construct. Mans
As described by our et al., Nature 336: 348-352 (1988), to prepare for the enrichment of ES clones with homologous recombinants, herpes simplex virus (H
The SV) thymidine kinase (TK) gene was included. Plasmid
The HSV TK gene was obtained from pGEM (TK) by digestion with EcoRI and HindIII. This TK DNA fragment was added to the EcoRI site of pGMT and Hind
Subcloned between the III site and the plasmid pGMT
-TK (Fig. 26c) was prepared.

Restriction digestion of DNA with XhoI and partial digestion with XbaI from the positive genomic phage clones to provide extensive regions of homology to the target sequence.
5.9 kb genome Xba I / Xho located 5'of J _H region
The I fragment was induced. This Xba, as shown in Figure 26a
The I site is not present in genomic DNA, but rather is derived from the phage sequence immediately adjacent to the cloned genomic heavy chain insert in the positive phage clone. Xb this fragment
Subcloning into aI / Xho I digested pGMT-TK generated plasmid pGMT-TK-J _H 5 '(Fig. 27d).

The final stage of production was the 2.8 kb Eco from pUC18 J _H -neo containing the neo gene and flanking genomic sequences.
Included excision of RI fragments. This fragment was blunt-ended with Klenow polymerase and similarly blunt-ended with pGMT-T.
Subcloned into the XhoI site of K-J _H 5 '. The resulting construct, J _H KO1 (FIG. 27e), contains a 6.9 kb genomic sequence flanking the J _H locus with a 2.3 kb deletion spanning the J _H region with the neo gene inserted therein. Figure 27f
Shows the structure of the endogenous heavy chain gene after homologous recombination with the targeting construct.

Example 11 Production and analysis of targeted ES cells Essentially as described [Robertson, EJ (1987).
Terato- carcinomas and Embryonic Stem Cells: A Pra
ctical Approach, EJ Robertson (Oxford: IRL Pr
ess), pp. 71-112], a mitotically inactive SNL76 / 7 feeder layer [McMahon and Bradley, Cell 62 : 1073-1085 (199).
0)] AB-1 ES cells were grown above. Generation of AB-1 derived chimeras demonstrated to have germline transduction ability of the ES genome prior to electroporation of ES cells by the targeting construct J _H KO1 as described in previous examples. Was used to determine the pluripotency of ES cells.

The heavy chain inactivation vector J _H KO1 was digested with Not I and the method described [Hasty et al., Nature 35.
0: 243-246 (1991)] and electroporated into AB-1 cells. 100 electroporated cells
Plates were plated at a density of 1-2 × 10 ⁶ cells / dish on mm dishes. After 24 hours, G418 (200 mg / ml active ingredient) and FIAU (0.5 m
M) was added to the medium and drug resistant clones were developed for 8-10 days. Clones were picked, trypsinized, split into two parts and expanded further. Half of the cells from each clone were then frozen and the other half analyzed for homologous recombination between the vector and the target sequence.

DNA analysis was performed by Southern blot hybridization. As stated [Laird et al., Nuc
l. Acids Res. 19 : 4293 (1991)] clone to DNA
Was isolated, digested with StuI, and labeled as probe A.
It was probed with the 500 bp EcoRI / Stu I fragment shown in 27f. This probe is a 4.7 kb Stu in the wild type locus.
The I fragment was detected, while the 3 kb band is characteristic of homologous recombination of the targeting vector and the endogenous sequence (see Figures 26a and 27f). 525 G418 screened by Southern blot hybridization and
Of the FITU double resistant clones, 12 were found to contain a 3 kb fragment characteristic of recombination with the targeting vector.
The fact that these clones display the expected target phenomenon at the J _H locus (as shown in Figure 27f) indicates that HindII
Confirmed by further digestion with I, Spe I and Hpa I.

Digested with HindIII, Spe I and Hpa I
Probe A to Southern blot of isolated DNA (see Figure 27f).
)) Hybridization is at the wild-type locus.
Of 2.3 kb,> 10 kb and> 10 kb, respectively.
For the targeted heavy chain locus,
Bands of 5.3 kb, 3.8 kb, and 1.9 kb are expected, respectively.
(See FIG. 27f). 12 detected by StuI digest
All of the positive clones are target J_HCharacteristic of genes
Putative HindIII, Spe I and Hpa I bands are shown. Addition
Therefore, use a neo-specific probe (Probe B, Figure 27f).
Southern blocks of StuI digests of all 12 clones
Analysis yielded only a 3 kb putative fragment,
Each contain a single copy of the targeting vector.
certified. Generation of mice with J _H deletion 3 of the targeted ES clones described in the previous chapter
Thaw and as described [Bradley, A. (1987)Teratocarcin
omas and EmbryonicStem Cells: A PracticalApproach,
 E.J. Robertson (Oxford: IRL Press), pp. 113-151
(1987)] C57B1 / 6J blastocysts and pseudopregnant females.
Transferred to the womb. ES cell line on a black C57B1 / 6J background
ES cell to chimera from the amount of agouti film coloring derived from
Visually identify the degree of contribution of Two target clones
Almost half of the progeny obtained from blastocyst injection in
No. 3 (ie, Agouti and black coloring) 3rd
Target clones did not give rise to chimeric animals. Them
The majority of chimeras contribute a significant contribution of ES cells to film coloring
Degree (70% or more). AB1 ES cells are an XY cell line
The majority of chimeras have male colonized female embryos.
It was male because it was transsexualized by ES cells. Male chimera
Were crossed with C57BL / 6J females to determine germline transmission of the ES genome.
The descendants for the presence of a dominant agouti coat color, which is a characteristic
Sympathize.

Chimeras from both of those clones consistently produced agouti progeny. Since the only copy of the heavy chain locus was targeted in the infused ES clone,
Each Agouti progeny has a 50% chance of inheriting the mutant locus
I had. Screening for target genes was performed by Southern blot analysis of StuI digested DNA from tail biopsy samples using the probe used to identify target ES clones (Probe A, Figure 27f). As expected, about the Agouti descendants
50% showed a hybridizing StuI band of approximately 3 kb in addition to the wild type band of 4.7 kb. This result demonstrates germline transmission of the targeted J _H locus.

To generate mice homozygous for the mutation, heterozygotes were mated and the progeny heavy chain genotype was determined as described above. As expected, three genotypes were derived from the heterozygous mating: wild-type mice with two copies of the normal J _H locus, heterozygotes with one target copy and one normal copy of the gene, and Mice homozygous for the J _H mutation. Deletion of the _JH sequence from those latter mice was confirmed by Southern blot hybridization using a probe specific for _JH (Probe C, Figure 26). 4.7 kb in DNA samples from heterozygous and wild-type offspring
Hybridization of the J _H probe to the fragment was observed, while no signal was detected in the sample from the J _H mutant homozygote. This demonstrates the generation of a novel mouse strain in which both copies of the heavy chain gene have been mutated due to the deletion of the _JH sequence.

Example 12 Heavy chain minilocus transgene A. A plasmid for cloning large DNA sequences
Construction of the Do Vector 1. The pGP1a plasmid pBR322 was digested with EcoRI and StyI and the following oligonucleotides: oligo-42 5'-caa gag ccc gcc taa tga gcg ggc
ttt ttt ttg cat act gcg gcc gct −3 ′ oligo-43 5′-aat tag cgg ccg cag tat gca aaa
aaa agc ccg ctc att agg cgg gct −3 ′.

The resulting plasmid pGP1a can be excised with a rare cutting enzyme NotI
Modify to construct NA constructs. It is a strong transcription termination signal [Ch
ristie et al . , Proc. Natl. Acad. Sci. USA 78 : 4180 (198
1)] downstream (with respect to the ampicillin resistance gene AmpR)
Contains a NotI restriction site. This termination signal reduces the potential toxicity of the coding sequence inserted into the NotI site by eliminating read-through transcription from the AmpR gene. In addition, this plasmid has a low copy number compared to the pUC plasmid because it retains the pBR322 copy number regulatory region. This low copy number also further reduces the potential toxicity of the insert and reduces selection for large inserts by DNA replication. Vector pGP1b, pGP1c, p
GP1d and pGP1f are derived from pGP1a and contain different polylinker cloning sites. The polylinker sequence is given below.

[0306]

[Chemical 1]

Each of these plasmids can be used to make large transgene inserts, which inserts
It can be excised with NotI so that the transgene DNA can be purified from the vector sequence before microinjection. 2. pGP1b pGP1a was digested with NotI and the following oligonucleotides: oligo-47 5'-ggc cgc aag ctt act gct gga tcc
tta att aat cga tag tga tct cga ggc −3 ′ oligo-43 5′-ggc cgc ctc gag atc act atc gat
taa tta agg atc cag cag taa gct tgc −3 ′.

The resulting plasmid, pGP1b, contains a short polylinker region flanked by NotI. This is Not
Facilitates the production of large inserts that can be excised by I. 3. pGPe The following oligonucleotides: oligo-44 5'-ctc cag gat cca gat atc agt acc
tga aac agg gct tgc −3 ′ oligo-45 5′-ctc gag cat gca cag gac ctg gag
cac aca cag cct tcc -3 'using the polymerase chain reaction technique, an immunoglobulin heavy chain 3'enhancer derived from rat liver DNA [S.
Petterson et al., Nature 344: 165-168 (1990)] was amplified.

The amplified product was digested with BamHI and Sph I, and pNNO3 digested with BamHI / Sph I (pNNO3 was the following restriction sites in the order listed: NotI, BamHI, NcoI, ClaI,
EcoRV, XbaI, Sac I, Xho I, Sph I, Pst I, Bgl
II, EcoRI, SmaI, KpnI, HindIII and NotI are pUC-derived plasmids containing a polylinker)
Cloned into. The resulting plasmid pRE3 was BamHI
And HindIII digested, and the insert containing the rat Ig heavy chain 3'enhancer digested with BamHI / HindIII
It was cloned into pGP1b. The resulting plasmid pGPe
(Figure 28 and (Chemical Formula 2) and (Chemical Formula 3)) contain several unique restriction sites into which the sequence can be cloned and then excised with the 3'enhancer by NotI digestion.

[0310]

Embedded image

[0311]

Embedded image

B. Small locus transgene expressing IgM
Construction of pIGM1 1. Isolation of J-μ constant region clone and construction of pJM1 Phage vector λEMBL3 / SP6 / T7 (Clonetech Laborato
ries, Inc., Palo Alto, CA), a human placental genomic DNA library cloned into a human heavy chain J region-specific oligonucleotide: oligo-15'-gga ctg tgt ccc tgt gtg atg ctt.
screened with ttg atg tct ggg gcc aag-3 'and the phage clone λ
1.3 was isolated. A 6 kb HindIII / KpnI fragment containing all six J segments as well as the D segment DHQ52 and the heavy chain J-μ intron enhancer was isolated from this clone. The same library was used for human μ-specific oligonucleotides: oligo-2 5'-cac caa gtt gac ctg cct ggt cac
The phage clone λ2.1 was isolated by screening with aga cct gac cac cta tga-3 '. A 10.5 kb HindIII / XhoI fragment containing the entire μ switch region and μ constant region exons was isolated from this clone. These two fragments were Kpn I / Xho
Ligated with pNNO3 digested with I, plasmid
We obtained pJM1. 2. A 4 kb Xho I fragment was isolated from pJM2 phage clone λ2.1. The fragment is a so-called Σμ element involved in the δ-related deletion of the μ gene in certain IgD-expressing B cells [H. Yasui
Et al. , Eur. J. Immunol. 19 : 1399 (1989); which is incorporated herein by reference], containing sequences immediately downstream of the sequence of pJM1. This fragment is treated with the Klenow fragment of DNA polymerase I and ligated with XhoI cut and Klenow treated pJM1. The resulting plasmid, pJM2 (Figure 29), lacks an internal XhoI site but retains 3'XhoI as a result of an incomplete reaction with the Klenow enzyme. pJM2 is a complete human J region,
Heavy chain J-μ intron enhancer, all μ switch regions and μ constant region exons, and two 0.4 kb direct repeats σ μ and Σ μ involved in the δ-related deletion of the μ gene.
Contains. 3. Isolation of D region clone and production of pDH1 The following human D region specific oligonucleotides: oligo-45'-tgg tat tac tat ggt tcg ggg agt
A human placental genomic library was screened for D region clones using tat tat aac cac agt gtc-3 '. Phage clone λ4.
1 and λ 4.3 were isolated. D elements D _K1 , D _N1 and D
_M2 [Y. Ichihara et al., EMBO J. 7: 4141 (1988)]
A 5.5 kb XhoI fragment was isolated from phage clone λ4.1. The upstream flanking 5.2 kb XhoI fragment containing D elements D _LR1 , D _XP1 , D _XP'1 and D _A1 was cloned into phage clone λ4.3.
Isolated from. Each of these D region XhoI fragments was labeled with SalI of the plasmid vector pSP72 (Promega, Madison, WI).
It was cloned into the site and the XhoI site joining the two sequences was destroyed. Then, the upstream fragment was excised with XhoI and SmaI, and the downstream fragment was excised with EcoRV and XhoI. The resulting isolated fragment was ligated with SalI-digested pSP2 to give plasmid pDH1. pDH1 contains at least 7 D segments and contains Xho I (5 ') and EcoRV
It contains a 10.6 kb insert that can be excised at (3 '). 4. pCOR1 plasmid pJM2 was digested with Asp718 (Kpn I isoschizomer) and the Klenow fragment of DNA polymerase I was used to fill in the overhanging ends. The resulting DNA is then C
The insert was isolated by digestion with laI. PD this insert
H1 XhoI / EcoRV insert and XhoI / ClaI digestion
It was ligated with pGPe to prepare pCOR1 (Fig. 30). 5. pVH251 Two human heavy chain variable region segments V _H 251 and V _H 105
10.3, including Humphries et al., Nature 331: 446 (1988)
The kb genomic HindIII fragment was subcloned into pSP72 to give plasmid pVH251. 6. pIGM1 plasmid pCOR1 was partially digested with XhoI and pVH251
The isolated Xho I / Sal I insert was cloned into the upstream Xho I site to create plasmid pIGM1 (FIG. 31).
pIGM1 contains two sequence elements such that all of the following sequence elements can be isolated in a single fragment containing no vector sequences by digestion with NotI and microinjected into the pronucleus of mouse embryos. A functional human variable region segment, at least 8 human D segments, all 6 human J _H segments, human μ enhancer, human σ μ element, human μ switch region, all human μ coding exons and human Σ μ element, rat heavy chain 3 ′ Contains with enhancer. C. Small locus transgene pH expressing IgM and IgG
Construction of C1 1. Isolation of γ constant region clones Oligonucleotides specific for the following human IgG constant region genes: oligo-29 5′-cag cag gtg cac acc caa tgc cca
The human genomic library was screened using tga gcc cag aca ctg gac-3 '. The phage clones λ29.4 and λ29.5 were isolated. 4 kb HindI of phage clone λ29.4 containing γ switch region
Using the II fragment, the phage vector Lambda FIX ^™ II
A human placental genomic DNA library cloned in (Stratagene, La Jolla, CA) was probed. The phage clone λSg1.13 was isolated. To determine the subclasses of the different γ clones, using the subclones of each of the above three phage clones as templates and the following oligonucleotides as primers: oligo-67 5'-tga gcc cag aca ctg gac-3 ' Was used to carry out a dideoxy sequencing reaction.

Both phage clones λ29.5 and λS γ1.13 were determined to be γ1 subclasses. 2. 7.8 kb of phage clone λ29.5 containing pγe1 γ1 coding region
The HindIII fragment was cloned into pUC18. The resulting plasmid pLT1 was digested with XhoI, treated with Klenow fragment and religated to destroy the internal XhoI site.
The resulting clone pLT1xk was digested with HindIII and the insert was isolated and cloned into pSP72 to create plasmid clone pLT1xks. Digestion of pLT1xks with polylinker XhoI site and BamHI site from human sequence
A 7.6 kb fragment containing the γ1 constant region coding exons was provided. This 7.6 kb XhoI / BamHI fragment together with the adjacent downstream 4.5 kb BamHI fragment from phage clone λ29.5
Cloning into XhoI / BamHI digested pGPe,
The plasmid pγe1 was created. pγe1 contains all of the γ1 constant region coding exons with a 5 kb downstream sequence linked to the rat heavy chain 3 ′ enhancer. 3. pγe2 γ1 switch region and pre-switch non-fertile transcript (steril
e trans-cript) (P. Sideras et al., International Immuno
l. 1: 631 (1989)] including the first exon and 5.3 kb Hi
The ndIII fragment was isolated from the phage clone λS γ1.13,
And polylinker XhoI near the 5'end of the insert
The plasmid clone pSγ1s was prepared by cloning into site-containing pSP71. cloning the XhoI / Sal I insert of pSγ1s into XhoI digested pγe1
A plasmid clone pγe2 was created (Fig. 32). pγe2
Is linked to the rat heavy chain 3'enhancer,
It contains all of the γ1 constant region coding exons as well as the upstream switch region and non-fertile transcript exons, along with a 5 kb sequence. 4. The pHC1 plasmid pIGM1 was digested with XhoI, the 43 kb insert was isolated and cloned into XhoI digested pγe2 to generate plasmid pHC1 (FIG. 31). pHC1 is
All of the following sequence elements can be isolated by digestion with NotI in a single fragment containing no vector sequences and microinjected into the pronucleus of mouse embryos to produce transgenic animals. Chain 3 '
2 functional human variable region segments, at least 8 human D segments, all 6 human _JH segments, human J-μ enhancer, human σ with enhancers
μ element, human μ switch region, all human μ coding exons, human Σμ element, and human γ1 constant region (including related switch region and non-fertile transcript related exons)
Contains. D. Small gene chain transgene pH expressing IgM and IgG
Construction of C2 1. Isolation of human heavy chain V region gene VH49.8 Human placenta genomic DNA library Lambda FIX ^TM II (S
tratagene, La Jolla, CA) to the following human VH1 family-specific oligonucleotide: oligo-49 5'-gtt aaa gag gat ttt att cac ccc
Screening was carried out using tgt gtc ctc tcc aca ggt gtc-3 '.

The phage clone λ49.8 was isolated and a 6.1 kb XbaI fragment containing the variable segment VH49.8 was added to pNNO3.
Subcloned in (polylinker ClaI site
The plasmid pVH49.8 was generated downstream of 49.8 and with the polylinker XhoI site upstream). Sequencing of the 800 bp region of this insert revealed that VH49.8 possesses an open reading frame and complete splicing and recombination signals, thus indicating that the gene is functional (Table 2). (Chemical formula 4) (Chemical formula 5).

[0315]

[Chemical 4]

[0316]

Embedded image

2. Human V _H subcloned into pV2 plasmid pUC12
IV family genes V _H 4-21 [I. Sanz et al., EMBO J. 8:
3741 (1989)] containing a 4 kb XbaI genomic fragment with SmaI and H
It was excised with indIII and treated with the Klenow fragment of polymerase I. The blunt ended fragment was cloned into ClaI digested Klenow treated pVH49.8.
The resulting plasmid, pV2, contains the human heavy chain gene VH49.8, ligated upstream of VH4-21 in the same orientation using a unique SalI site at the 3'end and a unique XhoI site at the 5'end of the insert. . 3. 0.7 kb XbaI / Hi together with 3.1 kb XbaI fragment upstream near pSγ1-5 '
The ndIII fragment (representing the sequence immediately upstream and adjacent to the fragment containing the 5.3 kb γ1 switch region in plasmid pγe2) was isolated from phage clone λSg1.13, and
It was cloned into pUC18 vector digested with HindIII / XbaI. The resulting plasmid, pSγ1-5 ′, is a sterile transcript found in B cells before switching to the γ1 isotype [P. Sideras et al., Internat.
ional Immunol. , 1: 631 (1989)] containing a 3.8 kb insert representing the sequence upstream of the start site. Since the transcripts are involved in the initiation of isotype switches, and upstream cis-acting sequences are often important for transcriptional regulation, they should be engineered to promote correct expression of non-fertile transcripts and associated switch recombination. Is included in the transgene construct. 4. The pVGE1 pSγ1-5 ′ insert was excised with SmaI and HindIII, treated with Klenow enzyme and ligated with the following oligonucleotide linker: 5′-ccg gtc gac cgg-3 ′. This ligation product was digested with SalI to give S
It was ligated into pV2 digested with alI.

The resulting plasmid pVP contains two functional human variable gene segments VH49.8 and VH4-21 (Table 2
And (see (Chemical Formula 5)), a 3.8 kb γ1 switch 5'flanking sequence linked downstream thereof. Partial digestion with SalI and
After complete digestion with XhoI, the pVP insert is isolated by purification of the 15 kb fragment on an agarose gel. The insert is then cloned into the XhoI site of pγe2 to create plasmid clone pVGE1 (Figure 33). pVGE1 is
It contains two human heavy chain variable gene segments upstream of the human γ1 constant gene and associated switch regions. Using the unique SalI site between the variable and constant regions,
The D, J and μ gene segments can be cloned. The rat heavy chain 3'enhancer is linked to the 3'end of the γ1 gene and the entire insert is flanked by NotI sites. 5. The pHC2 plasmid clone pVGE1 was digested with Sal I to give pIGM1
The Xho I insert was cloned into it. The resulting clone pHC2 (Figure 31) contains 4 functional human variable region segments, at least 8 D segments, 6 human J
All _H segments, human J-μ enhancer, human σμ
Element, human μ switch region, all human μ coding exons, human Σμ element, and human γ1 constant region (with 4 kb flanking sequence upstream of non-fertile transcript start site, the relevant switch region and non-fertile transcript-related exons Including) is included.

Those human sequences have all of the above sequence elements.
Linked to rat heavy chain 3'enhancer so that it can be isolated on a single chain containing no vector sequences by digestion with NotI and microinjected into the pronucleus of mouse embryos to give transgenic animals. To be done.
Using the unique XhoI site at the 5'end of the insert,
Additional human variable gene segments can be cloned into it to further increase the recombinant diversity of this heavy chain minilocus. E. FIG. The NotI insert of the transgenic mouse plasmids pIGM1 and pHC1 was isolated from the vector sequence by agarose gel electrophoresis. The purified insert was microinjected into the pronucleus of fertilized (C57BL / 6 x CBA) F2 mouse embryos and surviving embryos as described by Hogan et al. (B.
Hogan, F. Costantini and E. Lacy, Methods of Man
ipulating the Mouse Embryo, 1986, Cold Spring Harb
or Laboratory, New York) transferred to a pseudopregnant female. Mice developed from injected embryos were analyzed for the presence of transgene sequences by Southern blot analysis of tail DNA. The transgene copy number was assessed by band intensity compared to a control containing a known amount of cloned DNA.

Serum was isolated from these animals at 3-8 weeks of age and harvested by Harlow and Lane (E. Harlow and D. Lane, Antibodies: A Laboratory Manual, 1988, C).
human encoded by the transgene as described by (oldSpring Harbor Laboratory, New York).
The presence of IgM and IgG1 was assayed by ELISA. The wells of a microtiter plate are replaced with human IgM
Specific mouse monoclonal antibody (clone AF6, #
0285, AMAC, Inc. Westbrook, ME) and a mouse monoclonal antibody specific for human IgG (clone JL512, # 02).
80, AMAC, Inc. Westbrook, ME). Serum samples were serially diluted in the wells and affinity-isolated alkaline phosphatase-conjugated goat anti-human Ig (multivalent) with minimal cross-reactivity with mousse immunoglobulins by preadsorption was used. , The presence of specific immunoglobulins was detected.

Table 1 and FIG. 34 show an ELISA for the presence of human IgM and IgG1 in the sera of two animals developed from embryos injected with the transgene insert of plasmid pHC1.
The results of the assay are shown. All control non-transgenic mice tested by this assay were negative for human IgM and IgG expression. Two strains of mice containing the pIGM1 NotI insert (strains # 6 and 15) are human
It expresses IgM but not human IgG1. We have pHC1
Six lines of mice containing the insert were tested. As a result, 4 strains (strains # 26, 38, 57 and 122) express both human IgM and human IgG1, and 2 strains of mouse (strain # 19).
And 21) did not express detectable levels of human immunoglobulin.

PHC1 that did not express human immunoglobulin
Transgenic mice were so-called _GO mice, which developed directly from microinjected embryos and could be mosaic for the presence of the transgene. Southern blot analysis showed that many of those mice contained one or a few copies of the transgene per cell. Detection of human IgM in the serum of pIGM1 transgenic animals, and human IgM and IgG1 in the serum of pHC1 transgenic animals, function correctly in directing transgene sequences to VDJ ligation, transcription and isotype switching. Provide evidence. One animal (# 18) was negative for the transgene by Southern blot analysis and showed no detectable levels of human IgM or IgG1. The second animal (# 38) contains approximately 5 copies of the transgene as assayed by Southern blotting,
And it showed detectable levels of both human IgM and IgG1. The results of the ELISA assay for 11 animals developed from transgene-injected embryos are summarized in the table below (Table 1).

[0323]

[Table 1]

Table 1 shows the incorporated transgene DN
3 shows the correlation between the presence of A and the immunoglobulin encoded by the transgene in serum. Two of the animals found to contain the pHC1 transgene did not express detectable levels of human immunoglobulin.
They are both low copy animals and either do not contain a complete copy of the transgene or they have a genetic mosaic (pointed out by <1 copy per cell evaluated for animal # 21. Be done)
And the transgene-containing cells may not be transferred to the hematopoietic lineage. Alternatively, transgenes may be integrated in genomic regions that do not lead to their expression. Human IgM in serum of pIGM1 transgenic animals
For the detection of human IgM and IgG1 in the serum of pHC1 transgenic animals.
It is pointed out that it functions correctly in directing J-binding, transcription and isotype switching. F. Immunity derived from transgenic mouse spleen mRNA to evaluate the functionality of the pHC1 transgene with respect to the involvement of the human B cell receptor encoded by the transgene in cDNA clone VDJ ligation and class switching, and B cell development and allelic exclusion. The structure of the globulin cDNA clone was investigated. Examining the total diversity of heavy chains encoded by the transgene, focusing on D and J segment usage, addition of N regions, CDR3 length distribution, and the frequency of junctions leading to functional mRNA molecules. It was VH105
Transcripts encoding IgM and IgG incorporating VH251 and VH251 were examined.

Polyadenylated RNA was isolated from week 11 male second generation 57 lineage pHC1 transgenic mice.
This RNA was used to synthesize oligo dT initiated single stranded cDNA. The resulting cDNA was then used as a template for each of the four PCR amplification reactions using the following four synthetic oligonucleotides as primers: VH251 specific oligo-149 ctagct cga gtc caa gga gtc tgt gcc gag gtg.
cag ctg (g, a, t, c); VH105 specific oligo-150 gtt gct
cga gtg aaa ggt gtc cag tgt gag gtg cagctg (g, a,
t, c); Human γ1-specific oligo-151 ggc gct cga gtt c
ca cga cac cgt cac cgg ttc; and human μ-specific oligo-152 cct gct cga ggc agc caa cgg cca cgc tgc
tcg. Reaction 1 is VH2 using primers o-149 and o-151
Amplification of 51-γ1 transcript, reaction 2 primer o-1
49 and o-152 were used to amplify the VH251-μ transcript and reaction 3 was VH105-γ1 using primers o-150 and o-151.
Amplify the transcript, and reaction 3 uses primer o-150.
And o-152 were used to amplify the VH105-μ transcript.

The resulting 0.5 kb PCR product was isolated from an agarose gel. The μ transcript product was more abundant than the γ transcript product, consistent with the corresponding ELISA data (FIG. 40). The PCR product was digested with XhoI and cloned into plasmid pNN03. 9 from each of 4 PCR amplifications
Double-stranded plasmid DNA from the miniprep of one clone
Was isolated and a dideoxy sequencing reaction was performed.
Two of the clones were found to be deletions containing no D or J segment. They are normal RN
Since it could not be derived from the A splicing product, it probably resulted from the deletion introduced during PCR amplification. One of the DNA samples was found to be a mixture of two individual clones and the other three clones produced no readable DNA sequence (probably the DNA sample was not pure enough). It will be due to).

The DNA sequences of the VDJ junction from the remaining 30 clones are summarized in (Chemical Formula 6) (Chemical Formula 7). Each sequence is unique, indicating that the single pathway of gene rearrangement is not dominant or a single clone of transgene-expressing B cells is not dominant. The fact that no two sequences are similar indicates that the immense diversity of immunoglobulins can be expressed from a small minilocus containing only two V segments, 10 D segments and 6 J segments. It is also a hint. Both of the V segments contained in the transgene, all 6 J segments and 7 of 10 D segments are used for the VDJ junction.

In addition, both constant region genes (μ and γ1) are included in the transcript. The VH105 primer was found to be specific for VH105 in the reactions performed. Therefore, many of the clones from reactions 3 and 4 contained the VH251 transcript. In addition, the clone isolated from the PCR product of ligation reaction 3 was found to encode IgM rather than IgG; however, this was due to the PCR product from reaction 4 because the DNA was isolated on the same gel. May represent pollution. Adult peripheral blood lymphocytes (PB
L) to amplify the immunoglobulin heavy chain sequence and V
Similar experiments in which the DNA sequence of the DJ junction was determined were
Reported by Yamada et al . [ J. Exp. Med. 173: 395-407 .
(1991); which is incorporated herein by reference]. The data from this human PBL were compared to our data from pHC1 transgenic mice.

[0329]

[Chemical 6]

[0330]

[Chemical 7]

G. J-Segment Selection Table 2 compares the distribution of J-segments incorporated in transcripts encoded by the pHC1 transgene with those found in adult PBL immunoglobulin transcripts. Their distribution profiles are very similar, with the J4 segment being the dominant segment followed by the J6 segment in both systems. J2 is human P
It is the rarest segment in BL and in transgenic animals.

[0332]

[Table 2]

H. D segment selection 49% of clones analyzed by Yamada et al. (4 out of 82)
0) contained the D segment contained in the pHC1 transgene. The other 11 clones contained sequences that were not assigned to any of the known D segments by the authors. Two of the 11 unspecified clones appear to be derived from an inversion of the DIR2 segment contained in the pHC1 construct. Ichihara et al. [ EMBO J. 7: 4141
(1988)] and Sanz [ J. Immunol . 147:
1720-1729 (1991)].
da et al. [ J. Exp. Med. 171: 395-407 (1991)] were not taken into account. Table 2 shows the D segment distribution for pHC1 transgenic mice and human PBLs by Yamada et al.
Comparison with that observed for transcripts. Yamada
These data were re-edited to include DIR2 usage and exclude the D segment that is not in the pHC1 transgene.

Table 3 demonstrates that the distribution of D segment integration is very similar in transgenic mice and human PBLs. The two dominant human D segments DXP'1 and DN1 are also frequently found in transgenic mice. The biggest difference between the two distributions is that DHQ5 is higher in transgenic mice than in humans.
2 high frequency. The high frequency of DHQ52 is reminiscent of the D segment distribution in human fetal liver. Sanz is a heavy chain transcript
14% reported that they contained the DHQ52 sequence. If the D segment not found in pHC1 is excluded from the analysis, Sa
31% of fetal transcripts analyzed by nz contain DHQ52. This is comparable to the 27% we observed in pHC1 transgenic mice.

[0335]

[Table 3]

I. VDJ Junctional Functionality Table 4 was analyzed from pHC1 transgenic animals 30
The deduced amino acid sequence of the VDJ region from each clone is shown. The translation sequence is 23 out of 30 VDJ junctions (77
%) Is within the frame (in
-frame).

[0337]

[Chemical 8]

[0338]

[Chemical 9]

J. CDR4 length distribution table 4 shows in-frame VDJ in pHC1 transgenic mice.
CDR3 peptide length from transcripts with junctions compared to that in human PBLs. Human PBL data was also obtained from Yamada et al. Although the two distributions are similar, the distribution in transgenic mice is slightly smaller than that observed in human PBL CDR3.
It was biased towards peptides. The average length of CDR3 in transgenic mice is 10.3 amino acids. this is
Sanz [ J. Immunol . 147: 1720-1729 (1991)] human
It is substantially the same as the average size reported for the CDR3 peptide.

[0340]

[Table 4]

Example 13 Rearranged Heavy Chain Transgene A. Isolated rearranged human heavy chain VDJ segment isolated phage vector λEMBL3 / SP6 / T7 (Clonetech Laborato
ries, Inc., Palo Alto, CA) 2
Human leukocyte genomic DNA library was cloned into a 1 kb Pac of λ1.3 containing the human heavy chain J-μ intron enhancer.
Screen with the I / HindIII fragment. Positive clones were labeled with the following _VH- specific oligonucleotides: oligo-7 5'-tca gtg aag gtt tcc tgc aag gca.
tct gga tac acc ttc acc −3 ′ oligo-8 5′-tcc ctg aga ctc tcc tgt gca gcc
Test for hybridization with a mixture of tct gga ttc acc ttc agt-3 '.

Clones that hybridize with both the V and J-μ probes are isolated and the DNA sequence of the rearranged VDJ segment is determined. B. Generation of Rearranged Human Heavy Chain Transgene A fragment containing a functional VJ segment (transcriptional reading frame and splice signal) was inserted into plasmid vector pSP72 so that the XhoI site derived from the plasmid was adjacent to the 5'end of the insert sequence. Subcloning into. Functional VD
Subclones containing the J segment were cloned into XhoI and PacI (Pa
cI is a rare cleavage enzyme that recognizes a site near the J-μ intron enhancer) and the insert is cloned into XhoI / PacI digested pHC2 to obtain a functional VDJ segment, J-. μ intron enhancer, μ switch element, μ constant region coding exon and γ1 constant region (which is a non-fertile transcript-related sequence, γ
A transgene construct (including 1 switch and code exons). This transgene construct is excised with NotI and microinjected into the pronucleus of mouse embryos to produce transgenic animals as described above.

Example 14 Light Chain Transgene A. Preparation 1. Plasmid vector pGP1c Plasmid vector pGP1a plasmid vector was digested with NotI, and the following oligonucleotides: Oligo -81 5'-ggc cgc atc ccg ggt ctc gag gtc
gac aag ctt tcg agg atc cgc −3 ′ oligo-82 5′-ggc cgc gga tcc tcg aaa gct tgt
cga cct cga gac ccg gga tgc − 3 ′ is ligated into it. The resulting plasmid pGP1c is
XmaI, XhoI, SalI, Hind flanked by NotI sites
Includes polylinkers with III and BamHI restriction sites. 2. Plasmid vector pGP1d Plasmid vector pGP1a was digested with NotI and the following oligonucleotides: oligo-87 5'-ggc cgc tgt cga caa gct tat cga.
tgg atc ctc gag tgc −3 ′ oligo-88 5′-ggc cgc act cga gga tcc atc gat
aag ctt gtc gac agc −3 ′ is ligated into it. The resulting plasmid pGP1d is
SalI, HindIII, Cla I, B flanked by NotI sites
Includes polylinkers with amHI and XhoI restriction sites. B. Isolation of Jκ and Cκ clones Phage vector λEMBL3 / SP6 / T7 (Clonetech Laborato
ries, Inc., Palo Alto, CA) and a human placental genomic DNA library cloned into human κ light chain J region-specific oligonucleotide: oligo-36 5'-cac ctt cgg cca agg gac acg act.
Screened with gga gat taa acg taa gca-3 'and phage clone 13
Isolate 6.2 and 136.5. The 7.4 kb XhoI fragment containing the Jκ1 segment from 136.2 is isolated and subcloned into plasmid pNNO3 to create plasmid clone p36.2. Jκ segment 2 together with Cκ gene segment
A neighboring 13 kb Xho I fragment containing ~ 5 was phage cloned
Isolated from 136.5 and subcloned into plasmid pNNO3 to create plasmid clone p36.5.
When these two clones were put together, Jκ1 7.2 kb
It spans the region beginning upstream and ending 9 kb downstream of Cκ. C. Generation of rearranged light chain transgene 1. C for expressing rearranged variable segment
κ vector pCK1 13 kb Xh of plasmid clone p36.5 containing Cκ gene
The oI fragment is cloned into the Sal I site of plasmid vector pGP1c with the 5 kb end of the insert flanking the plasmid Xho I site along with 9 kb of downstream sequence. The resulting clone, pCK1, is able to accommodate the cloned fragment containing the rearranged VJκ segment at the unique 5'Xho I site. Then the transgene is
It is excised with otI and purified from the vector sequence by gel electrophoresis. The resulting transgene construct is human J
It may include the -Ck intron enhancer and may include the human 3'kappa enhancer. 2. Cκ vector containing heavy chain enhancer pCK2 mouse heavy chain J-μ intron enhancer [J. Banerji for expressing rearranged variable segment]
Et al., Cell 33 : 729-740 (1983)].
The 0.9 kb XbaI fragment of A was subcloned into pUC18 to create plasmid pJH22.1. This plasmid
It was linearized with SphI and the ends were filled in using Klenow enzyme. The Klenow treated DNA is then Hind
Digested with III and ligated with it the MluI (Klenow) / HindIII fragment of the phage clone λ1.3 (previous example) containing the human heavy chain μ intron enhancer [Hayday et al., Nature 307: 334-340 (1984)]. did.

The resulting plasmid, pMHE1, contains a single
It consists of the mouse and human heavy chain μ intron enhancer ligated together in pUC18 so that it will be excised on the BamHI / HindIII fragment. This 2.3 kb fragment was isolated and designated pGP1c
Clone in to create pMHE2. pMHE2 to Sal
It is digested with I and the 13 kb Xho I insert of p36.5 is cloned into it. The resulting plasmid, pCK2, contains pC2 except that the mouse and human heavy chain J-μ intron enhancers are fused to the 3'end of the transgene insert.
Same as K1. Different enhancers, namely the mouse or rat 3'κ or heavy chain enhancers [Meyer and Neuber, to regulate expression of the final transgene].
ger, EMBO J. 8: 1959-1964 (1989); Petterson et al., Na
ture 344: 165-168 (1990)] can be used to make similar constructs. 3. Isolation of rearranged kappa light chain variable segment Phage vector lambda EMBL3 / SP6 / T7 (Clonetech Laborato
ries, Inc., Palo Alto, CA) 2
Two human leukocyte genomic DNA libraries containing p36.5
Screening was done with the human kappa light chain J region containing the 3.5 kb XhoI / SmaI fragment. Positive clones were labeled with the following Vκ-specific oligonucleotides: oligo-65 5′-agg ttc agt ggc agt ggg tct ggg.
Hybridization with aca gac ttc act ctc acc atc agc-3 'was tested. A clone hybridizing with both the V and J probes was isolated and the DN of the rearranged VJκ segment was isolated.
Determine the A sequence. 4. Generation of transgenic mouse containing rearranged human light chain construct Fragment containing functional VJ segment (open reading frame and splice signal) was added to XhoI of vectors pCK1 and pCK2.
Subcloning into the site creates a rearranged kappa light chain transgene. The transgene construct is isolated from the vector sequence by digestion with NotI. The insert fragment purified on agarose gel is microinjected into the pronucleus of mouse embryos to produce transgenic mice. Animals expressing the human kappa chain are crossed with transgenic animals containing the heavy chain minigene (Example 14) to produce mice that fully express human antibodies.

Not all of the VJκ combinations are capable of forming stable heavy chain-light chain complexes with a wide spectrum of various heavy chain VDJ combinations, and therefore several different rearranged VJκ clones were used. Different light chain transgene constructs are made and bred with mice expressing the heavy chain minilocus transgene. From double transgenic (both heavy and light chain constructs) animals,
Peripheral blood, spleen and lymph node lymphocytes were isolated, stained with fluorescent antibodies specific for human and mouse heavy and light chain immunoglobulins (Pharmingen, San Diego, CA) and analyzed on a FACScan analyzer (Becton Dickinson, San Jose,
CA) to analyze by flow cytometry. The highest levels of human heavy / light chain complexes were produced on the surface of the greatest number of B cells and did not adversely affect immune cell compartment (assayed by flow cytometric analysis using B and T cell subset-specific antibodies). Sometimes) rearranged light chain transgene constructs are selected for the production of human monoclonal antibodies. D. Unrearranged light chain minilocus transgene
Construction of 1. Jκ for constructing a small locus transgene,
13 kb Cκ-containing Xho of Cκ-containing vector pJCK1p36.5
The I insert is treated with Klenow enzyme and cloned into HindIII digested and Klenow treated plasmid pGP1d. A plasmid clone is selected in which the 5'end of the insert is adjacent to the ClaI site from the vector. The resulting plasmid p36.5-1d is digested with Cla I and treated with Klenow. The Jκ1-containing 7.4 kb XhoI insert of p36.2 is treated with Klenow and cloned into ClaI digested and Klenow treated p36.5-1d. Select clones with the p36.2 insert in the same orientation as the p36.5 insert. This clone pJCK1 (FIG. 35) contains the complete human Jκ and Cκ regions, with 7.2 kb upstream and 9 kb downstream.

The insert also contains the human J-Cκ intron enhancer and may include the human 3'κ enhancer. The insert is flanked by unique 3'SalI sites for the purpose of cloning additional 3'flanking sequences, such as heavy or light chain enhancers. Unique XhoI
A site is placed at the 5'end of the insert for cloning purposes in the unrearranged VK gene segment. The unique SalI and XhoI sites are flanked by NotI sites used to isolate the final transgene construct from the vector sequence. 2. Isolation of unrearranged Vκ gene segment and generation of transgenic animals expressing human Ig light chain protein. T7 (Clon
human placenta genomic DNA cloned into etech Laboratories, Inc., Palo Alto, CA). Sequencing the variable gene segment from the resulting clone,
Select clones that appear to be functional. Criteria for determining functionality are to include the open reading frame, the complete splice acceptor and donor sequences, and the complete recombinant sequence.

D containing selected variable gene segments
The NA fragment is cloned into the unique XhoI site of plasmid pJCK1 to create a minilocus construct. The resulting clones are digested with NotI, the insert is isolated and microinjected into the pronucleus of mouse embryos to produce transgenic animals. The transgenes of those animals will undergo VJ binding during B cell development. Animals expressing the human kappa chain are crossed with transgenic animals containing the heavy chain minilocus to obtain mice that fully express human antibodies.

Example 15 Genomic Heavy Chain Human Ig Transgene This example illustrates the introduction of a human genomic heavy chain immunoglobulin transgene introduced into mouse germ cells by microinjection into zygotes or integration into ES cells. Describe cloning.

Marzluff, WF et al. (1985) Transcription
and Translation: A Practical Approach , BD Hamme
s and SJ Higgins, 89-129, IRL Press, Oxfor
Nuclei are isolated from fresh human placental tissue as described by d. Isolated nuclei (or PBS-washed human spermatocytes) were embedded in 0.5% low-melting agarose blocks, and nuclei were treated with 1 mg / ml proteinase K in 500 mM EDTA, 1% SDS for spermatocytes. Is dissolved in 500 mM EDTA, 1% SDS, 10 mM DTT at 1 mg / ml proteinase K at 50 ° C. for 18 hours. Proteinase K is inactivated by incubating the block in 40 μg / ml PMSF / TE for 30 minutes at 50 ° C. Next by M. Finney, Current Protocols in M
olecularBiology (F. Ausubel et al., John Wiley & Son
S., Aug. 4, 1988, eg Chapter 2.5.1), the DNA is digested with the restriction enzyme NotI in agarose.

The DNA digested with Not I was then digested with Anand
Et al . , Nuc. Acids Res . 17 : 3425-3433 (1989) and fractionated by pulsed field gel electrophoresis. Fractions rich in NotI fragment are assayed by Southern hybridization to detect one or more sequences encoded by this fragment. Such an array is
It contains a representative of all six V _H families with heavy chain D segment, J segment and γ1 constant region [This fragment was described by Berman et al. (1988), supra from HeLa cells.
Although identified as a kb fragment, we found it to be an 830 kb fragment from human placenta and sperm DNA]. Fractions containing this NotI fragment (Fig. 4
(See McCormick et al., Technique 2: 65).
-71 (1990)] ligated into the NotI site of vector pYACNN. The plasmid pYACNN is an Eco version of pYACneo (Clontech).
RI digested and oligonucleotide 5'-AAT TGC GG
It is prepared by ligation in the presence of CCGC-3 '.

Traver et al . , Proc. Natl. Acad. Sci. USA
86 : 5898-5902 (1989), the heavy chain N
The YAC vector containing the otI fragment is isolated. The cloned NotI insert is isolated from high molecular weight yeast DNA by pulsed field gel electrophoresis as described by M. Finney (supra). DNA is concentrated by the addition of 1 mM spermine and microinjected directly into the nuclei of single cell embryos as described above. Alternatively, DNA is isolated by pulsed field gel electrophoresis and introduced into ES cells by lipofection [Gnirke et al., EMBO J. 10 : 1629-1634 (1991)], or YACs are transformed into ES cells by spheroplast fusion. Introduce inside.

Example 16 85 kb S of human genomic DNA containing discontinuous genomic heavy chain Ig transgene V _H 6, D segment, J segment, μ constant region and part of γ constant region
The peI fragment is essentially a YAC as described in Example 1.
Isolated by cloning. Fragment from the germline variable region, such as the above-mentioned, including a V ₁ ~V ₅ of multicopy
A YAC containing a 570 kb NotI fragment, upstream of the 670-830 kb NotI fragment, is isolated as described above. (Berman et al. (1988),
Shown above are two 57's, each containing multiple V segments.
A 0 kb NotI fragment was detected. The two fragments are co-injected into the nucleus of mouse single cell embryos as described in Example 1.

Co-injection of two different DNA fragments typically results in the integration of both fragments at the same site in the chromosome. Therefore, at least one of each of the two fragments
About 50% of resulting transgenic animals containing copies
Has a V segment fragment inserted upstream of the constant region containing fragment. Of those animals, depending on the orientation of the 570 kb Not I fragment with respect to the position of the 85 kb Spe I fragment,
About 50% will perform V-DJ binding by DNA inversion and about 50% will perform V-DJ binding by deletion. Animals isolated from the resulting transgenic animal and shown by Southern blot hybridization to contain both transgenes (specifically, animals containing both multiple human V segments and human constant region genes). ) Are tested for their ability to express human immunoglobulins according to standard techniques.

Example 17 Functionally rearranged cells in transgenic B cells
Using antigen identification interest variant region sequences, the following genetic properties: J _H deletion homozygous endogenous owned chain locus for (Example 10); unrearranged human heavy chain Hemizygous for a single copy of the minilocus transgene (Examples 5 and 14); and hemizygous for a single copy of the rearranged human kappa light chain transgene (Examples 6 and 14), Immunize mice with Hallow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbo
r, New York (1988)].

After the immunization schedule, the spleen is removed and splenocytes are used to prepare hybridomas. Genomic DNA is prepared using cells from individual hybridoma clones that secrete antibodies that are reactive with the antigen of interest. A sample of genomic DNA is digested with several different restriction enzymes that recognize a unique 6 base pair sequence and fractionated on an agarose gel. Southern blot hybridization is used to identify two DNA fragments within the 2-10 kb range. One of the fragments is rearranged human heavy chain V
It contains a single copy of the DJ sequence, the other contains a single copy of the rearranged human light chain VJ sequence. The two fragments are size fractionated on an agarose gel and cloned directly into pUC18. The cloned insert is subcloned into heavy and light chain expression cassettes containing constant region sequences, respectively.

The plasmid clone pγe1 (Example 12) is used as a heavy chain expression cassette and the rearranged VDJ sequence is cloned into the XhoI site. The plasmid clone pCK1 is used as a light chain expression cassette and the rearranged VJ sequence is cloned into the XhoI site. The resulting clones are used together to transfect SP ₀ cells to produce antibodies that react with the antigen of interest [MS
Co et al . , Proc. Natl. Acad. Sci. USA 88 : 2869 (199
1)].

Alternatively, mRNA is isolated from the cloned hybridoma cells described above and used to synthesize cDNA. The expressed human heavy and light chain VDJ and VJ sequences are
Then, it is amplified by PCR and cloned [JW
Larrich et al. (1989) Biol. Technology , 7: 934-938].
After determining the nucleotide sequence of those clones, an oligonucleotide encoding the same polypeptide was synthesized and C. Queen et al . , Proc. Natl. Acad. Sci. USA.
84 : 5454-5458 (1989) to produce a synthetic expression vector. Immunization of Transgenic Animals with Complex Antigens The next experiment showed that transgenic animals can be successfully immunized with complex antigens, such as those on human erythrocytes, and the response kinetics observed in normal mice. Prove that they can respond with similar kinetics.

Blood cells are generally suitable immunogens and comprise many different types of antigens on the surface of red and white blood cells. Collect human blood tube from immunized One providers by human blood, functionally disrupted endogenous heavy chain locus (J _H D) and has and a human heavy chain minigene construct (HC1) Was used to immunize transgenic mice containing These mice are designated as line 112. Blood was washed, resuspended in 50 ml Hank's solution and diluted to 1 × 10 ⁸ cells / ml. Then use a 28 gauge needle and a 1 cc syringe to prepare 0.2 ml (2 x 10
⁷ cells) was intraperitoneally injected. This immunization protocol was repeated almost every week for 6 weeks. Serum antibody titers were monitored by drawing blood from the retro-orbital bleed, collecting serum and testing it for specific antibodies. Preimmune hemorrhage was taken as a control. At the very last immunization, three days before sacrificing the animals to obtain serum or hybridomas, one immunization with 1 × 10 ⁸ cells into the caudal vein was performed to increase the production of hybridomas. went.

[0359]

[Table 5]

Mice # 2343 and 2348 have the desired phenotype: human heavy chain small gene transgenic on heavy chain disruption background. Preparation of hybridomas Hybridomas were prepared by fusing mouse splenocytes of about 16-week-old transgenic mice (Table 5) immunized as described above with a fusion partner consisting of the non-secreting HAT-sensitive myeloma cell line X63 Ag8.653. Was produced. Hybridoma clones were cultured and hybridoma supernatants containing immunoglobulins with specific binding affinity for blood cell antigens were identified by, for example, flow cytometry. Flow Cytometry Flow cytometry was used to test serum and hybridoma supernatants. Erythrocytes from the donor were washed 4 times in Hank's solution and 50,000 cells were placed in 1.1 ml polypropylene microtubules. Cells were stained with supernatant from hybridoma or antisera with staining medium [phenol red or biotin (Irvine Scientific), 3% newborn bovine serum, 0.1%.
Incubation for 30 minutes on ice in 1 × RPMI medium without% sodium azide. Controls consisted of littermate mice with other genotypes. Then the cells are Sorvall RT60
It was washed by centrifuging in OB at 1000 rpm for 5-10 minutes at 4 ° C. After washing the cells twice, the fluorogenic reagents were used to detect antibodies on the cell surface. Tested with two monoclonal reagents. One is a mouse anti-human mu heavy chain antibody labeled with FITC (Pharmagen, San Diego, CA) and the other is a rat anti-mouse kappa light chain antibody labeled with PE (Becton-Dickenson, San Jose, CA). Both of these reagents gave similar results. Whole blood (red blood cells and white blood cells) and white blood cells alone were used as target cells. Both sets gave positive results.

Sera of transgenic mice and littermate controls were incubated with either red blood cells from donors or white blood cells from other individuals, washed, and then developed with anti-human IgM FITC labeled antibody and in a flow cytometer. analyzed. The results show that sera from mice transgenic for the human minilocus (mouses 2343 and 2348) show human IgM reactivity, whereas sera from all littermates (2344, 2345, 2346, 2347) do. Showed no sex. Normal mouse serum (NS) and phosphate buffered saline (PBS) were used as negative controls. Red blood cells were unfiltered and white blood cells were filtered to contain only lymphocytes. Lines are drawn on the x and y axes to give a comparison. Flow cytometry was performed on 100 supernatants from fusion 2348. Four supernatants showed positive reactivity with blood cell antigens.

Example 18 Endogenous mouse immunoglobulin with antisense RNA
Reduced expression A. Vector for expression of antisense Ig sequences 1. Construction of cloning vector pGP1h Vector pGP1b (described in previous examples) was transformed with Xho I and BamHI.
Digested with and the following oligonucleotides: 5'- gat cct cga gac cag gta cca gat ctt gtg aat t
cg -3 ′ 5′- tcg acg aat tca caa gat ctg gta cct ggt ctc g
The plasmid pGP1h was constructed by ligating with ag-3 '. This plasmid contains the following restriction sites: NotI, EcoRI, BglII, Asp7.
It contains a polylinker containing 18, Xho I, BamHI, Hind III, NotI.

2. Generation of pBCE1 0.8 of pVH251 (described in the previous example) containing the promoter-leader sequence exon, the first intron and part of the second exon of the human _VH- V family immunoglobulin variable gene segment. The kb XbaI / BglII fragment was replaced with Xba
The plasmid pVH251N was constructed by inserting it into the vector pNN03 digested with I / BglII.

Human growth hormone gene [hGH; Seeburg
(1982) DNA 1: 239-249] including the code exons 2.2
The kb BamHI / EcoRI DNA fragment was cloned into BamHI / EcoRI digested pGH1h. The resulting plasmid
Digested with BamHI and inserted the BamHI / EcoRI fragment of pVH251N in the same orientation as the hGH gene,
I made hgh.

The 0.9 kb XbaI fragment of mouse genomic DNA containing the mouse heavy chain J-μ intron enhancer [Banerji et al. (1983) Cell 33 : 729-740] was subcloned into pUC18 to create plasmid pJH22.1. This plasmid was linearized with SphI and the ends were filled in with Klenow enzyme. Then, the Klenow-treated DNA was added to HindII.
1.4 kb MluI of phage clone λ1.3 (Example above) digested with I and containing the human heavy chain J-μ intron enhancer [Hayday et al. (1984) Nature 307: 334-340].
The (Klenow) / HindIII fragment was ligated with it. The resulting plasmid, pMHE1, consists of the mouse and human heavy chain J-μ intron enhancers ligated together in pUC18 so that they can be excised in a single BamHI / HindIII fragment.

The BamHI / HindIII fragment of pMHE1 was transformed into BamHI /
It was cloned into HindIII-cut pVhgh to generate B cell expression vector pBCE1. This vector (Fig. 42
Contains a unique XhoI and Asp718 cloning site into which antisense DNA fragments can be cloned. Expression of those antisense sequences is directed by the upstream heavy chain promoter-enhancer combination, and the downstream hGH gene sequences provide in addition to intron sequences, polyadenylation sequences that facilitate expression of the transgene constructs. The antisense transgene construct made from pBCE1 can be separated from the vector sequence by digestion with NotI. B. IgM antisense transgene construct Oligonucleotide: 5'-cgc ggt acc gag agt cag tcc ttc cca aat gtc-
3 ′ 5′- cgc ctc gag aca gct gga atg ggc aca tgc aga-
The 3'was used as a primer for amplification of mouse IgM constant region sequences by polymerase chain reaction (PCR) using mouse spleen cDNA as a substrate. The resulting 0.3 kb PCR product was digested with Asp718 and XhoI, and Asp718 /
The antisense transgene construct pMAS1 was made by cloning into pBCE1 digested with Xho I. The purified NotI insert of pMAS1 alone or in combination with one or more alternative transgene constructs was microinjected into the pronucleus of half-day mouse embryos to generate transgenic mice. This construct is mouse IgM mR
It expresses RNA transcripts in B cells that hybridize with NA, thus down-regulating the expression of mouse IgM protein.

Double transgenic mice containing pMAS1 and a human heavy chain transgene minilocus such as pHC1 (produced by co-injection of both constructs or by mating single transgenic mice) It will express the Ig receptor encoded by the human transgene on a higher proportion of B cells than in mice transgenic for the heavy chain minilocus only.
The ratio of human to mouse Ig receptor expressing cells is due, in part, to factors promoting B cell differentiation and development and competition between the two populations for cells. Since the Ig receptor plays an important role in B cell development, mouse Ig receptor expressing B expressing reduced levels of IgM on the surface (due to mouse Ig-specific antisense down-regulation)
Neither the cells nor the cells expressing the human receptor will compete. C. Igκ antisense transgene construct Two oligonucleotides: 5'-cgc ggt acc gct gat gct gca cca act gta tcc-
3 ′ 5′- cgc ctc gag cta aca ctc att cct gtt gaa gct-
The 3'was used as a primer for amplification of mouse IgKappa constant region sequences by polymerase chain reaction (PCR) using mouse spleen cDNA as a substrate. The resulting 0.3 kb PCR product was digested with Asp718 and XhoI, and Asp718 /
The antisense transgene construct pKAS1 was generated by cloning into pBCE1 digested with Xho I. The purified NotI insert of pMAS1 alone or in combination with one or more alternative transgene constructs was microinjected into the pronucleus of half-day mouse embryos to generate transgenic mice. This construct is mouse Igκ mRN
It expresses RNA transcripts in B cells that hybridize with A, and thus mouse Ig as described above for pMAS1.
Down regulates expression of kappa protein.

Example 19 This example demonstrates the successful immunization and immune response in transgenic mice of the invention. Immunization of mice Blue mussel hemocyanin (Calbiochem, La Jolla, Cali) linked with more than 400 dinitrophenyl groups per molecule
fornia) (KLH-DNP) previously published method (Practical
Immunology, L. Hudson and FC Hay, Blackwell Sc
alum precipitation according to Orientific Publishing, p. 9, 1980). Alum-precipitated KLH-DNP 400 μg 10
Each mouse was injected ip with 100 μg of dimethyldioctadecyl ammonium bromide in 0 μl of phosphate buffered saline (PBS). Serum samples were collected 6 days later by retro-orbital sinus bleeding. Analysis of human antibody reactivity in serum Indirect enzyme linked immunosorbent assay (ELI
SA) was used to assess antibody reactivity and specificity. Several target antigens were tested to analyze antibody induction by the immunogen. Blue mussel hemocyanin (Calbiochem) to identify reactivity to protein components, bovine serum albumin to identify reactivity to haptens and / or modified amino groups, and KLH-DNP to identify reactivity to whole immunogens did. Human antibody binding to the antigen was detected by enzyme conjugates specific for IgM and IgG subclasses that have no cross-reactivity with mouse immunoglobulins.

Briefly, microtiter plates were antigen coated by drying 5 μg / ml protein in PBS overnight at 37 ° C. Serum samples diluted in PBS, 5% chicken serum, 0.5% Tween-20 were incubated in the wells for 1 hour at room temperature, then anti-human IgG Fc and IgG F (ab ')-horseradish in the same diluent. Incubated with peroxidase or anti-human IgM Fc-horseradish peroxidase. After 1 hour at room temperature, enzyme activity was assessed by addition of ABTS substrate (Sigma, St. Louis, Missouri) and read 30 minutes later at 415-490 nm. Human weight in the immune response in transgenic mice
Chain Involvement Figure 43 represents the response of 3 littermate mice to immunization with KLH-DNP. No. 1296 mouse has unrearranged human IgM and IgG transgenes,
It was homozygous for Ig heavy chain disruption. No. 1299 mice had the transgene on an undisrupted background, and No. 1301 mice did not inherit any of their gene sets. Another littermate, mouse 1297, carries the human transgene and was hemizygous for mouse heavy chain disruption. It was included in the experiment as a non-immunized control.

The results demonstrate that both human IgG and IgM responses were generated against the hapten in the context of conjugation to the protein. Human IgM also developed against KLH, but there was no significant level of human IgG present at this time. In pre-immune serum samples from the same mouse, the titer of human antibody against the same target antigen was not significant.

Example 20 This example demonstrates the successful immunization and immune response with human antigens in the transgenic mice of the invention, and shows that non-random somatic mutations in the variable region sequences of the human transgene Prove what will happen. Human immunoglobulin heavy chain against human glycoprotein antigen
Demonstration of an antibody response comprising the transgenic mice used in this experiment were generated by the introduction of a transgene at the J region resulting in loss of functional endogenous (mouse) heavy chain production (supra). It was homozygous for a functionally disrupted mouse immunoglobulin heavy chain locus. The transgenic mouse also contains at least one completely unrearranged human heavy chain minilocus transgene (HC1, supra), which transgene contains a single functional V _H gene (V
_H 251), the human μ constant region gene, and the human γ1 constant region gene. Transgenic mice that have been shown to express human immunoglobulin transgene products (supra) have been selected for immunization with human antigens, and the transgenic mice have the ability to generate an immune response to human antigen immunization. Proved. HC1-
3 mice of 26 strains and 3 mice of HC1-57 strain (above)
Was injected with human antigen.

100 μg of purified human carcinoembryonic antigen (CEA) insolubilized on alum was injected in incomplete Freund's adjuvant on day 0, and then on additional days 7, 14, 21 and 28. Alum-precipitated CEA in adjuvant was injected weekly. Serum samples were collected by retro-orbital hemorrhage each day prior to CEA injection. Equal volumes of sera from each of the 3 mice in each group were pooled for analysis.

Human μ chain-containing immunoglobulins and human γ chain-containing immunoglobulins bound to human CEA immobilized on microtiter plates were measured by an ELISA assay. The results of the ELISA assay for human μ chain-containing immunoglobulin and human γ chain-containing immunoglobulin are shown in FIGS. 44 and 45, respectively. By day 7, significant human μ chain Ig antibody titers were detected for both strains, and an increase was observed by day 21. For human γ chain Ig, the significant antibody titer was delayed, the former being HC1-57 strain on day 14 and the latter being HC1-
It was revealed on the 21st day in 26 strains. The antibody titer of human γ chain Ig continued to increase with time throughout the experiment. The observed human μ-chain Ig response is characteristic of the pattern observed with affinity maturation, when combined with the plateauing, then rising, rising γ-chain response. twenty one
Analysis of day samples showed a loss of reactivity to the irrelevant antigen, blue mussel hemocyanin (KLH). This points out that the antibody response was specifically directed against CEA.

These data show that animals transgenic for the unrearranged human immunoglobulin loci were (1) able to respond to human antigens (eg human glycoprotein, CEA), and (2) were observed. An isotype switch (class switch) as exemplified by a μ to γ class switch,
And (3) show that they represent the features of affinity maturation in the humoral immune response. In general, these data show that (1) human Ig transgenic mice have the ability to induce heterologous antibodies, and (2) a single transgene heavy chain variable region has the ability to respond to restricted antigens. , (3) response kinetics over time typical of primary and secondary response formation, (4) IgM to IgG class switch of humoral immune response encoded by the transgene, and (5) transgenic It is pointed out that animals have the ability to produce human sequence antibodies against human antigens. Somatic mutations in the human heavy chain transgene minilocus.
The proof HC1 HC1-57 line of transgenic mice containing multiple copies of the transgene were crossed with immunoglobulin Jukusariketsushitsu mice, including destruction both alleles and the endogenous mouse heavy chain comprises HC1 transgene A mouse was obtained (supra). The mice express human mu and gamma 1 heavy chains along with murine kappa and lambda light chains (supra). One of the mice was hyperimmunized against human carcinoembryonic antigen by repeated intraperitoneal injections over a period of 1.5 months. The mice were sacrificed and lymphoid cells were isolated from the spleen, groin and mesenteric lymph nodes, and Peyer's patches. The cells were combined and total RNA was isolated. The RN
First strand cDNA was synthesized from A and the following two oligonucleotide primers: 149 5'-cta gtc cga gtc caa gga gtc tgt gcc gag g
tg cag ctg (g / a / t / c) -3 '151 5'- ggc gct cga gtt cca cga cac cgt cac cgg t
Used as template for PCR amplification with tc-3 '.

The primers specifically amplify the VH251 / γ1 cDNA sequence. The amplified sequence was digested with XhoI and cloned into the vector pNN03. The DNA sequences of the inserts of the 23 random clones are shown in Figures 46-51; sequence mutations from the germline sequences are indicated and the dots indicate that the sequences are the same as germline. Comparison of the germline sequence of the VH251 transgene with the cDNA sequence revealed that three of the clones were completely unmutated and the other 23 contained somatic mutations.

One of the three unmutated sequences is out of frame (ou
t-of-frame) derived from VDJ ligation. Somatic mutations observed at specific locations occur with a frequency and distribution pattern similar to that observed in human lymphocytes [Cai et al. (1992) J. Exp. Med. 176: 1073; Are incorporated herein by reference]. The overall frequency of somatic mutations is about 1%; however, the frequency within CDR1 extends to about 5%, pointing to selections for amino acid changes that affect antigen binding. This demonstrates antigen-directed affinity maturation of human heavy chain sequences.

Example 21 This example demonstrates the successful formation of a transgene by co-introducing two separate polynucleotides that, when recombinant, form the complete human light chain minilocus transgene. . Rearranged by co-injection of two overlapping DNA fragments
Preparation of free light chain minilocus tiger down Sujin 1. unrearranged functional Vκ gene segments vk
65.3, vk65.5, vk65.8 and vk65.15 are isolated Vκ-specific oligonucleotides oligo-65 (5'-
agg ttc agt ggc agtggg tct ggg aca gac ttc act ctc
acc atc agc -3 ') using the phage vector λEMBL3
/ SP6 / T7 (Clonetech Laborat-ories, Inc., Palo Alto,
A human placental genomic DNA library cloned into CA) was probed. V from positive phage clones
The DNA fragment containing the kappa segment was subcloned into a plasmid vector.

Variable gene fragments from the resulting clones were sequenced and clones that appeared to be functional were selected. Criteria for determining functionality include: open reading frame, complete splice acceptor and donor sequences, and complete recombinant sequences. Four functional Vκ gene segments (vk65.3, vk65.5, vk65.8 and vk65.8, from four different plasmid clones isolated by this method and
The DNA sequence of vk65.15) is shown in FIGS. Four plasmid clones p65.3f, p65.5g1, p65.8 and p65.15
f) is explained below. (1 a) The 3 kb XbaI fragment of p65.3f phage clone λ65.3 was constructed such that the Sal I site derived from the vector was proximal to the 3'end of the insert and the BamHI site derived from the vector was 5'of the insert. Subcloned into pUC19 proximal to the'end.
The 3 kb BamHI / Sal I insert of this clone was subcloned into pGP1f to give p65.3f. (1 b) The 6.8 kb EcoRI fragment of the p65.5g1 phage clone λ65.5 was constructed such that the vector-derived Xho I site was proximal to the 5'end of the insert and the vector-derived SalI site was 3 of the insert. It was subcloned into pGP1f so that it was proximal to the'end. The resulting plasmid was named p65.5g1. (1 c) The 6.5 kb HindIII fragment of p65.8 phage clone λ65.8 was added to pSP7.
Cloning into 2 gave p65.8. (1 d) The 10 kb EcoRI fragment of p65.15f phage clone λ65.16 was added to pUC18.
Was subcloned into plasmid p65.15.3. The Vκ gene segment in the plasmid insert was
Mapped to a 4.6 kb EcoRI / HindIII small fragment and
Subcloned into pGP1f. The resulting clone p
65.15f has unique Xho I and Sal I sites located at the 5'and 3'ends of the insert, respectively. 2. pKV4 The Xho I / Sal I insert of p65.8 was cloned into the Xho I site of p65.15f to create plasmid pKV2. p6
A 5.5 g1 Xho I / Sal I insert was cloned into the Xho I site of pKV2 to create pKV3. pKV3 Xho I / Sa
The lI insert was cloned into the XhoI site of p65.3f to create plasmid pKV4. This plasmid contains a single 21 kb Xho containing four functional Vκ gene segments.
Contains the I / SalI insert. Not I can be used to excise the entire insert. 3. pKC1B (3 a) pKcor Two genes derived from human genomic DNA phage λ clone
The XhoI fragment was subcloned into a plasmid vector. The first fragment, 13 kb Jκ2-Jκ5 / Cκ
The containing fragment was treated with Klenow enzyme and cloned into HindIII digested and Klenow treated plasmid pGP1d. The 5'end of the insert is the vector-derived Cla
A plasmid clone (pK-31) close to the I site was selected. Second Xho I fragment, 7.4 kb containing Jκ1
DNA fragment of 3'insertion XhoI site is vector Sal
Xho I / Sal, as destroyed by ligation to the I site
It was cloned into pSP72 digested with I.

The obtained clone p36.2s was cloned with Jκ1 of 4.5.
Cla I site derived from the inserted fragment upstream of kb, and Jκ1 and J
It contains a ClaI site from the polylinker slightly downstream of the naturally occurring Xho I site during κ2. This clone
Digestion with Cla I liberates a 4.7 kb fragment, which is in the correct 5 '→ 3' orientation, pK-3 digested with Cla I.
5 human Jκ segments, human intron enhancer, Cκ, 4.5 kb of 5 '
A plasmid was created containing the flanking sequences. This plasmid pKcor contains unique flanking XhoI and SalI sites 5'and 3'to the insert, respectively. (3 b) pKcorB 4 kb BamHI fragment containing human 3'κ enhancer [Ju
. dde, JG and Max, EE, Mol Cell Biol 12 :. 5206
(1992); which is incorporated herein by reference] was cloned into pGP1f with the 5'end proximal to the Xho I site of the vector. The obtained plasmid p24Bf was digested with Xho I to obtain pKcor 17.7 kb Xho I / S.
The al I fragment was cloned into it in the same orientation as the enhancer fragment. The resulting plasmid, pKcorB, contains unique Xho I at the 5'and 3'ends of the insert, respectively.
And a Sal I site. (3 c) pKC1B The XhoI / SalI insert of pKcorB was cloned into the SalI site of p65.3f to create the light chain minilocus transgene plasmid pKC1B. This plasmid contains 5 Jκ segments, a human intron enhancer, a human Cκ, and a human 3′κ enhancer. this
The entire 25 kb insert can be isolated by NotI digestion. 4. The two NotI inserts from Co4 plasmids pKV4 and pKC1B were mixed in microinjection buffer at a concentration of 2.5 μg / ml each and placed in the pronuclei of half-day mouse embryos as described in the above example. Simultaneous injection. The resulting transgene animal contains a transgene insert (designated Co4, the recombinant product shown in Figure 56) in which the two fragments were integrated together. The 3'kb of the pKV4 insert and the 3'kb of the pKC1B insert are exactly the same. Several integration events indicate that homologous recombination occurred between the two fragments over the 3 kb consensus sequence. The Co4 locus will direct the expression of the repertoire of human sequence light chains in transgenic mice.

The previous description of the preferred embodiments of the invention is as follows:
It is provided for purposes of illustration and explanation. They are not intended to be exclusive, nor is it intended to limit the invention to the precise disclosed form. Many modifications and variations are possible in light of the above teaching. All publications and patent applications in this specification are herein incorporated by reference as if each publication or patent application was explicitly and individually indicated to be incorporated herein by reference. Incorporated into.

Example 22 This example demonstrates the successful production of a mouse hybridoma clone secreting a monoclonal antibody containing a human immunoglobulin chain encoded by the human Ig transgene and which is reactive with a specific immunogen. ing.

Mono Incorporating Human Heavy Chain Transgene Product
Generation of clonal antibody 1. Immunization of mice harboring the human heavy chain transgene Mice containing human heavy chains encoding the transgene homozygous for the knockout (ie, functional disruption) of the endogenous heavy chain gene ( see Example 20 above) were purified. Were immunized with human CEA, followed by splenocyte harvesting after a suitable period of immune response. Using conventional technology (Kohler
And Milstein, Eur. J. Immunol. , 6 : 511-519 (1976);
Harlow and Lane , “ Antibodies: Its Laboratory Manual , Cold.
Spring Harbor, to generate a New York (1988) refer) hybridomas were the murine spleen cells were fused with mouse myeloma cells. The mice used for immunization contain a single functional V _H gene (V _H251 ), human D and J segments, human μ constant region, and human γ1 constant region genes, _unrearranged in humans. It contained the heavy chain minilocus transgene. The transgenic line from which it was derived was designated HC1-57 (supra).

Purified human carcinoembryonic antigen (CEA) insolubilized on alum (Cyrstal Chem, Chicago, IL or
Scripps Labs. San Diego, CA) 100 μg in complete Freund's adjuvant injected at day 0 and then at 7, 14, 14, 21 and 28 days later with alum precipitation in incomplete Freund's adjuvant. Done C
EA was injected once a week. 20 μg of soluble CE
A was administered intravenously on day 83, then on day 92, 50 μg of alum-precipitated CEA in incomplete Freund's adjuvant was infused. A human heavy chain response to CEA was confirmed in serum samples prior to fusion of myeloma cells and splenocytes.

On day 95, animals were euthanized, spleens removed and P3X63-Ag with polyethylene glycol.
8.653 mouse myeloma cells (ATCC CRL 1580, America
n Type Culture Collection, Rockville, MD). Two weeks later, supernatants from fusion wells were screened by ELISA for the presence of antibodies that specifically react with CEA, including human heavy chain μ or γ constant region epitopes. Briefly, purified human CEA was coated onto PVC microtiter plates at 2.5 μg / ml, which was then coated with PBS, 0.5% Tween.
n incubated with culture supernatant diluted 1: 4 or 1: 5 in 5% chicken serum.

The plates were washed, followed by a subsequent goat antiserum conjugated with horseradish peroxidase specific for human IgG Fc or human IgM.
Rabbit antiserum specific to Fc5Mu (Jackson Immuno R
esearch, West Grove, PA) was added. After further washing, the presence of conjugate bound to the captured antibody was confirmed by the addition of ABTS substrate. Two independent fusion wells were found to contain an antibody with substantial avidity for CEA. ELIS after cloning
By A, both hybridomas were found to be positive for the presence of human μ chain and mouse κ chain. No mouse IgG or IgM was detected using a similar assay.

Subcloning of two independent parent hybridomas resulted in two clones designated 92-09A-4F7-A5-2 and 92-09A-1D7-1-7-1. Both strains were deposited at the ATCC Patent Culture Depositary under the Budapest Treaty and received ATCC designations HB11307 and HB11308, respectively. For culture supernatants from these cell lines, ELISA
Specificity was assessed by using A to test for reactivity against several purified target proteins. As shown in Figure 57, an ELISA assay to determine the reactivity of monoclonal antibodies against various antigens was performed using CEA and CEA-related antigen NCA-.
Only 2 was demonstrated to show significant reactivity, which represents a restricted development of reactivity for the variable regions of the heterohybrid immunoglobulin molecules.

Example 23 In this example, the rearranged human VDJ gene encoded by the human Ig minilocus transgene is used to encode a chimeric human / mouse Ig chain by, for example, a transswitch mechanism to produce an endogenous Ig constant region. It proves that it can be transcribed as a copy containing a gene. A trans-switch encoding a chimeric human-mouse heavy chain.
Identification of Zee transcripts RNA was isolated from hyperimmunized HC1 strain 57 transgenic mice homozygous for the endogenous heavy chain J segment deletion (supra). Taylor et al. (1993) Nucleic Acids Res. 2 included for reference in this document .
CDNA was synthesized according to 0 : 6287 and amplified by PCR using the following two primers: o-149 (human V _H251 ): 5′-CTA GCT CGA GTC CAA GGA GTC TGT GCC GAG GTG.
CAG CTG (G, A, T, C) -3'o-249 (mouse gamma): 5'-GGC GCT CGA GCT GGA CAG GG (A / C) TCC A (G / T)
The A GTT CCA -3 'oligonucleotide o-149 is specific for the variable gene segment V _H251 encoded by HC1, while o
-249 hybridizes to both mouse and human gamma sequences in the following specificity order: mouse γ1 = mouse γ2b = mouse γ3> mouse γ2a.
>> The DNA sequences from 10 randomly selected clones generated from the human γ1 PCR product were determined and these are shown in Figure 58. Two clones contained human VDJ and mouse γ1; four clones human VDJ and mouse γ2.
b was included. Also, four clones contained human VDJ and mouse γ3. These results indicate that within a fraction of transgenic B cells, the transgene-encoded human VDJ was recombined into the endogenous mouse heavy chain locus by class switching or similar recombination. It represents.

Example 24 This example describes a method for screening hybridoma pools to distinguish clones encoding chimeric human / mouse Ig chains from clones encoding and expressing human Ig chains. For example, in a pool of hybridoma clones made from transgenic mice homozygous for the endogenous heavy chain locus interrupted at the J region and containing the human Ig heavy chain transgene, transswitched human VDJ-mouse A hybridoma clone encoding a constant region heavy chain can be identified and isolated from a hybridoma clone that expresses human VDJ-human constant region heavy chain. Hybridoma screen for removing chimeric Ig chains
The learning screening process involves two steps that can be performed alone or optionally in combination:
(1) Preliminary ELISA-based screen and (2) Secondary molecular characterization of hybridoma candidates. Preferably, for the initial identification of hybridoma candidates expressing human VDJ region and human constant region, a preliminary EL
A screen based on ISA is used.

Hybridomas showing positive reactivity with antigens (eg, immunogens used to elicit antibody responses in transgenic mice) were screened for mouse μ, γ, κ and λ and human μ, Tested with a panel of monoclonal antibodies that react specifically with γ and κ.
Only hybridomas that are positive for human heavy and light chains and negative for mouse chains are identified as candidate hybridomas that express human immunoglobulin chains. Thus, it can be seen that the hybridoma candidate has reactivity with a particular antigen and has an epitope characteristic of the human constant region.

RNA was isolated from hybridoma candidates,
Used to synthesize the first strand cDNA.
The cDNA of this first strand is then transformed into (single-stranded DN
RNA ligase (ligating A) is used to ligate to a unique single-stranded oligonucleotide of predetermined sequence.
The ligated cDNA is then amplified in two reactions by PCR with two sets of oligonucleotide primers. Set H (heavy chain) contains oligos that specifically anneal to either human μ or human γ1 (depending on the results of the ELISA) and oligos to the oligo-X sequence. In this way, a bias against detection of special V segments, including mouse V segments that may be translocated into the human mini locus, is prevented. The second set of primers, set L (light chain), contains oligos that specifically anneal to human kappa and oligos that specifically anneal to oligo-X. PC
The R product is molecularly cloned and its several DNA sequences determined to ascertain whether the hybridoma is producing the only human antibody based on sequence comparisons against the human and mouse Ig sequences.

Example 25 This example demonstrates the production of transgenic mice harboring the human light chain (κ) minilocus. A fragment containing 13 kb of XhoI Jκ2-Kκ from a human κ minilocus transgenic mouse KC1 (isolated from a human genomic DNA phage library by hybridization to a κ-specific oligonucleotide as described above) phage clone with Klenow enzyme. To produce pK-31.
It was cloned into the Klenow-treated HindIII site of GP1d. This disrupted the insert XhoI site and placed the only polylinker-derived XhoI site at the 5'end next to Jκ2. A unique polylinker-derived ClaI site is located between this XhoI site and the inset sequence, while a unique polylinker-derived SalI site is located at the 3'end of the insert.

7.5 kb Xh containing Jκ1 and upstream sequences
The oI fragment was likewise isolated from a human genomic DNA phage clone (which was isolated from a human genomic DNA phage library by hybridization to a kappa-specific oligonucleotide as described above). This 7.5 kb XhoI fragment was added to pSP
It was cloned into the SalI site of 72 (Promega, Madison, Wisconsin) thus disrupting both XhoI sites and placing the polylinker ClaI site 3 ′ of Jκ1. The resulting clone was digested with ClaI and contained 4.7 kb of upstream sequence and 4.7 containing Jκ1.
A kb fragment was released.

This 4.7 kb fragment was designated as pK-31.
Was cloned into the ClaI site of p. The remaining unique 5'XhoI site is derived from the polylinker sequence. Human VκIII not rearranged
6.5 kb XhoI / SalI DN containing gene segment 65.8 (plasmid p65.8, Example 21)
The A fragment was cloned into the XhoI site of pKcor to generate plasmid pKC1. pKC1
Of the NotI insert were microinjected into mouse fetuses on day 1/2 to generate transgenic mice. Two independent pKC1-induced transgenic lines were established and used to breed mice containing both the heavy and light chain mini loci. These strains KC1-67
3 and KC1-674 each show approximately 1 transgene by Southern blot hybridization.
And estimated to include 10-20 copies of integration. To excise the 2.3 kb insert containing both the KC1e mouse and human heavy chain J-μ intron enhancer, Bam
Plasmid pMHE1 using HI and HindIII
(Examples 13 and 18) were digested. This fragment was treated with Klenow and the SalI linker (New England Bi
olabs, Beverly, Massachusetts) and pKC
It was cloned into the unique 3'SalI site of 1 to generate plasmid pKC1e. pKC1e Not
The I insert was microinjected into mouse fetuses on day 1/2 to generate transgenic mice. Four independent pKC1e-induced transgenic lines were established and used to breed mice containing both heavy and light chain mini loci. These strains KC1e-139
9, KC1e-1403, KC1e-1527 and KC
1e-1536 was obtained by Southern blot hybridization to give transgenes of about 20-50,
It was estimated to include the incorporation of 5-10, 1-5 and 3-5 copies. pKC2 not been rearranged human VκIII gene segments 6
A 6.8 kb XhoI / SalI DNA fragment containing 5.5 (plasmid p65.5gl, Example 21) was cloned into the unique 5'XhoI site of pKC1 to generate plasmid pKC2. This minilocus transgene contains two distinct functional VκIII gene segments. PK in mouse fetus on day 1/2
C2 NotI insert was microinjected to generate transgenic mice. Five independent pKC2-induced transgenic lines were established and used to breed mice containing both heavy and light chain mini loci. These lines KC2-1573, KC2-157
9, KC2-1588, KC2-1608 and KC2-
1610 is about 1-5, 10-5 of transgene by Southern blot hybridization, respectively.
It was estimated to include the integration of 0,1-5,50-100 and 5-20 copies.

Example 26 This example shows that transgenic mice bearing the human kappa transgene are capable of generating an antigen-induced antibody response forming antibodies containing a functional human kappa chain. There is. Transgenic mice containing antibody-responsive HC1-57 human heavy chain and KC1e human kappa transgene linked to human Igκ light chain were immunized with purified human soluble CD4 (human glycoprotein antigen. Polystyrene latex particles (Polysciences, Warrington,
Purified human CD4 insolubilized by conjugation to PA) (NEN Research product, Westwoo
d, MA) 20 μg ip at day 0 in saline with dimethyldioctadecyl ammonium bromide (Cal bio chem, San Diego, CA), then 20 and 34 days later.
Further injections were given on the day.

Retro-orbital bleeds were taken on days 25 and 40 and screened by ELISA for the presence of antibodies against CD4 containing human IgM or human IgG heavy chains. Briefly, 2.5 μg / ml purified human CD4 was coated on a PVC microtiter plate, PBS, 0.5% Tween-20, 5%.
Were incubated with the culture supernatant diluted 1: 4/1: 5 in chicken serum. The plates were washed and then horseradish peroxidase-conjugated goat antiserum or human IgM specific for human IgG Fc
Rabbit antiserum specific for Fc5Mu (Jackson Immuno
Research, Westr Grove, PA).

After additional washing with the addition of ABTS substrate, the presence of conjugate bound to the captured antibody was determined. Human μ Reactive with Antigen in Both Hemorrhages
Was detected, but γ reactivity was basically undetectable.
On day 40, goat anti-human kappa peroxidase conjugate (Si
Samples were also tested for antigen-reactive human kappa chain using the same assay (Gma, St. Louis, MO). CD at this point
A kappa reactivity binding 4 was detected. The results of the assay are shown in Figure 59.

Example 27 This example is homozygous for the functionally disrupted mouse heavy and light chain loci (heavy and kappa chain loci), yet functional human heavy chain and functional human It shows the successful generation of mice that harbor in parallel a human light chain transgene and a human heavy chain transgene that can be productively rearranged to encode a light chain. Such mice are called "0011" mice, where the first two double-digit two
Two zeros represent the lack of functional heavy and light chain loci in this mouse, and the next two-digit one indicates that this mouse is hemizygous for the human heavy chain and human light chain transgenes. It means that. This example shows that such 0011 mice are capable of generating a specific antibody response against a predetermined antigen, and that isotype switching may be involved in such an antibody response. 0011/0012 mice: endogenous Ig knockout +
Human Ig Transgene A functionally disrupted endogenous heavy chain locus lacking a functional J _H region, which also harbors a human HC1 transgene such as the HC1-26 transgenic mouse strain described above (J
Mice that were homozygous for HD ++ or JHΔ ++) were treated with a functionally interrupted endogenous kappa chain locus lacking a functional J _H region (here JKD +).
+ Or JKΔ ++, see FIG. 9) and inbred with mice homozygous for JHD ++ / JKD +.
Mice homozygous for a functionally interrupted heavy and kappa chain locus (heavy chain / kappa chain knockout) containing the HC1 transgene called + were generated.

Such mice were produced by inbreeding and selected based on genotype as assessed by Southern blot of genomic DNA. HC1-26
These mice, referred to as + / JKD ++ / JHD ++ mice, were transformed into mice harboring the human kappa chain transgene (lines KC2-1610, KC1e-1399 and KC).
1e-1527; see Example 25), homozygous for the functionally disrupted heavy and light chain loci and yet with the HC1 transgene and KC2 or KC1.
Southern blot analysis of genomic DNA was used to identify offspring mice that were hemizygous for the e-transgene.

Such mice were designated by number and were identified with respect to their genotype using the following abbreviations: HC1-26 + is hemizygous for human heavy chain minilocus transgene integration of the HC1-26 strain. JHD ++ represents homozygous for J _H knockout; JKD ++ represents homozygous for Jκ knockout; KC2-1610 + is strain KC2-16.
10 represents the hemizygosity for the KC2 human kappa transgene integrated as in 10; KC1e-152
7+ represents the hemizygosity for the KC1e human kappa transgene integrated in the same way as for strain KC1e-1527; KC1e-1399 + represents strain KC1e−.
KC1e human kappa integrated as in 1399
Represents a half-junction for a transgene.

The individual descendants obtained as a result are designated by numbers (for example, 6295, 6907, etc.).
And evaluated for the presence of the J _H knockout allele, the Jκ knockout allele, the HC1-26 transgene and the kappa transgene (KC2 or KC1e), respectively, at each locus being either hemizygous (+) or homozygous (++). ) Was determined to be either. Table 10 shows some number designations, sex and genotypes of offspring mice.

Table 6 ID number Sex Ig code Genotype 6295 M 0011 HC1−26 +; JHD ++; JKD ++; KC2 −1610+ 6907 M 0011 HC1−26 +; JHD ++; JKD ++; KC1e-1527 + 7086 F 0011 HC1−26 +; JHD ++; JKD ++ KC1e-1399 + 7088 F 0011 HC1-26 +; JHD ++; JKD ++; KC1e-1399 + 7397 F 0011 HC1-26 +; JHD ++; JKD ++; KC1e-1527 + 7494 F 0012 HC1-26 +; JHD ++; JKD ++; KC2-1610 ++ 0011 HC1-26 +; JHD ++; JKD ++; KC1e-1399 + 7648 F 0011 HC1-26 +; JHD ++; JKD ++; KC2 -1610+ 7649 F 0012 HC1-26 +; JHD ++; JKD ++; KC2 -1610 ++ 7654 F 0011 HC1-26 +; JHD ++ JKD ++; KC2 -1610 + 7655 F 0011 HC1 -26+; JHD ++; JKD ++; KC2 -1610 + 7839 F 0011 HC1 -26+; JHD ++; JKD ++; KC1e-1399 + 7656 F 0001 HC1 -26-; JHD ++; JKD ++ 610 + 7C2; F 1100 Co1-241-; JHD +; JKD +

We removed spleens from 6 week old female mice. By Southern blot hybridization, mouse # 7655 showed that HC1 (strain 26) and K
C2 (strain 1610) transgene is hemizygous for uptake and JHΔ and JκΔ in mouse μ and κJ regions.
It was determined to be homozygous for the targeted deletion. Mouse # 7656 was cloned into KC2 (strain 1610) by Southern blot hybridization.
It was found to be hemizygous for transgene integration and homozygous for the JHΔ and JκΔ targeted deletions of the mouse μ and κJ regions. Mouse # 7777 was determined by Southern blot hybridization to be hemizygous for the JHΔ and JκΔ targeted deletions of the mouse μ and κJ regions. Due to the recessive nature of these deletions, this mouse should be phenotypically wild type.

Development of endogenous Ig chains in mice
Present (1) wild type mouse (7777), (2) 0001 mouse harboring the human light chain transgene (7656) in a homozygous for heavy chain and kappa knockout alleles and (3) homozygous for heavy chain and kappa knockout alleles To select explanted lymphocytes from 0011 mice that harbor the human light chain transgene and the human heavy chain transgene (7655) at the junction, either human μ, mouse μ, human κ, mouse κ, or mouse λ was selected. FACS analysis with a panel of reactive antibodies was used.

We use Mishell and Shiigi (Mishell,
BB & Shiigi.SM (eds) Selected in Cellular Immunology
The way it was done . Single cell suspensions were prepared from spleens and erythrocytes were lysed with NH ₄ Cl as described by WH Freeman & Co., New York, 1980). Lymphocytes are stained with the following reagents: propidium iodide (molecular probe, Eugene, OR), FITC-conjugated anti-human IgM
(Clone G20-127; Pharmingen, San Diego, CA), FITC-conjugated anti-mouse IgM (clone R6-60.2; Pharmingen, San Diego, CA),
Phycoerythrin-conjugated anti-human Igκ (clone H
P6062; Cal Tag, South San Francisco, C
A), FITC-conjugated anti-mouse Igλ (clone R
26-46; Pharmingen, San Diego, CA), FIT
C-conjugated anti-mouse B220 (clone RA3-6B
2; Pharmingen, San Diego, CA), and Cy-chromoconjugated anti-mouse B220 (clone RA3-6B).
2: Pharmingen, San Diego, CA). We are the FAC
Stained cells were analyzed using a Scan flow cytometer and LYSIS II software (Becton Dickinson, San Jose, CA).

Macrophages and residual erythrocytes are excluded by gating on the forward and side scatter. Dead cells are eliminated by gating out propidium iodide positive cells. The flow cytometry data in FIGS. 60 and 61 confirm Southern blot hybridization data, and mouse # 7655 is human μ
And human kappa, and relatively little, if any, murine mu or murine kappa. Nevertheless, a significant fraction of B cells (about 70-80%)
Hybrid Ig consisting of human heavy chain and mouse lambda light chain
It seems to express the receptor.

FIG. 60 shows the relative distribution of B cells expressing human μ or mouse μ on the cell surface; 0011 mouse (7655) lymphocytes are positive for human μ,
Relatively lacking mouse μ; 0001 mouse (7
656) lymphocytes do not express many human μ or mouse μ; wild type mouse (7777) lymphocytes do not express mouse μ.
But lacks human μ.

Figure 61 shows the relative distribution of B cells expressing human kappa or mouse kappa on the cell surface; 0011 mouse (7655) lymphocytes are positive for human kappa but lack relative mouse kappa. 0001 mouse (7656) lymphocytes do not express many human kappa or mouse kappa; wild-type mouse (7777) lymphocytes express mouse kappa but lack human kappa.

FIG. 62 shows the relative distribution of B cells expressing mouse λ on the cell surface; 0011 mouse (765).
5) Lymphocytes are positive for mouse λ; 0001
Mouse (7656) lymphocytes do not express significant mouse lambda; wild type mouse (7777) lymphocytes do not express mouse lambda.
, But at a relatively low level compared to the 0011 mouse (7655).

FIG. 63 shows the relative distribution of B cells positive for endogenous mouse λ compared to human κ (transgene encoded). The upper left panel shows the results of cells from wild-type mice lacking the human transgene (s) with functional endogenous heavy and light chain alleles; the cells are positive for mouse lambda. The illustration on the upper right shows the κ knockout background (JKD +
+) Cells from mice harboring the integration of the human K transgene of the KC1e-1399 strain (# 5822); the cells are positive for human K or mouse λ in roughly proportional amounts.

The lower left panel shows cells from a mouse (# 7132) harboring the human κ transgene integration of the KC2-1610 strain with a κ knockout background (JKD ++): human κ rather than mouse λ. And more cells were positive, indicating that the KC2-1610 transgene integration was not as efficient as the KC1e-1399 transgene integration. The lower right panel shows cells from mice that harbor the human kappa minilocus transgene (KCo4) and lack the functional endogenous mouse kappa allele.

The data presented in Figure 63 also demonstrate phenotypic variability between transgenes. Such variability can be achieved by individual transgenes and / or transgenics expressing one or more phenotypic characteristics (eg isotype switch, high level expression, low mouse Ig background) resulting from the integrated transgene. It indicates that it is desirable to make a choice for the lineage. Generally, single or multiple transgenes are formed separately to form multiple individual transgenic lines that differ according to (1) the transgene, (2) the site of transgene integration, and / or (3) the genetic background. Gene species (eg pKC1e, p
KC2, KCo4) are used.

Individual transgenic lines are tested for the following desirable parameters: (1) ability to enhance immune response to a predetermined antigen, (2) isotype switch within the transgene-encoded constant region. Frequency and / or endogenous (eg mouse) Ig
Transswitch frequency for constant region genes,
(3) expression level of transgene-encoded immunoglobulin chain and antibody, (4) endogenous (eg, mouse)
Expression levels of immunoglobulins, immunoglobulin sequences, and (5) frequency of productive VDJ and VJ rearrangements. Typically, a transgenic line is selected that produces maximal concentrations of transgene-encoded (eg, human) immunoglobulin chains: Preferably, the selected line is at least about in the serum of the transgenic animal. 40 μg
/ Ml of transgene-encoded heavy chain (eg human μ
Or human gamma) and / or at least about 100 μg / ml of a transgene-encoded light chain (eg, human kappa).

Mice were examined for expression of human and mouse immunoglobulin chains in non-immunized sera of mice and in serum after immunization with a specific antigen, human CD4. Figure 64 shows four separate genotypes.
4 shows the relative expression of human μ, human γ, mouse μ, mouse γ, human κ, mouse κ and mouse λ chains present in the sera of non-immunized 0011 mice (nt = test Human κ is the most abundant light chain, and human μ and mouse γ (putatively the product of transswitch) are the most abundant heavy chains, but there is variability between strains. However, this demonstrates the utility of the selection step to identify advantageous genotype combinations that allow expression of the human chain while minimizing expression of the mouse chain. Mouse # 6907 and 7088 show a human μ to human γ isotype switch (cis switch within the transgene).

FIG. 65 shows human μ (huμ), human γ (huγ), human κ (huκ), mouse μ (msμ), mouse γ (ms) in mice of various 0011 genotypes.
γ), mouse κ (msκ), and mouse λ (msλ) serum immunoglobulin chain levels are shown. Specific antibody response in 0011 mice 0011 mice (# 6295) were immunized with an immunogenic dose of human CD4 according to the following immunization regimen: 0
Day 1, intraperitoneal injection of 100 μl of CD4 mouse immune serum;
On day 1, inject 20 μg of human CD4 (American Bio-Tech) on latex lymphocytes with DDA in 100 μl; on day 15, 20 μg of human CD4 on latex lymphocytes with DDA in 100 μl (American Bio-Tech). Tech); on day 29, 20 μg human CDA (American Bio-Tec on latex lysate with DDA in 100 μl).
h) infusion; on day 43, 20 μg of human CD4 (American B) on latex lymphocytes with DDA in 100 μl.
io-Tech).

FIG. 67 shows human μ in anti-CD4 response,
Figure 3 shows the relative antibody response to CD4 immunization at 3 and 7 weeks demonstrating the presence of human kappa and human gamma chains. Human γ-chain is present at significantly increased abundance in serum at 7 weeks and has a cis-intraheavy chain transgene with a temporal relationship similar to that of the isotype switch in wild-type animals.
This indicates that a switch (isotype switch) has occurred.

Figure 68 shows a schematic assortment of various human heavy and light chain transgenes.

Example 28 This example provides a targeted knockout of the mouse lambda light chain locus. Unlike the targeted inactivated Ig heavy and kappa light chain loci of the mouse lambda light chain locus , the mouse V
The λJλ and Cλ gene segments are not organized into three groups arranged in a 5 ′ → 3 ′ array, but instead are interspersed. The most 5'portion is composed of two V segments (Vλ2 and VλX), and these two segments are followed by 3'directions, each linked to their own J segment (Jλ2Cλ2 and pseudogene Jλ4Cλ4). The two constitutive domain exons that were created follow. Next comes the most widely used V segment (Vλ1), which is followed by a second cluster of constant region exons (Jλ3Cλ3).
And Jλ1Cλ1) follow. Overall, the loci span about 200 kb and the spacing between the two clusters is -20-90 kb.

Expression of the lambda locus was superior to that of Jλ
Rearrangement of Vλ2 or VλX up to 2 and only rarely Jλ
An additional 3'rearrangement for 3 or Jλ1 is involved.
Vλ1 can recombine with both Jλ3 and Jλ1.
Thus, it is possible to mutate the lambda locus in order to sufficiently eliminate recombination and expression of the locus.

The distance between the two lambda gene clusters is through the generation of a single dense targeted deletion, as used in inactivating the mouse Ig heavy and kappa light chain loci. Thus making it difficult to inactivate the expression of the locus. Instead, a small single deletion that would delete the expressed lambda light chain spans approximately 120 kb spanning Jλ2Cλ2 to Jλ1Cλ1 (Figure 69). This removes all of the lambda constant region exons as well as the Vλ1 gene segment, ensuring inactivation of the locus.

A replacement-type targeting vector (Thomas and Capecehi (19
87) In the above article ) is constructed. This marker is embedded within a genomic lambda sequence that flanks the deletion to provide homology to the lambda locus,
Similarly, HSV is added at one end of the homology region to allow enrichment for cells that have homologously integrated the vector.
It may also include the -tk gene. Since it has been reported that the use of isogenic targeting vectors for targeted chromosomal DNA enhances the efficiency of homologous recombination, the lambda locus genomic clone sequence is homologous to the targeted ES strain. Obtained by screening the genotype system 129 / Sv genomic phage library.

[0421] Targeting vectors that differ in length of homology to the lambda locus are constructed. The first vector (Vector 1 in FIG. 70) has a total of about 8-12.
It contains a marker gene flanked by kb lambda locus sequences. For targeting events where the replacement vector mediates the addition or detection of a few kb of DNA, this has been demonstrated to be in the homology range above and beyond (Hasty et al. (1991), supra ; Thomas; et al. (199
2) In the above article ). About 4 more flanking lambda arrays
A vector with 0-60 kb is also constructed (vector 2 in Figure 70). A human Ig mini locus of at least 80 kb was routinely cloned and plasmid vector pG
Propagated in P1 (Taylor et al. (1993) ibid.
Middle ) An alternative approach for inactivation of the lambda locus utilizes two independent mutations within the same ES cell, eg two constant region clusters or two V region loci mutations. .

Since both constant regions are contained within ~ 6 kb of each DNA, one of the V loci spans ~ 19 kb, so the targeting vector is J
λ2Cλ2 / Jλ4Cλ4 and Jλ3Cλ3 / Jλ1C
Constructed to independently delete the λ1 locus. As shown in FIG. 70, each vector contains a selectable marker within the PGK expression cassette surrounded by a total of ~ 8-12 kb of lambda locus genomic DNA flanking each deletion (eg, neo or pa
c). In order to enrich for homologous recombination events by positive-negative selection, H was added to the targeting vector.
It is possible to add the SV-tk gene.

ES cells are sequentially targeted with the two vectors so that clones are generated that support the deletion of one of the constant region genes. These clones are then sequentially targeted with the two vectors to generate clones that support the deletion of one of the constant region loci, after which they are cloned into the remaining functional constant region clusters. Target to create a deletion. Since both targeting events are thus directed to the same cell, it is preferable to use different selectable markers for the two targeting. In the schematic example shown in Figure 70, one of the vectors contains the neo gene and the other contains the pac (puromycin N-acetyltransferase) gene.

A third potentially dominant selectable marker is the hyg (hygromycin phosphotransferase) gene. Both the pac and hyg genes are PGs that have been successfully used to target the neo locus into the Ig heavy and kappa light chain loci.
It can be inserted into a K expression construct. Since the two lambda constant domain clusters are closely linked,
It is important that the two mutations are on the same chromosome.
There is a 50% chance of mutating the same allele, preferably by two independent targeting events, the linkage of mutations being that simultaneous during the breeding (crossing) of the chimera derived from doubly targeted ES cells. Established by separation.

Example 28 This example provides a targeted knockout of the mouse heavy chain locus. Targeted inactivation of the mouse heavy chain locus Gene targeting in ES cells to delete at least one and preferably substantially all of the mouse heavy chain locus constant region genes is shown in FIG. 70. A homologous recombination gene targeting transgene with the defined structure is used. FIG. 70 shows a general overview of the targeting transgene. Segment (a) is a cloned genomic DNA sequence upstream (ie, adjacent to the J _H gene) of the constant region gene (s) to be deleted; segment (b) is pgk-
Contains a positive selectable marker such as neo; segment (c) is cloned downstream of the constant region gene (s) to be deleted (ie, distal to the constant region gene (s) and _JH gene) Genomic DNA
Is a sequence; and segment (d), which is also optional, is
It contains a negative selectable marker gene (eg HSV-tk). Figure 71 is an " immunoglobulin gene ", Honjo T. A.
lt, FW and Rabbits TH (eds) Academic Press, NY (198
9) Shows a map of the mouse heavy chain locus taken from p129.

The targeting transgene is shown in FIG.
And the (1) (a) segment is the 11.5 kb insert of clone JH8.1 (Ch
en etal. (1993) Int. Immunol. 5 : 647) or an equivalent portion containing a sequence of at least about 1 to 4 kb upstream of the mouse Cμ gene, and (2) (b) segment is as described above. pgk-neo, and the segment (3) (c) is 5'-gtg ttg cgt gta tca gct gaa acc ta.
A 4-6 kb insert isolated from a phage clone of the mouse Ca gene isolated by screening a mouse genomic clone library with an end-labeled oligonucleotide having the sequence g aaa cag ggt gac cag -3 'or 167 shown in 72
It contains a 4 bp sequence and segment (4) (d) contains the HSV-tk expression cassette described above.

Alternatively, the first targeting is a homology region or constant region gene (s), a first selectable positive marker gene (pgk-neo) and HSV-t.
k Stepwise deletion of one or more heavy chain constant region genes containing segments (a) and (c) homologous to sequences flanking the negative selection marker is performed. Thus, the (a) segment may comprise at least about 1-4 kb of sequence homologous to the region upstream of Cγ3,
The (c) segment may include a sequence of at least about 1 to 4 kb homologous to the region upstream of Cγ2a.

This targeting transgene is Cγ
3, Cγ1, Cγ2b and Cγ2a genes are deleted. This first targeting transgene is introduced into ES cells and the properly targeted recombinants are selected (eg at G418) to produce a properly targeted C region deletion. Negative selection for loss of the HSV-tk cassette is then made (eg g
anciclovir or FIAU). The resulting properly targeted C-deleted recombinant of FIG. 1 has a heavy chain locus lacking the Cγ3, Cγ1, Cγ2b and Cγ2a genes.

The second targeting transgene is
Homology region, ie, constant region gene (s), positive selection marker gene of the second species different from the first species (eg gp
t or pae) and HSV-tk containing segments (a) and (c) homologous to sequences flanking the negative selectable marker. Thus, the (a) segment is
At least about 1 to 4 homologous to the region upstream of Cε
kb sequence can be included, and (c) segment is Cα
Can contain at least about 1-4 kb of sequence homologous to the region upstream. This targeting transgene deletes the Cε and Cα genes.

This second targeting transgene is introduced into the properly targeted C-region recombinant ES cells obtained from the first targeting event. Cells that are properly targeted for the second knockout event (ie, by homologous recombination at the second targeting transgene) are selected by a selective agent specific for the second positive selectable marker gene (eg,
mycophenolic acid for selection on gpt; pa
Puromycin for selecting for c). Negative selection for loss of the HSV-tk cassette is then made (eg, ganciclov).
with ir or FIAU). These resulting properly targeted second round C region recombinants have a heavy chain locus lacking the Cγ3, Cγ1, Cγ2b, Cγ2a, Cε and Cα genes.

Properly targeted first or second round recombinant ES cells lacking one or more C region genes were used for blastocyst injection as described above and chimeric mice were generated. Produce. Germline transmission of the targeted heavy chain allele is established and the resulting founder mice are bred (mated) to generate mice homozygous for the C region knockout. Such C-region knockout mice have several advantages over J _H knockout mice: In one example, C-region knockout mice contain between the human heavy chain transgene and the endogenous heavy chain locus constant region. Has a reduced ability (or is completely absent) of undergoing a transswitch of C. pneumoniae, thus reducing the frequency of chimeric human / mouse heavy chains in transgenic mice.
Although mu and delta are also frequently deleted by homologous targeting, knockout of the mouse gamma gene is preferred.

C-region knockouts can be performed in conjunction with other targeted lesions within the endogenous mouse heavy chain locus: to eliminate productive VDJ rearrangements of the mouse heavy chain locus, among others. And it is possible to combine the J _H knockout with the C region deletion to eliminate or reduce the transswitch between the human heavy chain transgene and the mouse heavy chain locus. For some embodiments, it is possible to produce mice that are specifically deficient in one or more C region genes of the endogenous heavy chain locus but carry some other C region genes. May be desirable. For example, if the chimeric human / mouse IgA confers an advantageous phenotype and does not substantially interfere with the desired utility of the mouse, it may be preferable to carry the mouse Cα gene to allow transswitch production of this IgA. There is.

Example 29 This example demonstrates ex vivo depletion of lymphocytes expressing endogenous (mouse) immunoglobulin from lymphocyte samples obtained from transgenic mice harboring the human transgene. Lymphocytes expressing mouse Ig are selectively depleted by specific binding to anti-mouse immunoglobulin antibodies lacking substantial avidity for human immunoglobulins encoded by the transgene (s). Ex vivo Depletion of Mouse Ig-Expressing B Cells Mice homozygous for the human heavy chain minilocus transgene (HC2) and human light chain minilocus transgene (KCo4) were C57BL / 6 (B6) syngeneic mice. 2211 mice (ie, homozygous for the functional endogenous mouse heavy chain locus, homozygous for the functional endogenous mouse light chain locus, and one of the human heavy chain transgenes). Mice with one copy and one copy of the human light chain transgene. Such 2211 mice also express B6 major and minor major histocompatibility antigens. These mice are primed with an immunogenic dose of antigen and splenocytes are separated after about 1 week.

Standard methods (Wyso cki et al. (1978). Pro
c.Natl.Acad.Sci. (USA) 75; 2844; MACS magnetic cell sorting, solid-phase linked antibody-dependent cell separation according to MACS magnetic cell sorting, Miltenyi Biotec Inc, Sunnyvale, CA), followed by:
Substantially cell lysis of an antibody-dependent complement-mediated to remove residual cells positive relates mouse Ig (Contact the "cellular immunology
Selected Ways to Kick ", Mishell BB & Shiigi SM (ed
s), WHFreeman and Company, New York, 1980, p211
~ 212) remove B cells positive for mouse Ig. The remaining cells in the depleted sample (eg, B cells positive for human Ig, T cells), preferably in combination with additional anti-mouse Ig antibody to the developing B cells, SCID / B6 or RAG / B6. Mice are injected intravenously. The reconstituted mouse is then further immunized with an antigen to obtain antibodies and affinity matured cells for producing hybridoma clones.

[0435] The method for fully produce human antibodies in the production somatic chimeric mice of fully human antibodies in Example 30 Somatic chimeras are described. These mice have human immunoglobulin (Ig) heavy and light chain transgenes,
Embryonic stem (ES) cells lacking functional mouse Ig heavy and kappa light chain genes are generated by introducing them into blastocysts from RAG-1 or RAG-2 deficient mice.

RAG-1 and RAG-2 deficient mice (Mo
mbaerts et al. (1992) Cell 68 : 869; Shinkai et a
l. (1992) Cell 68 : 855) lacks mouse B and T cells due to the inability to initiate the VDJ rearrangement and assemble the gene segment encoding Igs and the T cell receptor (TCR). This defect in the production of B and T cells can be complemented by injection of wild-type ES cells into blastocysts derived from RAG-2 deficient animals.
The resulting chimeric mice produce mature B and T cells that are fully derived from the injected ES cells (Chen et al. (1993) Proc. Natl. Acad. Sci. USA 90 :
4528).

In all of the chimeric B and / or T cells:
Genetic engineering of injected ES cells is used to introduce defined mutations and / or exogenous DNA constructs. Chen et al. (1993), Proc.Natl.Acad.Sci. USA 9
0: 4528-4532) Produced chimera producing T cells in the absence of B cells when injected into RAG blastocysts and produced ES cells with homozygous inactivation of the Ig heavy chain locus (Original unclear). Transfection of the rearranged mouse heavy chain into mutant ES cells results in the rescue of B cell development and regeneration of both B and T cells within the chimera.

It is possible to generate chimeric mice that express fully human antibodies in the absence of mouse Ig heavy chain or kappa light chain synthesis. The human Ig heavy and light chain constructs are introduced into ES cells that are homozygous for inactivation of both the mouse Ig heavy and kappa light chain genes. At this time, ES cells are injected into blastocysts derived from RAG2-deficient mice. The resulting chimera does not express the mouse Ig heavy chain and kappa light chain genes but expresses the human Ig gene infused ES
It contains B cells exclusively derived from the cells. Inactivation of immunoglobulin heavy chain and kappa light chain genes
Generation of homozygous ES cells was performed according to known procedures (Chen et al. (1993) EMBO ).
J. 12: 821; and Chen et al. (1993) Int. Immunol.
Op.cit), by targeted deletion of IgJ _H and the J k / C kappa sequences in ES cells, they deactivated the Ig
Mice bearing heavy and kappa light chain loci were generated. The two mutant strains of mice were bred together to generate homozygous strains for inactivation of both Ig loci. This double mutant system was used for the induction of ES cells. The protocol used was basically that described by Robertson (198).
7. In “Teratocarcinoma and embryonic stem cells; one practical approach”.

P71-112. Edited by EJ Robertson, IRL Press). Briefly, spontaneous mating of synzygous double mutant mice generated blastocysts. Conceptual females were ovariectomized at 2.5 days gestation and "delayed" blastocysts were washed out of the uterus at 7 days gestation and cultured on feeder cells to help maintain their undifferentiated state. . Stem cells from the inner cell mass of blastocysts identifiable by their morphology were removed, dissociated and passaged on feeder cells. Cells with normal karyotype will be identified and the male cell line will be tested for their ability to generate chimeras and contribute to the germ cells of the mouse. Male ES cells are preferred over female lines because male chimeras can produce much more progeny. Human into ES cells lacking mouse Ig heavy chain and kappa light chain
Introduction of Ig genes Human immunoglobulin heavy and light chain genes are introduced into mutant ES cells either as minilocus constructs such as HCS and KC-CO4 or as YAC clones such as J1.3P. Transfection of ES cells with human Ig DNA is performed by techniques such as electroporation or lipofection with cationic lipids.
A selectable marker is linked to the construct or ES to allow selection of ES cells that have taken up human DNA.
Co-transfect cells with the construct. Since the mutant ES cells contain the neomycin phosphotransferase (neo) gene as a result of the gene targeting event that generated Ig gene inactivation, the hygromycin phosphatase was introduced to introduce the human Ig gene into the ES cells. Phosphotransferase (hyg)
Alternatively, various selectable markers are used, such as puromycin N-acetyltransferase (pac).

Human Ig heavy and light chain genes were selected for different selections.
Simultaneously in mutant ES cells using selectable markers
Or sequentially. Transfection
After selection, select cells with the appropriate selectable marker,
Frozen to confirm and analyze integration of human gene sequences
Drug resistant colonies are expanded for NA analysis. Chimera generation As described (Bradley, A. (1987),
And embryonic stem cells: one practical approach ”, p113
~ 151, edited by E.J. Robertson, IRL Press) RAG-2 embryo
ES clones containing human Ig heavy and light chain genes in blastocysts
Infused and transferred into the uterus of a pseudopregnant female.
Presence of human antibodies by ELISA assay of serum samples
Screen offspring for. Human monoclona
Positive animals are used for immunization and production of antibody.

Example 31 This example demonstrates the homologous recombination in ES cells of a targeted frameshift mutation into the mouse heavy chain locus leading to the deletion of B cells undergoing switch recombination. Describe the introduction of. Frameshifted mice are a suitable host for hosting non-mouse (eg, human) transgenes encoding human sequence immunoglobulins.

New frameshifted mice were introduced, among others, to express non-mouse (eg, human) sequence immunoglobulins encoded by heavy chain transgene (s) and / or light chain transgenes. Class-switched affinities from the transgene can be used for isolation of hybridomas expressing mature human sequence antibodies. A frameshift is introduced into one of the four mouse JH gene segments and into the first exon of the mouse μ gene. The two frameshift mutations introduced compensate each other and thus the expression of fully functional murine mu heavy chains when B cells use frameshifted JH for functional VDJ ligation. enable. None of the other three JH segments can be used for functional VDJ joining because of uncompensated frameshifting in the remaining JH genes. Alternatively, compensating frameshifts can be engineered into multiple mouse JH genes.

Mice homozygous for the compensated and frameshifted immunoglobulin heavy chain allele show near-physiological levels of peripheral B cells and near-physiological levels of serum IgM, including both mouse and human mu. Have. However, B cells recruited into germinal centers frequently undergo class switching to non-μ isotypes. Such a class switch in B cells expressing the endogenous murine mu chain leads to the expression of uncompensated frameshifted mRNA, as the rest of the non-μC _H genes do not have a compensated frameshift. The resulting B cells do not express the B cell receptor and are deleted. Therefore,
B cells expressing mouse heavy chains are deleted once they have reached the stage of differentiation where isotype switching occurs. However, it does not include isotype limited frameshifting, which has the power of an isotype switch,
B cells expressing heavy chains encoded by non-mouse (eg, human) transgenes have the potential for further development, including isotype switching and / or affinity maturation.

Thus, mice undergoing frameshift have an impaired secondary response with respect to the mouse heavy chain (μ), but a significant secondary response with the transgene-encoded heavy chain. When a heavy chain transgene capable of undergoing a class switch is introduced within this mutant background, the non-IgM secondary response is dominated by the B cell expressing transgene. It is thus possible to isolate affinity matured human sequence immunoglobulin expressing hybridomas from these frameshifted mice. Furthermore, frameshifted mice generally have immunoprotective levels of mouse IgM that may be advantageous if the human heavy chain transgene can only encode a limited range of variable region capacities.

To make hybridomas that secrete human sequence monoclonal antibodies, transgenic mutant mice were immunized and their spleens were fused with a myeloma cell line and the resulting hybridomas were encoded by the transgene. To screen for expression of human non-μ isotypes. In addition, frameshifted mice will contain a functional μ-switch sequence adjacent to the transcribed VDJ that serves as an active substrate for the cis switch (Gu et al, (1993) Cell 73 ; 1155),
Thus, it may be advantageous over JH-deficient mice because it reduces transswitched B cell levels expressing chimeric human / mouse antibodies. Construction of frameshift vector Two separate frameshift vectors are constructed. One of these vectors was used to introduce two nucleotides at the 3'end of the mouse J4 gene segment, the other vector was derived from the 5'end of exon 1 of the mouse mu gene from these same nucleotides. Used to delete two nucleotides. 1. JH vector A 3.4 kb Xh covering mouse heavy chain J region and μ intron enhancer in a plasmid vector containing herpes thymidine kinase gene and neomycin resistance gene under the control of phosphoglycerate kinase promoter.
Subcloning the oI / EcoRI fragment (tk / neo cassette, Hasty et al., (1991) Natu
re 350 : 243). This clone is then used as a substrate to generate two different PCR fragments using the following oligonucleotide primers: o-A1 5'-cca cac tct gca tgc tgc aga agc tt.
t tct gta -3 'o-A2 5'-ggt gac tga ggt acc ttg acc cca gt
a gtc cag -3 'o-A3 5'-ggt tac ctc agt cac cgt ctc ctc ag
a ggt aag aat ggc ctc-3'o-A45'-agg ctc cac cag acc tct cta gac ag
Oligonucleotides o-A1 and o-A2 are used to amplify a 1.2 kb fragment digested with caac tac-3 'SphI and KpnI. Oligonucleotides o-A3 and o-A4 are used to amplify a 0.6 kb fragment digested with KpnI and XbaI. These two digested fragments are then combined with SphI / XbaI.
Clone into plasmid A digested with to generate plasmid B.

Plasmid B contains a 2 nucleotide insertion at the end of J4 and a new KpnI site upstream of the insertion. KpnI as a diagnostic marker for insertion
Use parts. To increase the efficiency of homologous recombination, additional flanking sequences can be cloned into the 5'XhoI and 3'EcoRI sites of the plasmid. The resulting plasmid was then digested with SphI or another restriction enzyme with a single site within the insert and electroporated into embryonic stem cells, which were then subjected to Hasty et al (1991). ), And select in G418 as described in the above . Homologous recombinants were identified by Southern blot hybridization and then selected by FIAU as described by Hasty et al, with 2 base pair insertions and JH.
A deleted subclone containing only the new KpnI site within 4 is obtained. These were identified by Southern blot hybridization of KpnI digested DNA,
Confirmed by DNA sequence analysis of PCR amplified JH4 DNA.

The resulting mice have the unmutated sequence: ie TGGGGTCAAGG A ACCTCAGTCACCGTCTCCTCAG. gtaagaatggcctctcc ... TrpGlyGlnGlyThrSerValThrValSerSerGlu is a mutant sequence, that is, ... TGGGGTCAAGG T ACCTCAGTCACCGTCTCCTCAG AG gtaagaatggcctctcc ... A vector containing a two base pair deletion at the 5'end of the first mu exon using in vitro mutagenesis similar to that described above to engineer a two base pair insertion into the mu exon 1 vector JH4 gene segment. PCR products and genomic subclones are assembled to produce In order to further mark the mutation, a new XmnI was created by changing A to G.
The site is also introduced downstream.

The sequence of the mu gene that has not been mutated is: ctggtcctcag AG AGTCAGTCCTTCCC A AATGTCTTCCCCCTCGTC ... GluSerGlnSerPheProAsnValPheProLeuVal The sequence of the mu gene that has been mutated is: XmnI ... ctggtcctcag The homologous recombination vector containing the AGTCAGTCCTTCCC G AATGTCTTCCCCCTCGTC ... SerGlnSerPheProAsnValPheProLeuVal mutant sequence is linearized and electroporated into an ES cell line containing the JH4 insert.
Homologous recombinants are identified from neomycin resistant clones. These homologous recombinants containing a frameshift insertion on the same chromosome as the JH insertion are identified by Southern blot hybridization of KpnI / BamHI digested DNA. Mutant 9 from wild type 11.3kb
Link the JH4 insertion with a new KpnI site that reduces the size of the J-μ intron containing KpnI / BamHI up to kb. Then, using FIAU, the inserted t
The resulting clones are selected for deletion of the k / neo cassette. Clones containing the mutant μ exon are identified by Southern blot hybridization of DNA digested with XmnI. Mutations are confirmed by DNA sequence analysis of PCR amplified mu exon 1 DNA. Generation of frame-shifted mice Next, 2 base pair insertions in JH4 and 2 in mu exon 1
An ES cell line containing both base pair deletions is introduced into a blastocyst stage embryo that is inserted into the body of a pseudopregnant female to generate a chimera. The chimeric animal is bred to obtain germline transmission and the resulting animal is bred to homozygous to express the mouse heavy chain homozygous for the compensated frameshifted heavy chain locus. Mutant animals with impaired secondary humoral immune responses in B cells are obtained.

A frame I of the mouse heavy chain transgene, eg pHC1 or pHC2, was frameshifted.
Mice harboring such human transgenes in mice carrying the gH locus can be bred to the mouse heavy chain frameshift background by cross breeding. Via inbreeding and backcrossing, homozygous at the murine IgH locus to the mu-compensated and frameshifted murine IgH allele (ie compensated in-frame expression of mu-chain only but not non-mu-chain in mice) And harboring at least one integrated copy of a functional human heavy chain transgene (eg pHC1 or pHC2). Such mice may optionally include a knockout of the endogenous mouse kappa and / or lambda genes as described above, and optionally a human or other non-mouse light chain transgene (eg, pK
C1e, pKC2, etc.).

Alternatively, the human transgene (s) (heavy chain and heavy chain and heavy chain (s) are arranged so that the frameshift compensated by the frameshift within the transgene constant region gene (s) is contained in the transgene J gene (s). (/ Or light chain) may include a compensatory frame shaft, a transswitch for an endogenous constant region gene is uncompensated and produces truncated or nonsense production; such uncompensated transswitch B-cells expressing different immunoglobulins are selected and depleted.

Example 32 Inactivation of Endogenous Heavy Chain by D Region Excision This example shows a positive-negative for replacing mouse germline immunoglobulin heavy chain D region with a non-functional rearranged VDJ segment. The selective homologous recombination vector will be described. The resulting allele functions in B cells as a normal non-productive allele, with the allele heavy chain class switch thus undergoing reduction of transswitch levels to the active transgene locus. D region targeting construct The 8-15 kb DNA fragment upstream of the mouse D region is DFL16.1 listed in Gen Bank.
An oligonucleotide probe containing approximately 50 contiguous nucleotides of the published sequence for the segment is used to isolate and subclone from a mouse-based 129 phage library. DFL16.1 is the upstream D segment (ie, proximal to the V region gene cluster and distal to the constant region gene cluster).

Similarly, a 9.5 kb BamHI fragment containing the first two coated exons of the JH3, JH4, Eμ, Sμ and μ constant regions was transformed into mouse strain 129.
Isolate from genomic phage library and subclon. Next, mouse hybridomas (all types)
5 to 10 kb of the rearranged VDJ is isolated and a synthetic linker containing a stop codon is inserted within the J segment.
The internal linker of J is preferred over the out-of-frame VDJ ligation because V substitution rearrangements are possible.

PGKneo these three fragments
Assembled with the positive selection cassette and the PGKHSVtk negative selection cassette to form a positive-negative selection vector for deleting the mouse D region in 129-induced ES cells (eg AB1) by homologous recombination. The targeting vector is itself a relocated 5-10 kb rearranged V ligated to a 9.5 kb BamHI fragment.
It is formed by ligating the 8-15 kb DNA fragment to a positive selection cassette (eg PGKneo) which is itself ligated to DJ. The negative selection cassette (eg PGKHSVtk) is then ligated at either end of the targeting construct. The construction of such a D region targeting vector is shown schematically in FIG.

The D region targeting construct was labeled AB1.
Transfer into ES cells and perform positive and negative selection as described above to clone properly targeted ES cells. Properly targeted ES cell clones are used for blastocyst injection to generate chimeric mice. Chimeric mice are bred to produce founder mice that harbor heavy chain alleles inactivated by the D region. Inbred offspring are produced to produce homozygotes lacking a functional endogenous heavy chain locus.

Using such homozygotes, human Ig
Transgene (eg pHC1, pHC2, pKC
2, pKC1e, KCo4) and cross-breeding into mice harboring (by backcrossing to homozygotes lacking a functional D region) and lacking a functional endogenous heavy chain locus and human weight ( Generate mice bearing the (chain) transgene (and preferably also the human light chain transgene).

In embodiments in which some functional endogenous light chain loci (eg, the lambda locus) remain, in general,
The transgene contains transcriptional control sequences that direct high level expression of human light chain (eg, κ) polypeptides, thus allowing the transgene locus to effectively compete with the remaining endogenous light chain (eg, λ) locus. Is preferred. For example, the Co4 kappa light chain transgene is generally favored over pKC1 for its ability to effectively compete with the endogenous λ locus in transgenic animals.

Example 33 This example describes expansion of the human light chain transgene V gene potency range by co-injection of a human kappa light chain mini-locus with a yeast artificial chromosome containing a portion of the human V kappa locus. For co-injection of Vκ-containing YAC DNA and κ mini-locus
Introduction of functional human light chain V segment by unamplified YAC DN from clone 4x17E1 of the commonly available ICRF YAC library.
As A, a YAC clone of about 450 kb containing a part of the human Vκ locus was obtained (Larin et al. (1991) Proc . Nat) .
l.Acad.Sci (USA) 88 : 4123; Imperial Cancer Rese
arch Fund, Genome Research Institute, London, UK). 450kb YA without standard amplification by standard Parffield gel electrophoresis according to manufacturer's specifications
C clones were separated (CHEFDR-II, electrophoresis cell, Bio-Rad Laboratories, Richmond, CA). Six individual pulsed field gels were stained with ethidium bromide and the gel material containing the YAC clone DNA was excised from the gel and then poured into a new triangular gel tray (low melting point in standard gel buffer). Embed this in an agarose gel. Standard electrophoresis buffer plus 2M NaO
Using a narrow agarose gel with Ac at the apex,
The resulting triangular gel (6 excised Y
(Including an AC-containing gel block) was extended. The gel was then placed in an electrophoresis chamber immersed in standard gel buffer.

The Y-shaped gel former has a current (flow?)
Rise above the surface of the buffer so that the flow is limited to the narrow and high salt gel area. NaOA in buffer
to prevent diffusion of c throughout the high salt gel section,
I placed an acrylic block. The YAC DNA was then electrophoresed from the original excised (embedded) excised gel into a narrow high salt gel section. At the transition from the low-salt gel to the high-salt gel, there is a decrease in resistance that effectively stops the migration of DNA at the apex of the triangular gel.

After electrophoresis and staining with ethidium bromide, the concentrated YAC DNA was excised from the rest of the gel and the agarose was separated by GELase (Epi Center Technolog).
ies, Madison, Wisconsin). Next, cesium chloride is added to the resulting YAC-containing liquid,
A density of 1.68 g / ml was obtained. This solution for 36 hours 37
Centrifuge at 000 rpm to remove all contaminants from YAC
The DNA was isolated. The resulting 0.5 ml fraction of the density gradient was separated and separated with 5 mM Tris (pH 7.4),
Peak D for 5mM NaCl, 0.1M EDTA
The NA fraction was dialyzed.

After dialysis, the resulting YAC D
Concentration of a 0.65 ml solution of NA was found to contain 2 μg / ml DNA. This YAC DNA is 2
0: 1: 1 (microgram YAC4 × 17E1: KC
1B: KV4) in the ratio of plasmids pKC1B and p
Mixed with purified DNA insert from KV4.
The resulting 2 μg / ml solution was injected into the pronuclei of half-day B6CBF2 embryos and 95 surviving microinjected embryos were transferred into the oviducts of pseudopregnant females. Twelve mice developed from microinjected embryos were born.

Example 34 This example demonstrates a class switch, immunization in an immunized transgenic mouse homozygous for an inactivated endogenous immunoglobulin locus and containing an HC1 or HC2 heavy chain transgene. Describe cell mutations and B cell development.

To demonstrate that the human sequence germline-located minilocus can replace the authentic locus, we used a system containing the human germline-located IgH transgene to lack endogenous IgH. Mouse strains were bred. The two transgene miniloci HC1 and HC2 have 1 and 4 functional variable (V) segments, 10 and 16 diversity (D) segments, all 6 joining (JH) segments, and both μ, respectively.
And a γ1 constant region segment. The mini locus is
Closely linked to the coding segment --JH-
Includes human cis-acting regulatory sequences such as the μ intron enhancer and μ and γ1 switch sequences.

They also contain an additional enhancer element derived from the 3'end of the rat IgH locus. We used the HCI and HC2 transgenic mice as described ( supra ) JH segment (J
HD mice) and are therefore bred with stem cell-derived mutant mice that are unable to undergo functional heavy chain rearrangement. The resulting transgenic-JHD mice contain B cells that are dependent on the introduced heavy chain sequences. Immunizations and hybridomas We immunized mice by intraperitoneal injection of 50-100 μg of antigen. Human carcinoembryonic antigen (CEA; Crystal Chem, Chicago, IL), chicken egg white lysozyme (HEL; Pierce, Rockford, IL) and keyhole limpet hemocyanin (KLH; Pierce, Ro) were used as antigens.
ckford, IL) was included. For the first injection, we mixed the antigen with complete Freund's adjuvant and for subsequent injections with incomplete Freund's adjuvant (Gibc
BRL, Gaithersburg, MD) was used. The splenocytes were treated with non-secretory mouse myeloma P3X63-Ag8.653 (ATCC,
CRL1580).

We assayed serum samples and hybridoma supernatants by ELISA for the presence of specific and non-specific antibodies containing human heavy chain sequences. For detection of non-specific antibodies, we used a human heavy chain isotype-specific antibody (mouse MAbα human IgG1, clone HP606.
9, Cal bio chem, La Jolla, CA; mouse MAbα human IgM, clone CH6, The Binding Site, Birmingh
am, UK) coating microtiter wells,
Peroxidase-conjugated antiserum (horseradish peroxidase-conjugated affinity purified fab fragment from polyclonal goat α human IgG (fc), c
at # 109-036-098; affinity-purified horseradish peroxidase-conjugated polyclonal rabbit alpha human IgM (fc), cat # 309.
-035-095.

Jackson Immuno Research, West Grove, P
A) was used to develop. For detection of antigen-specific antibodies, we coated microtiter wells with antigen and developed with peroxidase-conjugated human heavy chain isotype-specific sera. We use hydrogen peroxide and 2,2'-azino-bis- (3-ethylbenzthiazoline-b-sulfonic acid, Sigma Chem. Co., St. Louis,
Bound peroxidase was detected by incubation with Mo). The reaction product is measured by absorption at 415 nm and corrected for absorption at 490 nm. Flow Cytometry We prepared single cell suspensions from spleen, bone marrow and peritoneal cavity and lysed red blood cells with NH ₄ Cl as described by Mishell and Shiigi. Lymphocytes were stained with the following reagents: phycoerythrin-conjugated anti-mouse Igκ (clone X36; Becton Dickinson, San.
Joss. CA), FITC-conjugated anti-mouse IgD (clone SBA1, Southern Biotech, AL), FITC-conjugated anti-mouse CD5 (clone 53-7.3; Becton).
Dickinson, San Jose. CA), FITC-conjugated anti-mouse Igλ (clone R26-46); Pharmingen, Sa
n Diego, CA) and Cy-chromoconjugated anti-mouse B
220 (clone RA3-6B2; Pharmingen, San Di
ego, CA), we have a FACScan clone hemocytometer and LYSIS II software (Becton Dickinson, Sa
n Jose. CA) were used to analyze the stained cells. Gating on the forward and side scatterers eliminates most macrophages, neutrophils and residual red blood cells. B Cell Compartment Rescue HC1 Transgenic-In the abdominal cavity of JHD animals,
Approximately one-quarter of the normal levels of CD ⁵⁺ B cells and conventional CD ^{5 −} B cells are found. Transgenic peritoneal CD5 ⁺ B cells are similar to the described so-called B-1 cells in normal animals: they are larger than conventional B and T lymphocytes and are traditionally found in the spleen. Express lower levels of B220 than other B cells,
It contains a higher proportion of cells expressing lambda light chain. Over 90% of splenic B cells express κ, while up to 50% of peritoneal B cells
The cells express λ. Thus, while conventional B cell levels are evenly reduced in all tissues, B- reported to be much more potent in self-renewal.
Level 1 appears to be normal in HC1 transgenic-JHD animals. In class-switch transgenic-JHD mice, repetitive exposure to antigen results in the production of human γ1 as well as μ antibodies. We performed human CE in transgenic-JHD mice at weekly intervals.
A for 4 weeks with antigen-specific IgM and Ig
Serum levels of G1 were monitored (Figure 74). In the first week,
There is a detectable IgM response but no IgG1 response. However, the IgG1 response is greater than the IgM response after 2 weeks and continues to increase while the IgM response remains relatively homeostatic. This pattern, an early IgM response followed by an IgG response, is typical of a secondary immune response, in which the cis-acting sequences contained within the transgene lead to a class switch cytokine. Suggesting that they may be responding to. We considered three possible mechanisms for the expression of non-μ isotypes, each of which has been discussed in the literature. These mechanisms are: alternate splicing that does not involve the deletion of the μ gene; “δ-type” switches that involve the deletion of the μ gene via homologous recombination between flanking repeats; and the switch region. Is non-homologous recombination between. The results of our experiments, described below, represent a switch region recombination model.

Two types of non-deletion alternating splicing mechanisms can be invoked to explain the isotype shift. First, it is possible to express a single transcript from the transgene that covers both μ and γ1; this transcript can be alternatively spliced in response to cytokines elicited by exposure to antigen. It was Alternatively, a sterile transcript starting upstream of the cytokine-induced γ1 could be trans-spliced to the μ transcript. If any of these mechanisms were responsible for the expression of the human γ1 sequence, one would expect to be able to isolate hybridomas expressing both μ and γ1. However, although hundreds of hybridomas expressing either human μ or human γ1 have been screened, no such dual producer (μ ⁺ , γ1 ⁺ ) hybridomas were found. This means that the expression of γ1 is accompanied by the deletion of the μ gene.

The deletion of the μ gene is due to non-homologous recombination between the μ and γ1 switch regions, or two flanking 400 bp direct repeats (σμ and Σμ) contained within the HC1 and HC2 transgenes. Can be mediated by homologous recombination between. Deletion recombination between σμ and Σμ has been reported to be responsible for the IgD ⁺ , IgM ⁻ phenotype of some human B cells. The first mechanism, heterologous switch recombination, should produce a switch product of variable length, while the second mechanism, σμ / Σμ recombination, should always produce the same product. We performed Southern blot analysis of genomic DNA isolated from a total of 3 hybridomas, one expressing μ and two expressing γ1 (FIG. 75). Genomic rearrangements are found upstream of the transgene γ1 only in two of the γ1 switch regions (FIG. 76). Furthermore, for all observed structures, σμ
Is incompatible with homologous recombination between and Σμ. Thus, our results are consistent with the model for γ1 isotype expression mediated by deletional non-homologous recombination between the transgene-encoded μ and γ1 switch regions. In addition to transswitch human γ1, mouse γ is found in the serum of HC1 and HC2 transgenic-JHD mice.
Mouse γ-expressing hybridomas were also obtained from these animals. Since non-transgenic homozygous JHD animals do not express detectable levels of mouse immunoglobulin, HC1 and HC2 transgenic-JHD animals.
The expression of mouse γ in animals appears to be due to the transswitch phenomenon. Not all of the transgenic hybridomas we analyzed express both those expressing either mouse or human constant region sequences. Therefore, it is unlikely that the trans-splicing mechanism is involved. CDNA of transswitch product
PCR amplification to isolate the clones was used to determine the nucleotide sequence of 10 of the resulting clones (Figure 77). The 5'oligonucleotide during PCR amplification is specific for transgene-encoded VH251 and the 3'oligonucleotide is mouse γ1, γ
It is specific for the 2b and γ3 sequences. Examples of transswitch products incorporating all three of these mouse constant regions can be found. Somatic mutations About 1% of the nucleotides in the variable region of the transswitch product shown in Figure 7 are not germline encoded. This is probably due to somatic mutations. Since the mutated sequences were translocated to the endogenous locus, the cis-acting sequences leading to these mutations could be located anywhere 3'from the mouse gamma switch. However, as discussed below, somatic mutations within the VDJ segment that did not undergo such translocation are also observed. And this result shows that the sequence required for heavy chain somatic mutation is contained in the transgene.

To determine whether the HC1 and HC2 constructs contained sufficient cis-acting sequences for somatic mutations in transgenic-JHD mice, we determined that two independent HC1 CDNA clones derived from the transgenic line and one HC2 line were isolated and partially sequenced. It can be seen that some of the γ1 transcripts from transgenic-JHD mice contain V regions with extensive somatic mutations. The frequency of transcripts with these mutations appears to increase with repeated immunizations. 78 and 79
Shows two sets of cDNA sequences: one set is RN
H immunized with a single injection of antigen 5 days prior to separating A
C1 (strain 26) transgenic-derived from JHD mouse;

The second set was HC1 (strain 26) transgenic-hyperimmunized by injecting the antigen 3 times on different days starting 5 months before RNA isolation.
Has been derived from JHD mice; the second set is RNA
HC1 (line 26) hyperimmunized by injecting antigen 3 times on different days starting 5 months before
Transgenic-derived from JHD mice (textual duplication). Only two of the 13 V regions from day 5 post-exposure contain any non-germline coding nucleotides. Each of these Vs contained only a single nucleotide change, giving an overall somatic mutation frequency of less than 0.1% for this sample. In contrast, none of the 13 V sequences from hyperimmunized animals were fully germline,
The overall somatic mutation frequency is 1.6%.

Μ and γ1 isolated from a single tissue sample
A comparison of the transcripts indicates that the frequency of somatic mutations is higher than that of the transgene copy that underwent class switching. We hyperimmunized CH1 strain 57
Forty-seven independent μ and γ1 cDNA clones from transgenic-JHD mice were isolated and partially sequenced (FIGS. 80 and 81). Most of the μcDNA clones have not been modified in relation to germline sequences, while more than half of the γ1 clones contain a large number of non-germline encoded nucleotides. Although γ1-expressing cells are quite different from μ-expressing cells, the two processes are not necessarily linked, but class switch and somatic mutation occur within the same subpopulation of B cells.

Extensive somatic mutations of the VH251 gene are not found in non-hyperimmunized CH1 transgenic mice, but have not been subjected to specific experiments.
Significant somatic mutations were found in the VH56p1 and VH51p1 genes in 2 transgenic mice. HC in non-immunized females at 9 weeks of age
Spleen and lymph node RNA from 2 transgenic animals
Separated. We individually amplified γ1 transcripts that incorporate each of the four V regions within the HC2 transgene using V and γ1 specific primers. The relative yield of each of the specific PCR products is VH56p1 >>VH51p1> VH4.
21> VH251. This technique, although not strictly quantitative, may represent a bias in the use of V segments in HC2 mice.

FIG. 82 shows 23 randomly picked γ1 cDNA sequences derived from PCR amplification with an equimolar mixture of all four V-specific primers. Again, VH56p1 (19/23 clone)
There is a bias towards. Furthermore, the VH56p1 sequence shows a significant somatic mutation with an overall frequency of 2.1% within the V gene segment. Examination of the CDR3 sequences revealed that although 17 out of 19 individual VH56p1 clones were unique, only 7 of them were unique.
Derived from only two different VDJ recombination events,
I understand that. Therefore, it appears that VH56p1-expressing B cells are likely selected by endogenous pathogens or self-antigens in animals that have not undergone specific experiments. It may be appropriate for this same gene to be over-represented within human fetal competence.

Summary Upstream cis-acting sequences define the functionality of individual switch regions and are required for class switching. HC1
Our observation that class switches within the transgene are largely restricted to cells involved in the secondary response and do not occur randomly across the entire B cell population, suggesting that the minimal inclusion involved with the transgene. It suggests that the sequence is sufficient. The γ sequence contained in this construct is only 11 upstream of the initiation site of γ1 sterile transcription.
Since it starts at 6 nucleotides,
The switch control area is dense.

Our results show that these important cis-acting regulatory elements are either tightly linked to individual γ genes or associated with 3'heavy chain enhancers contained within the HC1 and HC2 transgenes. It is proved that it is. We derived from translocation to an endogenous locus because the HC1 and HC2 inserts undergo a transgene autonomic class switch that can serve as a marker for sequences likely to undergo somatic mutations I was able to easily find a highly mutated copy that was non-trivial. We found somatic mutated gamma transcripts in three independent transgenic lines (two HC1 lines and one HC2 line). Therefore, it is unlikely that sequences flanking the transgene integration site will affect this process: instead, the transgene sequences are sufficient to direct somatic mutations.

Example 35 This example describes the production of hybridomas from mice homozygous for the inactivated endogenous immunoglobulin locus and containing transgene sequences encoding human sequence heavy chain and human sequence light chain. Describe. The hybridomas described herein secrete monoclonal antibodies containing human sequence heavy chains and human sequence light chains and bind to a predetermined antigen expressed on T cells. This example also demonstrates the ability of the mouse to produce human sequence antibodies in response to the human-derived immunogen human CD4, and such sources as sources for producing hybridomas secreting human sequence monoclonal antibodies reactive with human antigens. It also demonstrates the suitability of the mouse. A. HC1 transfection immunized with human CD4 antigen
Human Ig monoclonal antibody derived from nick mouse
Generation of the body harbors a transgene that is homozygous for the functionally disrupted J _H locus and that can be rearranged to encode the human sequence heavy chain and rearrangeable to encode the human sequence light chain. Transgenic mice were immunized. Mouse genotype is HC1-26 ⁺ KCl
e-1536 ⁺ J _H D ^{+ / +} J _K D ⁻ , which is homozygous for mouse heavy chain inactivation, and the presence of HC1 human sequence heavy chain transgene and KCle human sequence light chain transgene germline copy. Was represented.

Mice were immunized with a variant of the EL4 cell line (ATCC) expressing a mouse-human hybrid CD4 molecule encoded by a stably transfected polynucleotide. Expressed CD
The four molecules contain a substantially human-like CD4 sequence. 100
with 1 μl complete Freund's adjuvant (CFA)
Approximately 5 × 10 ⁶ cells in 00 μl PBS were introduced into mice on day 0 via intraperitoneal injection. 7th day,
Inoculation was repeated on the 14th, 21st, 28th, 60th and 77th days, and on the 18th, 35th and 67th days.
A test bleed was given on the day. On day 81, the spleen was removed and approximately 1.2 x 10 by standard method (PEG fusion).
About 7.2 × 10 ⁷ splenocytes were fused to ⁷ fusion partner cells (P3 × 63Ag8.653 cell line: ATCC), and RPMI1640 15% FCS, 4 mM glutamine, 1 mM sodium pyruvate was added to HAT and P.
The cells were cultured in the medium supplemented with SN medium. A number of fusions were performed.

Hybridomas were grown and purified recombinant soluble human sequence CD4 (Amer expressed in CHO cells
ican Bio-Technologies, Inc. (ABT), Cambridge, MA)
And / or the supernatants were tested using ELISA for binding to commercially available sources such as CD4 obtained from NEN-DuPont. The ABT samples are human CD4 V ₁ ~
It contained a purified 55 kD human CD4 molecule consisting of the V ₃ domain. Recombinant human sequence CD4 (produced in CHO-K1 cells) was adsorbed to the assay plate and used to capture the antibody from the hybridoma supernatant, then the captured antibody was used as human μ , Human kappa, human gamma, mouse mu or mouse kappa were assessed for binding to a panel of antibodies.

One hybridoma was subcloned from its culture plate well called 1F2.
The 1F2 antibody bound to the ABT CD4 preparation was positive for human μ and human κ and negative for human γ, mouse γ and mouse κ. B. HC2 immunized with human CD4 and human IgE
Human Ig monoc derived from transgenic mouse
Production of Ronal Antibodies The heavy chain transgene HC2 is shown in Figures 78 and 79 and has been described above (see Example 34).

The human light chain transgene KCo4, depicted in Figure 68, is generated by co-integration of two individually cloned DNA fragments at a single site within the mouse genome. Fragment has four functional V
_It contains a _K segment, a 5J segment, a C _K exon and intron and both enhancer elements downstream (see Example 21) (Meyer and Neuberger (1989), EM.
BO J. 8: 1959-1964; Judde and Max (1992), Mol.Cel.
Biol. 12: 5206-5216). Since the two fragments share a common 3 kb sequence (see Figure 67),
These can potentially be integrated into genomic DNA as flanking 43 kb transgenes after homologous recombination between overlapping sequences.

It has been established that such recombination events frequently occur at the time of microinjection of overlapping DNA fragments (Pieper et al. (1992), Nucleic Acids Re.
It s 20:. 1259-1264). Co-injected DNA also tended to co-integrate into the zygote and the sequences contained within the individually cloned fragments were
It will be linked by DNA rearrangements during cell development. Table 11 shows that the transgene inserts from at least two of the transgenic lines are functional. An example of VJ ligation incorporating each of the four transgene-encoded V segments and each of the 5J segments is represented in this set of 36 clones.

[0481]

[Table 6]

The table above shows the use of human light chain V and J segments in KCo4 transgenic mice. This table contains the human kappa sequences shown,
The number of PCR clones amplified from the cDNAs from the two transgenic lines is shown. w Individual KCo4
Transgenic mouse (mouse # 8490, 3m
o. , Male KCo4 strain 4437; mouse # 8867,
2.5 mo. CDNA was synthesized using spleen RNA isolated from a female, KCo4 strain 4436). Ck-specific oligonucleotide 5'TAG AAG GAA TTC AGC AGG CA
CACA ACA GAG GCA GTT CCA 3 ', as well as two Vk-specific oligonucleotides 5'AGC TTC TCG AGCTCC TGC T
GC TCT GTT TCC CAG GTG CC 3'and 5'CAG CTT CTC G
The cDNA was amplified by PCR using a 1: 3 mixture of AG CTC CTG CTA CTC TGG CTC (C, A) CA GAT ACC 3 '. The PCR product was digested with XhoI and EcoRI and cloned into a plasmid vector. Partial nucleotide sequences of 18 randomly picked clones from each animal were determined by the dideoxycentration method. The sequence of each clone was compared to the germline sequence of the unrearranged transgene.

Twenty-three light chain minilocus positive and eighteen heavy chain positive mice developed from injected embryos. Human heavy and kappa chains in the absence of functional mouse heavy and kappa light chain loci
To obtain a mouse containing a light chain, an endogenous mouse heavy chain (system J
HD) and mice containing targeted mutations in the kappa light chain locus (line JCKD) were used to breed these mice and their progeny. These mice contain only λB cells.

Table 7 shows that somatic mutations occur within the variable regions of the transgene-encoded human heavy chain transcripts of transgenic mice.
23 cDNAs from HC2 transgenic mice
Clones were partially sequenced to determine the frequency of non-germline coding nucleotides within the variable region. The data only includes the sequences of V segment codons 17-94 from each clone, not the N region. RNA was isolated from the spleen and lymph nodes of mouse 5250 (HC2 strain 2550 hemizygous, JHD.
Homozygous), as described (reference) single-stranded cD
NA was synthesized and the γ transcript was amplified by PCR. Amplified c
The DNA was cloned into a plasmid vector and 23 randomly picked clones were partially sequenced by the dideoxy read-end method. The frequency of PCR-induced nucleotide changes is estimated as <0.2% from the constant region sequences.

[0485]

[Table 7]

Flow Cytometry We use a FACScan flow cytometer and LYSI.
SII Software (Becton Dickinson, San Jose. CA)
Were used to analyze the stained cells. Spleen cells were stained with the following reagents; Probium iodide (Molecular probe, Eugene, OR), Phycoerythrin-conjugated α-human Igκ (clone HP6062; Caltag, S. San Fran).
cisco, CA), Phycoerythrin-conjugated α-mouse Igκ (clone X36; Becton Dickinson, San Jos
e. CA), FITC-conjugated α-mouse Igλ (clone R26-46; Pharmingen, San diego, CA), F
ITC-conjugated α-mouse Igμ (clone R6-6
0.2; Pharmingen, San diego, CA), FITC-conjugated α-human Igμ (clone G20-127; Phar
mingen, San Diego, CA), and Cy-chromoconjugated anti-mouse B220 (clone RA3-6B2; Phar.
mingen, San Diego, CA). Human Ig transgene expression Figure 83 shows a flow cytometric analysis of splenocytes from KCo4 and HC2 mice that are homozygous for both JHD and JCKD mutations. Human sequence HC
The 2 transgene rescued B cell development within the JHD mutation background and restored the relative number of B220 ⁺ cells in the spleen to approximately half that of wild-type animals. These B cells expressed cell surface immunoglobulin receptors with transgene-encoded heavy chains.
The human KCo4 transgene was also functional and competed well for the intact endogenous lambda light chain locus. Approximately 95% of B splenocytes in JHD / JCKD homozygous mutant mice containing both heavy and light chain human transgenes (double transgenic) were completely human cell surface I.
Expressed gMκ.

Serum Ig levels were measured by ELISA as follows.
Determined by: Human μ: Mouse Mabα human IgM
(Clone CH6, binding site, Birmingham, UK) coated and peroxidase-conjugated rabbit alpha human IgM (fc) (cat # 309-035-095, Ja
Microtiter wells developed at cksson Immuno Research, West Grove, PA). Human γ: mouse MAb
α human IgG1 (clone HP6069, Calbiochem,
La Jolla, CA) coated peroxidase-conjugated goat α human IgG (fc) (cat # 109)
-036-098, Jackson Immuno Research, West Gr
ove, PA) developed microtiter wells.

Human κ: mouse Mabα human Igκ (ca
t # 0173, AMAC, Inc, Igκ (cat # A716
4, Sigma Chem. Co., St. Louis, MO) coated microtiter wells. Mouse γ: goat α mouse IgG (cat # 115-006-071, Jackson
Immuno Research, West Grove, PA) coated microtiter wells. Mouse λ: rat MAb
α mouse Igλ (cat # 02171D, Pharmingen,
San Diego, CA) coated peroxidase-conjugated rabbit alpha mouse IgM (fc) (cat #
309-035-095, Jackson Immuno Research, W
Microtiter wells developed at est Grove, PA). The bound peroxidase was treated with hydrogen peroxide and 2,2'-azino-bis-13-ethylbenzthiazoline-6-sulfonic acid, Sigma Chem. Co., St. Louis, M.
Detection by incubation with O 2). The reaction product is measured by absorption at 415 nm.

Double transgenic mice also express fully human antibodies in serum. Figure 84 shows measured serum levels of immunoglobulin proteins for 18 individual double transgenic mice that are homozygous for heavy and kappa light chain inactivation derived from several different transgenic founder animals. . We have found detectable levels of human μ, γ1 and κ. We have now shown that expressed human γ1 results from authentic class switching by genomic recombination between the transgene μ and γ1 switch regions. Moreover, we found that class switching within the transgene was achieved by somatic mutation of the heavy chain variable region.

In addition to human immunoglobulin, we have also found mouse γ and λ in serum. The presence of the mouse lambda protein is expected as the endogenous loci are completely intact. We have shown elsewhere that mouse gamma expression is the result of transswitch recombination of the transgene VDJ segment into the endogenous heavy chain locus. Initially wild-type heavy chain allele and rearranged VDJ
This trans-switching phenomenon, which has been established for the transgene (Durdik et al. (1989) , Proc.Natl.Acad.S
ci.USA 86:. 2346-2350; Gerstein et al (1990), fine
Cell 63: 537-548) occurs within the mutant JHD background because the downstream heavy chain constant region and its respective switch element are still intact.

Human I in double transgenic mice
The serum concentration of gMκ is about 0.1 mg / ml, and there is little variation between animals or strains. However, human γ
1. Mouse γ and mouse λ levels range from 0.1 to 10 micrograms / ml. The variation in gamma levels seen between individual animals may be the result of the fact that gamma is a constitutive homeostatic region. Expression is likely dependent on factors such as animal health, exposure to antigen and possibly MHC type. Mouse lambda serum levels are the only parameter that appears to correlate with individual transgenic lines.

The lowest number of transgene copies per integration (approximately 1-2 copies) KCo4 strain 4
436 mice have the highest endogenous lambda levels, while K
4437 mice of Co4 strain (10 to 10 times per integration)
Copy) has the lowest λ level. This is consistent with one model in which the κ transgene is followed by a relocation of endogenous λ, where serum λ levels are not selected and instead reflect the relative size of the precursor B cell pool. Hold.
Transgene loci containing multiple light chain inserts may have the opportunity to undergo one or more VJ recombination events, increasing the probability that one of them will be functional. Thus, the high copy lineage will have a smaller potential lambda cell pool. To test the ability of transgenic B cells to participate in the immune response with human CD4 and IgE , we immunized double transgenic mice with human protein antigens and tested serum levels of antigen-specific immunoglobulins by ELISA. Was measured. Polystyrene lymphocytes in complete Freund's adjuvant by intraperitoneal injection (cat # 08226, Polysciences Inc.,
50 μg recombinant sCD4 (cat, # 013101, American Bio-T) covalently linked to Warrington, PA
Mice were immunized with echnologies Inc., Cambridge, MA). Each of the mice is homozygous for disruption of the endogenous mu and kappa loci and human heavy chain transgene HC
Two lines 2500 and human kappa light chain transgene KCo4
Hemizygous for line 4437.

Methods Serum samples were diluted into recombinant sCD4 coated microtiter wells. Peroxidase-conjugated rabbit alpha human IgM (fc) (Jackson Im
muno Research, West Grove, PA) or peroxidase-conjugated goat anti-human Igκ (Sigma, St. Louis, M)
O) was used to detect human antibodies.

Figure 85 shows the primary response of transgenic mice immunized with recombinant human soluble CD4.
All four immunized animals were antigen-specific human IgM
Responses are shown at week 1. CD4 specific serum antibodies contain both human mu heavy chain and human kappa light chain. To assess the ability of the HC2 transgene to participate in the secondary response,
We hyperimmunized transgenic mice by repeatedly injecting antigen and monitored the heavy chain isotype of the elicited antibodies. Human heavy chain transgene HC2
And mice homozygous for the human kappa light chain transgene KCo4 were treated on day 0 with 25 μg of human IgEk (The Binding Site, Bir) in complete Freund's adjuvant.
(Mingham, UK). Thereafter, the mice were injected with IgEκ in Freund's incomplete adjuvant about every other week. Serum samples were diluted 1:10 and antigen-specific ELISAs were performed on human IgE, λ coated plates.

FIG. 86 shows a standard time course of immune response from these animals. We injected double-transgenic mice with human IgE in complete Freund's adjuvant and then boosted weekly with IgE in incomplete Freund's adjuvant. The early human antibody response was IgMκ, followed by the appearance of antigen-specific human IgGκ. The induced serum antibodies in these mice were
It did not show any reactivity to human IgM or BSA. Human IgG development over time, (text missing).

We also tested the ability of the heavy chain transgene to undergo class switching in vitro; animals in purified form of splenic B cells that were hemizygous for the same heavy chain construct (HC2, line 2550) were tested. , LPS and recombinant mouse IL-4 in the presence of human IgM to human I
Switch to gG1. However, the in vitro switch is not compatible with LPS and recombinant mouse IL-2 or LP.
S did not happen in the presence of its own.

Human IgM expressing cells are found in the spleen, lymph nodes, peritoneum and bone marrow of double transgenic / double knockout (0011) mice. Although the abdominal cavity contained a normal number of B cells, the absolute number of transgenic B cells in the bone marrow and spleen was about 10-5 of the normal value.
It is 0%. A decrease may result as a result of the delay in transgene-dependent B cell development. Double transgenic / double knockout (00
11) Mice also express fully human antibodies in serum, with the levels of human μ, γ1 and κ in these mice being significant. Expressed human γ
1 results from an authentic class switch by genomic recombination between the transgene μ and γ1 switch regions.

In addition, class switching within the transgene is accompanied by somatic mutations in the heavy chain variable region encoded by the transgene. In addition to human immunoglobulins, mouse μ and mouse λ are found in these mice. Mouse mu expression appears to be the result of transswitch recombination in which the transgene VDJ gene is integrated within the endogenous mouse heavy chain locus. The transswitch originally found in the literature for wild-type heavy chain alleles and the rearranged VDJ transgene was found in our J because the mouse downstream heavy chain constant region and its respective switch element are still intact. _H ^-/- happens in the background.

To demonstrate the ability of transgenic B cells to participate in the immune response, we immunized 0011 mice with human protein antigen and monitored serum levels of antigen-specific immunoglobulins. Initial human antibody response is Ig
M, followed by expression of antigen-specific human IgG (FIGS. 86 and 88). The delay before the appearance of human IgG antibodies is consistent with the link between the secondary response to antigen and class switching.

In transgenic mice immunized with human CD4, human IgG against the CD4 antigen
Reactivity was detectable at serum concentrations ranging from 2 × 10 ^{-2 to} 1.6 × 10 ^-4 . Identification of anti-human CD4 hybridoma Transgenic mice homozygous for human heavy chain transgene HC2 and human kappa light chain transgene KCo4 were immunized on day 0 with 20 μg of recombinant human CD4 in complete Freund's adjuvant. The mice were then injected with CD4 in incomplete Freund's adjuvant at approximately one week intervals. Figure 88 shows human antibody response to human CD4 in serum of transgenic mice. Serum samples were diluted 1:50 and antigen specific ELISA on human CD4 coated plates
Was performed. Each line represents the measured value of an individual sample. Black circles represent IgM and white circles represent IgG.

Line # 7494 (0012; HCl-2
6+; JHD ++; JKD ++; KC2-1610 +
+) Mice were immunized with human CD4 on days 0, 13, 20, 28, 33 and 47, to give anti-human CD4 antibody consisting of human κ and human μ or γ. Produced. On day 28, human μ and human κ were found in serum. By day 47, the serum response to human CD4 included human μ and human γ and human κ. On day 50, splenocytes were fused with P3 × 63-Ag8.653 mouse myeloma cells and cultured. Forty-four of 700 wells (6.3%) contained human γ and / or κ anti-human CD4 monoclonal antibody. Three of these wells were confirmed to contain human gamma anti-CD4 monoclonal antibody but lacked human kappa chain (possibly expressing mouse lambda). Nine primary wells contained fully human IgMκ anti-CD4 monoclonal antibody and were selected for further characterization. One such hybridoma expressing a fully human IgMκ anti-CD4 monoclonal antibody was cloned into 2C1.
It was called 1-8.

The primary hybridoma was cloned by the limiting dilution method, and human μ and κ reactive with CD4 were cloned.
The secretion of monoclonal antibody was evaluated. Five out of nine hybridomas remained positive in the CD4 ELISA. These human I for human CD4
The specificity of the gMκ monoclonal antibody is ovalbumin,
It was demonstrated by its lack of reactivity with other antigens including bovine serum albumin, human serum albumin, keyhole limpet hemocyanin and carcinoembryonic antigen.

To determine whether these monoclonal antibodies could recognize CD4 on the cell surface (eg native CD4), the supernatants of these five clones were also tested for reactivity with the CD4 + T cell line, SupT1. . Four of the five human IgMκ monoclonal antibodies reacted with these CD4 + cells. These IgMκ
To further ensure the specificity of the monoclonal antibody,
Freshly isolated human peripheral blood lymphocytes (PBL) were stained with these antibodies. Supernatants from clones derived from 4 out of 5 primary hybrids were CD4 +
It bound only to lymphocytes and not to CD8 + lymphocytes (Fig. 87).

FIG. 87 shows IgMκ anti-CD with human PBL.
4 shows the reactivity of the monoclonal antibody. Human PBL supernatants from each clone or isotype-matched negative control monoclonal antibody followed by PE conjugated mouse anti-human CD4 monoclonal antibody (top) or F
Incubation was with either mouse anti-human CD8 Ab conjugated to ITC (bottom row). FIT respectively
Any bound human IgMκ was detected with mouse anti-human μ conjugated to C or PE. One of the clones, 2C
Representative results are shown for 11-8 (right side) and control IgMκ (left side). As expected, negative control IgM
κ did not react with T cells and goat anti-human μ reacted with approximately 10% PBL, presumably human B cells. IgMκ
High levels of production of anti-CD4 monoclonal antibodies and excellent growth are important factors in selecting clonal hybridoma cells for development. Data from one of the hybridomas, 2C11-8, indicates that up to 5 pg / cell / day can be produced (Figure 89). Second
Similar results were found for the clones of. As is generally observed, production increases dramatically as cells enter stationary phase growth. FIG. 89 shows cell growth and secretion of human IgMκ anti-CD4 monoclonal antibody in small scale culture. Duplicate cultures were seeded at a rate of 2 × 10 ⁵ cells / ml in a total volume of 2 ml. Cultures were harvested every 24 hours for the next 4 days. Cell growth was determined by counting viable cells, and IgMκ production was quantified by ELISA for all human μ (pictured above). Ig
Production per cell per day was calculated by dividing the amount of Mκ by the number of cells (bottom panel).

FIG. 90 shows epitope mapping of human IgMκ anti-CD4 monoclonal antibody. IgMκ anti-CD4 monoclonal antibody 2C11-8 (HB 11
A competitive binding flow cytometry experiment was used to localize the epitope recognized by 668). For these studies, the mouse anti-CD4 monoclonal antibody Leu, which binds to non-overlapping unique epitopes on CD4
3a and RPA-T4 were used. Decreasing concentration of RPA
-CD preincubated with either TA or Leu-3a and subsequently stained with 2C11-8
PE Fluorescence of 4+ cells, PE-conjugated goat anti-human Ig
Detected by M. Human IgMκ anti-CD by Leu3a
There was concentration-dependent competition for the binding of the 4 monoclonal antibody 2C11-8, but not for its binding by RPA-T4. Thus, 2C11-8
The epitope recognized by is the monoclonal antibody L
It was similar or identical to the epitope recognized by eu3a, but completely different from that recognized by RPA-T4.

In summary, we have human IgM that reacts specifically with native human CD4 and can be used to discriminate human PBLS into CD4 ⁺ and CD4 ⁻ subpopulations.
Multiple hybridoma clones secreting the kappa monoclonal antibody were produced. At least one of these antibodies
One binds to or near the epitope constituted by the monoclonal antibody Leu3a. Monoclonal antibodies directed against this epitope have been shown to inhibit mixed leukocyte responses (Engl.
eman et al., J. Eyp. Med. (1981) 153: 193). The chimeric version of the monoclonal antibody Leu3a has shown some clinical efficacy in patients with mycosis fungoides (Knox et al. (1991) Blood 77:20).

We also isolated a hybridoma cell line from one mouse that responded to human CD4 immunization. Five of the cloned hybridomas secrete human IgGκ (human γ1 / human κ) antibodies that bind to recombinant human CD4 and do not cross-react with a panel of other glycoprotein antigens (as measured by ELISA) To do. IgGκ hybridoma, 4E4.2 and 2
The rates of binding and dissociation of the immunizing human CD4 antigen to the monoclonal antibody secreted by two of C5.1 were measured. The experimentally derived binding constant (Ka) is approximately 9 × for antibodies 4E4.2 and 2C5.1, respectively.
Was 10 ⁷ M ^-1 and 8 × 10 ⁷ M ^-1. These Ka
Has been used in clinical trials by others ( Chen et al. (1993) Int. Immunol. 6 : 647).
It falls within the scope of mouse IgG anti-human CD4 antibody.

[0508] Here, B expressing human immunoglobulin
We conclude that cells undergo development and respond to antigens in the context of the mouse immune system. Antigen reactivity leads to immunoglobulin heavy chain isotype switching and variable region somatic mutations. We have also demonstrated that conventional hybridoma technology can be used to obtain monoclonal human sequence antibodies from these mice. Thus, these transgenic antibodies are the source of human antibodies against human target antigens.

The transgenic mice of the invention are immunized with a human antigen selected from Tables B and C below. The transgenic mouse produces a human antibody that binds to the human antigen. B cells from immunized transgenic mice are used to generate hybridomas that secrete monoclonal antibodies against the selected human antigens.

[0510] Table B human CD antigens membrane components CD1a gp49 CD1b gp45 CD1c gp43 CD2 CD58 (LFA-3) , which is sign recognition receptor, gp50 epitope CD2R be limited to CD2 active T CD3 CD3 - complex (5 chain), gp / P 26, 20, 16 CD4 ClassII / HIV receptor, gp59 CD5 gp67 CD6 gp100 CD7 gp40 CD8 class 1 receptor, gp32, αα or αβ dimer CD9 p24 CD10 gp100, CALLA CD11a LFA-1, gp180 / 95 CD11b C3bi receptor, gp155 / 95 CD11c gp150 / 95 CDw12 p90-120 CD13 gp150 CD14 gp55 CD15 3-FAL, X-hapten CD16 FcRIII, gp50-65 CDw17 lactosylceramide CD18 β chain for CD11a, b, c CD19 gp95 CD20 p37 / 32 CD21 C3d / EBV-Rec. (CR2), p140 CD22 gp135, homology to myelin-related gp (MAG) CD23 FcεRII, gp45-50 CD24 gp41 / 38 CD25 IL-2R β chain, gp55 CD26 gp120 CD27 p55 (dimer) CD28 gp44 CD29 VLA β-antigen β1-chain, Plt GPIIa CD30 gp120, Ki-1 CD31 gp140, Plt, GPIIa CDw32 FcRII, GP40 CD33 gp67 CD34 gp105-120 CD35 CR1 CD36 gp90, Plt GPIV CD37 gp40-52 CD38 p45 CD39 gp70-100 CD40 gp50, homologous to NGF receptor CD41 Plt GPIIb-IIIa complex and GPIIb CD42a Plt GPIX, gp23 CD42b Plt GPIb, gp135 / 25 CD43 leukosialin, gp95

Table C CD44 Pgp-1, gp80-95 CD45 LCA, T200 CD45RA restricted T200, gp220 CD45RB restricted T200 CD45RO restricted T200, gp180 CD46 membrane cofactor protein (MCP), gp66 / 56 CD47 gp47-52, N-linked glycan CD48 gp41, PI-linked CDw49b VLA-α2 chain, Plt GPIa CDw49d VLA-α4 chain, gp150 CDw49f VLA-α6 chain, Plt GPIc CDw50 gp148 / 108 CDw51 VNR-α chain CDw52 -1, gp21-28 CD53 gp32-40 CD54 ICAM-1 CD55 DAF (decyl retention factor) CD56 gp220 / 135, NKH1, N-CAN isoforms CD57 gp110, HNK1 CD58 LFA-3, gp40-65 CD59 gp18- 20 CDw60 NeuAc-NeuAc-Gal- CD61 Integrin β3-, VNR-β chain, Plt GPIIIa CD62 GMP-140 (PADGEM), gp140 CD63 gp53 CD64 FcRI, gp75 CDw65 ceramide-dodecasaccharide 4C CD66 phosphoprotein gp180-200 CD67 P100, PI-linked CD68 gp110 CD69 gp32 / 28, AIM CDw70 Ki-24 CD72 gp43 / 39 CD73 p69 CD74 gp41 / 35/33 CDw75 P53 CD76 gp85 / 67 CD77 Globotria aosylceramide (Gb3) C Dw78

Table B above shows a cluster of differentiation of humans for immunizing transgenic mice of the invention and generating hybridomas producing monoclonal antibodies against human antigens.
It is a list of examples of (CD). Table C above is a list of examples of human non-CD antigens for immunizing transgenic mice of the invention and generating hybridomas that produce monoclonal antibodies against human antigens.

The transgenic mice of the invention are listed in Table D.
Immunize with a human-pathogen or antigen selected from
Transgenic mice produce antibodies that bind to human pathogens. B cells from immunized transgenic mice are used to generate hybridomas secreting monoclonal antibodies against the selected human pathogens.

Table D OKT3 OKT4 Tumor Necrosis Factor IgE Her-2 IL-2 Receptor (p55, p75) Lym-1 Fibrin α-Phetoprotein My9 Insulin Insulin Receptor Carcinoembryonic Antigen (CEA) Glucose Transfer Protein β-Interferon γ -Interferon tissue plasminogen activator (PBA) α-interferon nerve growth factor (NGF) endothelial growth factor (EGF) NGF receptor EGF receptor platelet-derived growth factor (PDGF) PGDF receptor insulin-like growth factor (IGF-1) IGF- 1 receptor Ciliary neurotrophic factor (CNFF) CNFF receptor Brain-derived neurotrophic factor (BDNF) BDNF receptor Neuronal differentiation factor (NDF) NDF receptor Heparin-binding neurotrophic factor (HBNF) HBNF receptor Transforming growth factor α (TGF-α) TGF-α receptor Transforming growth factor β (TGF-β) TGF-β receptor Bone growth factor-1 (BMP-1, OMP-1) BMP-1 receptor Bone growth Factor-2 (BMP-2, OMP-2) BMP-2 receptor interleukin-1 (IL-1) IL-1 receptor interleukin-3 (IL-3) IL-3 receptor interleukin-4 (IL-4) ) IL-4 receptor interleukin-6 (IL-6) IL-6 receptor interleukin-8 (IL-8) IL-8 receptor interleukin-12 (IL-12) IL-12 receptor interleukin-13 (IL) -13) IL-13 receptor Granulocyte / macrophage colony stimulating factor (GM-CS)
F) Granulocyte colony stimulating factor (G-CSF) Stem cell factor (SCGF) IL-1β converting enzyme GM-CSF receptor G-CSF receptor Thrombomodulin complement factor complement receptor Protein C cadherin gap junction protein ULA-4 ICAM- 1 LFA-3 ELAM P-selectin E-selectin multidrug resistance protein (MDR) atrial natriuretic peptide T-cell receptor (TCR) peptide substance P angiotensin converting enzyme (ACE) common acute lymphocytic leukemia antigen (CALLA) B16 (Melanoma antigen) Villous stimulating hormone (FSH) Luteinizing hormone (LH)

Table D, above, is a list of examples of human pathogens and their antigens for immunizing transgenic mice of the present invention and generating hybridomas producing monoclonal antibodies against the human pathogens.

Table E HIV-1 and HIV-2 viral antigens (gp120,
HIV protease HIV RNase H cytomegalovirus (CMV) and CMV virus antigen (gH, gp, etc.) human T-cell lymphotropic virus-1 (HTLV-2)
And its surface antigen human T-cell lymphotropic virus-2 (HTLV-2)
And surface antigens hepatitis A virus and virus antigens hepatitis B virus and virus antigens (eg, HBsAg, HBc
Ag, HBeAg) Hepatitis C virus and virus antigens Hepatitis D virus and virus antigens Hepatitis X virus and virus antigens Human papilloma virus (HPV) and virus antigens Varicella zoster virus and virus antigens Herpes simplex I virus and virus antigens Herpes simplex virus II Viruses and virus antigens Ebola (or Ahaburg) virus and virus antigens Japanese encephalitis B virus (JBE) and virus antigens California encephalitis virus and virus antigens Gray medullitis virus and virus antigens Coxsackie virus and virus antigens Renovirus and virus antigens Rabies ( Rhabdoviridae) virus and virus antigens Influenza (Orthomyxoviridae) virus and virus antigens Paramyxoviridae (Paline Flue The 1~5, mu
mps) virus and antigen measles virus and virus antigen respiratory syncytial virus (RSV) and virus antigen variola virus (smallpox) and virus antigen adenovirus (masterdenovirus) and virus antigen Epstein-Barr (mononucleosis) virus And virus antigen Plasmodium falcipa
rum) and viral antigens Corynebacterium diphtheria and surface antigens and toxins Corynebacterium haemolyticum and surface antigens and toxins Salmonella spp. and surface antigens and LPS Shigella spacese and surface antigens and LPS Proteus mirabilis speice and surface antigens and LPS Pseudomonas aeruginosa and surface antigens Vibrio cholerae and surface antigens and toxins Vibrio parahaemolytics and surface antigens and toxins Nigeria meningitidis and surface antigens Niseria gonorrhoeae and surface antigens Bacillus anthracis and surface antigens Clostridium botulinum And surface antigens and toxins Clostridium tetani and surface antigens and toxins Clostridium perfringens and surface antigens and toxins hemophiles Influenza and surface antigens Treconema paridium and surface antigens Listeria monocytogenes and surface antigens Nocardia Asteroides and surface antigens Pneumocystis carnie and surface antigens Pasteurella leukotoxin Staphylococcus enterotoxins and hyaluronidases and coagulase Streptococcus (β-hemolysis) And hemolysin and streptokinase Gram-negative lipopolysaccharide complex (LPS)

Table E above is a list of examples of human pathogens and their antigens for immunizing transgenic mice of the present invention and generating hybridomas that produce monoclonal antibodies to the human pathogens. Although the invention has been described in some detail by way of illustration for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be made within the scope of the claims.

[Brief description of drawings]

FIG. 1 is the complementarity determining regions CDR1, CDR2 and CDR3 and the framework regions FR1 and FR in unrearranged genomic DNA and in mRNA expressed from the rearranged immunoglobulin heavy chain gene.
2, represents FR3 and FR4.

FIG. 2 represents the human λ chain locus.

FIG. 3 represents the human kappa chain locus.

FIG. 4 represents the human heavy chain locus.

FIG. 5 represents a transgen construct containing a rearranged IgM gene linked to a 25 kb fragment containing human γ3 and γ1 constant regions, followed by a 700 bp fragment containing the rat chain 3 ′ enhancer sequence. .

FIG. 6 is a restriction map of the human kappa chain locus, representing fragments that can be used to form light chain transgens by in vivo homologous recombination.

FIG. 7 shows the production of pGP1.

FIG. 8 shows the constitution of the polylinker contained in pGP1.

FIG. 9 shows the fragments used to make the human heavy chain transgenes of the present invention.

FIG. 10 shows the production of pHIG1 and pCON1.

FIG. 11 shows that pREG2 was formed to form pREG2.
The human Cγ1 fragment inserted into RE3 (rat enhancer 3 ′) is shown.

FIG. 12 shows the production of pHIG3 ′ and pCON.

FIG. 13 shows a fragment containing a human D region segment used for producing the transgen of the present invention.

FIG. 14 shows the production of pHIG2 (D segment containing plasmid).

FIG. 15 shows a fragment containing the human Jκ and human Cκ gene segments used to make the transgens of the invention.

FIG. 16 shows the structure of pEμ.

FIG. 17 shows the generation of pKapH.

FIG. 18 shows the construction of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin heavy chain locus.

FIG. 19 shows the construction of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin heavy chain locus.

FIG. 20 shows the construction of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin heavy chain locus.

FIG. 21 shows the construction of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin heavy chain locus.

FIG. 22 shows the construction of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin heavy chain locus.

FIG. 23 shows the generation of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin light chain locus.

FIG. 24 shows the generation of a positive-negative selection vector for functionally disrupting the mouse endogenous immunoglobulin heavy chain locus.

FIG. 25 shows the structure of a vector for targeting κ chain.

FIG. 26 shows the structure of a mouse heavy chain targeting vector.

FIG. 27 shows the structure of a mouse heavy chain targeting vector.

FIG. 28 shows a map of the vector pGPe.

FIG. 29 shows the structure of vector pJM2.

FIG. 30 shows the structure of vector pCOR1.

FIG. 31 shows pIGM1, pHC1 and pHC.
2 shows two transgene constructs.

FIG. 32 shows the structure of pγe2.

FIG. 33 shows the structure of pVGE1.

FIG. 34 shows the assay results of human Ig expression in pHC1 transgenic mice.

FIG. 35 shows the structure of pJCK1.

FIG. 36 shows the production of synthetic heavy chain variable regions.

FIG. 37 shows the heavy chain minilocus construct pIC.
₁ is a schematic representation of M ₁ , pHC1 and pHC2.

FIG. 38 shows the heavy chain minilocus construct pIGG1.
And a schematic representation of the kappa light chain minilocus constructs pKC1, pKVe1 and pKC2.

FIG. 39 depicts a strategy for rearranging a functionally rearranged light chain gene.

FIG. 40 represents serum ELISA results.

FIG. 41 represents the results of an ELISA assay on sera from 8 transgenic mice.

FIG. 42 is a schematic representation of plasmid pBCE1.

FIG. 43 shows that KLH-DNP (37A), KL
K by measuring IgG and IgM levels specific for H (37B) and BSA-DNP (37C)
Fig. 3 shows the immune response of the transgenic mouse of the present invention to LH-DNP.

FIG. 44 shows ELISA data demonstrating the presence of antibodies that bind to human carcinoembryonic antigen (CEA) and comprise human μ chains; each panel shows mice on the indicated days post-immunization. Figure 3 shows the reciprocal serial dilution from pooled serum samples obtained from.

FIG. 45 shows ELISA data demonstrating the presence of an antibody that binds to human carcinoembryonic antigen (CEA) and comprises a human γ chain; each panel shows mice on the indicated days post-immunization. Figure 3 shows the reciprocal serial dilution from pooled serum samples obtained from.

FIG. 46. 23 generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA).
Aligned variable region sequences of individual randomly selected cDNAs,
Shown relative to the germline transgene sequence (top row). In each row, the nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). The regions corresponding to the heavy chain CDR1, CDR2 and CDR3 are indicated. Nucleotides that are not encoded by germ cells are shown in lowercase. The deduced amino acid sequence is given below the nucleotide sequence using the conventional one-letter notation.

FIG. 47. 23 produced from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA).
Aligned variable region sequences of individual randomly selected cDNAs,
Shown relative to the germline transgene sequence (top row). In each row, the nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). The regions corresponding to the heavy chain CDR1, CDR2 and CDR3 are indicated. Nucleotides that are not encoded by germ cells are shown in lowercase. The deduced amino acid sequence is given below the nucleotide sequence using the conventional one-letter notation.

FIG. 48. 23 generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA).
Aligned variable region sequences of individual randomly selected cDNAs,
Shown relative to the germline transgene sequence (top row). In each row, the nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). The regions corresponding to the heavy chain CDR1, CDR2 and CDR3 are indicated. Nucleotides that are not encoded by germ cells are shown in lowercase. The deduced amino acid sequence is given below the nucleotide sequence using the conventional one-letter notation.

FIG. 49. 23 produced from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA) 23
Aligned variable region sequences of individual randomly selected cDNAs,
Shown relative to the germline transgene sequence (top row). In each row, the nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). The regions corresponding to the heavy chain CDR1, CDR2 and CDR3 are indicated. Nucleotides that are not encoded by germ cells are shown in lowercase. The deduced amino acid sequence is given below the nucleotide sequence using the conventional one-letter notation.

FIG. 50. 23 generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA).
Aligned variable region sequences of individual randomly selected cDNAs,
Shown relative to the germline transgene sequence (top row). In each row, the nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). The regions corresponding to the heavy chain CDR1, CDR2 and CDR3 are indicated. Nucleotides that are not encoded by germ cells are shown in lowercase. The deduced amino acid sequence is given below the nucleotide sequence using the conventional one-letter notation.

FIG. 51. 23 generated from mRNA obtained from lymphoid tissue of HC1 transgenic mice immunized with human carcinoembryonic antigen (CEA) 23
Aligned variable region sequences of individual randomly selected cDNAs,
Shown relative to the germline transgene sequence (top row). In each row, the nucleotide changes to the germline sequence are displayed above the changes in the deduced amino acid sequence (if any). The regions corresponding to the heavy chain CDR1, CDR2 and CDR3 are indicated. Nucleotides that are not encoded by germ cells are shown in lowercase. The deduced amino acid sequence is given below the nucleotide sequence using the conventional one-letter notation.

Figure 52 shows the data of Figure 40 in histogram format; deduced amino acid residue positions are shown as the ordinate (left is amino-terminal direction, right is carboxy-terminal direction) and sequence variation. Is shown as abscissa.

FIG. 53 shows the nucleotide sequence of a human DNA fragment designated vk65.5 containing the Vκ gene segment; the Vκ coding region deduced amino acid sequence is also shown; splicing sequences and recombination signal sequences (heptamer / (Nonamar) is shown in the box.

FIG. 54 shows the nucleotide sequence of a human DNA fragment designated vk65.8 that contains the Vκ gene segment; the Vκ coding region deduced amino acid sequence is also shown; splicing sequences and recombination signal sequences (heptamer / (Nonamar) is shown in the box.

FIG. 55 shows the nucleotide sequence of the human DNA fragment designated vk65.15 containing the Vκ gene segment; the Vκ coding region deduced amino acid sequence is also shown; splicing sequences and recombination signal sequences (heptamer / (Nonamar) is shown in the box.

FIG. 56 shows formation of light chain minilocus by homologous recombination between two co-injected overlapping fragments.

FIG. 57: CEA and non-CEA showing specificity of antigen binding
ELI for monoclonal antibody reactive with antigen
The result of SA is shown.

Figure 58: 10 C's amplified by PCR to amplify transcripts containing human VDJ and mouse constant region sequences.
The DNA sequence of DNA is shown.

FIG. 59: ELISA for various dilutions of sera obtained from mice bearing both the human heavy chain minilocus transgene and the human kappa minilocus transgene.
Results show that the mice were immunized with human CD4 and the data shown represent antibodies that are reactive with human CD4 and have human kappa, human mu, or human gamma epitopes, respectively. ing.

FIG. 60 shows the relative distribution of lymphocyte staining for human μ or mouse μ as determined by FACS for 3 mouse genotypes.

FIG. 61 shows the relative distribution of lymphocyte staining for human kappa or mouse kappa, as determined by FACS for the three mouse genotypes.

FIG. 62 shows the relative distribution of lymphocyte staining for mouse λ, as determined by FACS for 3 mouse genotypes.

FIG. 63 shows the relative distribution of lymphocyte staining for mouse λ or human κ as determined by FACS for 4 mouse genotypes.

FIG. 64 shows the amount of human μ, human γ, human κ, mouse μ, mouse γ, mouse κ, and mouse λ chains in unimmunized serum.

FIG. 65: Nonimmunized 0 of various genotypes
Figure 9 shows a scatter plot showing the amount of human μ, human γ, human κ, mouse μ, mouse γ, mouse κ, and mouse λ chains in the serum of 011 mice.

FIG. 66 shows anti-human CD4 titer in 0011 mice.

FIG. 67: Antibody containing human μ, human γ, or human κ chain in anti-CD4 antibody in serum collected at 3 or 7 weeks after immunization of 0011 mice with human CD4. Indicates the titer.

Figure 68: Human heavy chain mini locus transgene PHC
1 and PHC2, and light chain minilocus transgenes pKC1, pKCle and a schematic diagram of the light chain minilocus transgene created by homologous recombination between PKC2 and Co4 at the indicated sites.

Figure 69 shows a linkage map of the murine lambda light chain locus taken from Storb et al. (1989) supra. The stippled boxes represent pseudogenes. 1 schematically shows inactivation of the mouse λ locus by homologous gene targeting.

FIG. 70 schematically shows the structure of a homologous recombination targeting transgene for deleting genes such as heavy chain constant region genes.

Figure 71: " Immunoglobulin gene ", Honjo, T, Al
t, FW, and Rabbits TH (eds) Academic Press, NY (19
89) Shows a map of the BALB / c mouse heavy chain locus taken from p129. Structural genes are indicated by closed boxes in the upper line. The second and third lines indicate the restriction site with the displayed symbols.

FIG. 72 shows the nucleotide sequence of the mouse heavy chain locus α constant region gene.

Two bp in FIG. 73 mouse heavy chain locus J ₄ in a gene
The structure of the frame shift vector (plasmid B) for introducing a frame shift is shown.

FIG. 74 shows the isotype-specific response of transgenic animals during hyperimmunization. The relative levels of reactive human μ and γ1 are shown by a colorimetric ELISA assay (y-axis). We have shown that by intraperitoneal injection of CEA in Freund's Adjuvant three transgenic animals of male HC1 strain 57 (# 1991, # 235) at age 7-10 weeks in a homozygous JHD background.
6, # 2357). The figure depicts binding of 250-fold dilutions of pooled serum (collected prior to each injection) to CEA coated microtiter wells.

FIG. 75 shows expression of the transgene-encoded γ1 isotype mediated by class switch recombination. The genomic structure of the integrated transgene in two different human γ1 expressing hybridomas is μ and γ1.
Consistent with recombination between switch regions. 75
Figure 3 shows a Southern blot of PacI / SfiI digested DNA isolated from three transgene expressing hybridomas. From left to right: clone 92-09A-5H1-
5, human γ1 ⁺ / μ ⁻ ; clone 92-90A-4G2
-2, human γ1 ⁺ / μ ^-; clone 92-09A-4F
7-A5-2, human γ1 ⁻ , μ ⁺ . All three hybridomas were JH hemizygous for HCl-57 incorporation.
D-dissection is derived from a homozygous 7 month old mouse (mouse # 1991). The blot shows a 2.3 kb BglII spanning the 3'half of the human γ1 switch region.
/ SfiI DNA fragment hybridized with a probe derived from the DNA fragment. No switch product was found in the μ expressing hybridomas, but the two γ1 expressing hybridomas 92-09A-5H1-5 and 92-09A-4G2-2 showed 5.1 kb and 5.3 kb of PacI / SfiI, respectively. Includes switch products that result in fragments.

FIG. 76 is a diagram of two possible deletion mechanisms capable of producing a μ to γ1 class switch. The human μ gene is flanked by 400 bp direct repeats (σμ and Σμ) that can be recombined to delete μ. Class switching by this mechanism always produces a 6.4 kb PacI / SfiI fragment, whereas class switching by recombination between the μ and γ1 switch regions shows size variation between individual switch events. P between 4kb and 7kb
Generates an acI / SfiI fragment. The two γ1-expressing hybridomas examined in FIG. 75 appear to undergo recombination between the μ and γ1 switch regions.

Figure 77 shows a chimeric human / mouse immunoglobulin heavy chain produced by transswitch. The cDNA clone of the transswitch product is a mixture of spleen and lymph node RNA isolated from hyperimmunized HC1 transgenic-JHD mice (# 2357; see legend to FIG. 74 for animal and immunization plan descriptions). Was produced by reverse transcription and PCR amplification. The partial nucleotide sequences of 10 randomly picked clones are shown. Lowercase letters represent the encoded germline, uppercase letters represent nucleotides that cannot be assigned to known germline sequences. These may be somatic mutations, N nucleotides or truncated D segments. The double-sided type represents the mouse γ sequence.

FIG. 78 shows that the rearranged VH251 transgene undergoes somatic mutations in hyperimmunized ones.

79: IgG from CH1 strain 26 mice showing primary reaction to antigen in FIG. 78 and secondary reaction in FIG. 79.
Partial nucleotide sequence of heavy chain variable region cDNA clone. Germline sequences are shown at the top; nucleotide changes from germline are represented for each clone. One cycle shows identity with the germline sequence,
Uppercase letters indicate that no germline source was identified. The sequences are organized according to the use of the J segment. The germline sequence for each of the J segments is shown. Lower case letters within the CDR3 sequences represent identities to known D segments contained within the HCl transgene. The assigned D segment is represented at the end of each array. Unallocated array is N
It can be derived from the addition of regions or somatic mutations. Or, in some cases, they may simply be too short to distinguish a random N nucleotides from a known D segment. FIG. 78 Primary Response: 13 randomly picked VH251-γ1 cDNA clones.
Four-week-old female HCl strain 26-JHD mice (# 2
599) was injected once with KLH and complete Freund's adjuvant. Spleen cell RNA was isolated after 5 days.
The overall frequency of somatic mutations within the V segment is 0.0
6% (2 / 3,198 bp). Secondary response of FIG. 79:
13 randomly picked VH251-γ1 cDNA clones. Two-month-old female HCl strain 26-JHD mice (# 3204) were injected with HEL and Freund's adjuvant three times over one month (primary injection was complete adjuvant and booster immunizations at 1 and 3 weeks). Incomplete adjuvant); RN of spleen and lymph nodes after 4 months
A was separated. The overall frequency of humoral mutations within the V segment is 1.6% (52 / 3,198 bp).

FIG. 80 shows that extensive somatic mutations are restricted to the γ1 sequence: somatic mutations and class switches occur within the same population of B cells. Hyperimmunized against CEA (see Figure 68 for immunization schedule) H
Cl strain 57 transgenic-JHD mice (#
2357) VH2 isolated from spleen and lymph node cells
51 Partial nucleotide sequence of the cDNA clone. Figure 80: IgM: 23 randomly picked VH251-
μcDNA clone. Nucleotide sequence of a 156 bp segment containing residues around CDRs 1 and 2. Overall level of somatic mutation is 0.1% (5 / 3,744 bp)
Is.

FIG. 81: Randomly picked VH251-γ1 cDNA clone of IgG: 23. Nucleotide sequence of a segment containing CDRs 1-3 and surrounding residues. Overall frequency of somatic mutations within the V segment is 1.1%
(65 / 5,658 bp). For comparison with the μ sequence in FIG. 80; mutation frequency for the first 156 nucleotides is 1.1% (41 / 3,588 bp).
See legends in FIGS. 80 and 81 for explanation of symbols.

FIG. 82. VH51P1 and VH56P1 show extensive somatic mutations in non-immunized mice.
Female HC2 strain 25 unimmunized at 9 weeks of age
50 transgenic-JHD mice (# 525
Partial nucleotide sequence of IgG heavy chain variable region cDNA clone from 0). The overall frequency of somatic mutations in the 19VH56p1 segment is 2.2% (101 /
4,674 bp). The overall frequency of somatic mutations within a single VH51p1 segment is 2.0% (5/24
6 bp). See legend to FIGS. 78 and 79 for explanation of symbols.

FIG. 83. Double transgenic mice with an endogenous Ig locus analyzed contain human IgMκ positive B cells. FACS of cells isolated from spleens of 4 mice with different genotypes. Left column: control mouse (#
9944, 6 week old female, JH +/-, JCκ + /
-; Heterozygous wild-type mouse heavy and kappa-light chain loci,
Non-transgenic). Column 2: Human heavy chain transgenics (# 9877, 6 weeks old female JH-/-, J.
Cκ − / −, HC2 strain 2550 ±; HC homozygous for the disrupted mouse heavy and κ-light chain loci, HC
2 Semi-junction for transgene). Column 3: Human κ-
Light chain transgenic (# 9878, 6 weeks old female JH − / −, JCκ − / −, KCo4 strain 4437+;
Homozygous for the disrupted mouse heavy and kappa-light chain loci and hemizygous for the KCo4 transgene). Right column: double transgenic (# 9879, 6 week old female, JH-/-m JCκ-/-, HC2 strain 2550+, KCo4 strain 4437+; disrupted mouse heavy and κk-light chain loci. Homozygous for, and hemizygous for the HC2 and KCo4 transgenes). Upper row: mouse λ light chain (x axis) and human κ light chain (y
Axis) splenocytes stained for expression. Second stage: Human μ
Splenocytes stained for expression of heavy chain (x-axis) and human kappa light chain (y-axis), Stage 3: For mu mu heavy chain (x-axis) and murine kappa light chain (y-axis) expression. Spleen cells stained. Bottom: Histogram of splenocytes stained for expression of mouse B220 antigen (logarithmic fluorescence: x-axis: cell number: y
axis). For each of the two color plates, the relative number of cells in each of the displayed inlays is given as a percentage of the e-parameter gate based on propidium iodide staining and light scattering. The fraction of B220 + cells in each of the samples shown at the bottom is given as a percentage of the lymphocyte light scatter gate.

Figure 84 Secreted immunoglobulin levels in the serum of double transgenic mice. 18 individual HC2s homozygous for endogenous heavy and kappa-light chain locus disruptions
/ KCo4 mouse γ and λ from double transgenic mice and human μ, γ and κ. Mouse: (+) HC
2 lines 2550 (up to 5 HC2 copies per integration), KCo4 line 4436 (1 to 1 integration per install)
2 copies of KCo); (○) HC2 line 2550,
KCo4 line 4437 (up to 10 KCo4 copies per integration); (x) HC2 line 2550, KCo
4 lines 4583 (up to 5 KCo4 per installation
Copy); (□) HC2 strain 2572 (30-50 HC2 copies per integration, KCo4 strain 4437;
(△) HC2 system 5467 (20-3 for each installation)
0 HC2 copy, KCo4 line 4437.

FIG. 85 shows human antibody response to human antigens. FIG.
5: Primary response to recombinant human soluble CD4. Human IgM and human kappa light chain levels have been reported in pre-bleed (O) and post-immunization (-) sera from four double transgenic mice.

FIG. 86: Switch to human IgG occurs in vivo. To inhibit non-specific cross-reactivity 1.
Human IgG (circles) with peroxidase-conjugated polyclonal anti-human IgG used in the presence of 5 μ / ml extra IgE, κ and 1% normal mouse serum.
Was detected. Human kappa light chains (squares) were detected with peroxidase-conjugated polyclonal anti-human kappa reagent in the presence of 1% normal mouse serum. Representative results from one mouse (# 9344; HC2 strain 2550, KCo4 strain 4436) are shown. Each point represents the average of duplicate wells minus background absorption.

FIG. 87: Human PBL using hybridoma supernatant that discriminates human CD4 + lymphocytes from human CD8 + lymphocytes
2 shows a FACS analysis of

Figure 88: Human alpha in serum of transgenic mice
-Indicates CD4IgM anf IgG.

FIG. 89: α-human CD4 of transgenic mice
Hybridoma monoclonal, 2C11-8 was RPA
-Shows competitive binding experiments comparing TA and Leu-3A monoclonals.

Figure 90: I of cultured 2C11-8 hybridoma
Production data for g expression are shown. The transgenic mice of the invention are immunized with a human antigen selected from Tables B and C below. The transgenic mouse produces a human antibody that binds to the human antigen. B cells from immunized transgenic mice are used to generate hybridomas that secrete monoclonal antibodies against the selected human antigens.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 21/08 9358-4B // C12N 15/02 ZNA (C12N 5/10 C12R 1:91) ( C12N 5/00 B C12R 1:91)

Claims (41)

[Claims] 1. At least one of a homozygous pair of a functionally disrupted endogenous heavy chain allele, a homozygous pair of a functionally disrupted endogenous light chain allele, and a heterologous immunoglobulin heavy chain transgene. Transgenic mouse comprising one copy and at least one copy of a heterologous immunoglobulin heavy chain transgene, which immunizes with an antigen followed by an antibody response. 2. The functionally disrupted endogenous heavy chain allele is a J _H region homologous recombination knockout, and the functionally disrupted endogenous light chain allele is a J _k region homologous recombination knockout. And the heterologous immunoglobulin heavy chain transgene is an HC1 or HC2 human minigene transgene, and the heterologous light chain transgene is
The transgenic mouse according to claim 1, which is a KC2 or KCle human κ transgene and the antigen is a human antigen. 3. The transgenic mouse according to claim 1, wherein the antibody response includes a population of antibodies containing human μ chain-containing immunoglobulin and human γ chain-containing immunoglobulin. 4. The antibody response comprises a population of antibodies, the population comprising: -an antibody comprising heavy and light chains essentially composed of human sequence variable regions and human sequence constant regions; and 2. An antibody comprising a heavy chain basically composed of a human sequence variable region and a mouse sequence constant region.
The transgenic mouse according to 1. 5. The transgenic mouse according to claim 4, wherein the mouse sequence constant region is a mouse γ constant region. 6. The transgenic mouse of claim 1, wherein the heterologous antibody comprises a population of heterologous immunoglobulins that specifically bind to human CD4 with a dissociation constant of about 8 × 10 ⁷ M ^−1. . 7. HC1-26 +; JHD ++; JKD ++; KC2-1610 ++; HCl-2
6+; JHD ++; JKD ++; KC2-1610 +; HCl-26 +; JHD ++; JKD ++; KCle-
The transgenic mouse according to claim 2, having a genotype selected from the group consisting of 1527+; and HCl-26 +; JHD ++; JKD ++; KCle-1399 +. 8. HCl-26 +; JHD ++; JKD ++; KCle-1527 + and HC
l-26 +; JHD ++; JKD ++; having a genotype selected from the group consisting of KCle-1399 +, the antibody response, human μ chain-containing immunoglobulin, human γ chain-containing immunoglobulin and,
The transgenic mouse according to claim 2, which comprises a population of antibodies each comprising a chimeric immunoglobulin that comprises a heavy chain consisting essentially of a human variable region and a mouse constant region. 9. (1) A homozygous functionally disrupted endogenous heavy chain locus comprising at least one mouse constant region gene capable of transswitching, comprising a functional switch recombination sequence, and ( 2) A transgenic mouse comprising a genome comprising a human heavy chain transgene comprising a functional switch recombination sequence which can be rearranged to encode a functional human heavy chain variable region and which is capable of undergoing a transswitch. 10. The transgenic mouse of claim 9, further comprising a human light chain transgene that can be rearranged to encode a functional human light chain variable region and express a human sequence light chain. 11. The transgenic mouse of claim 9, further comprising a homozygous functionally disrupted endogenous light chain locus. 12. A serum comprising an antibody comprising a chimeric heavy and light chain consisting of a human sequence variable region encoded by a human transgene and a mouse constant region sequence encoded by an endogenous mouse heavy chain constant region gene. The transgenic mouse according to claim 11. 13. The transgenic mouse according to claim 12, wherein the serum further contains an antibody containing human heavy chains and light chains consisting essentially of human variable regions and human constant regions. 14. A transgenic mouse comprising serum having a detectable amount of a chimeric heavy chain encoded by a sequence generated by a transswitch between a human transgene and an endogenous mouse heavy chain constant region gene. 15. Producing a human sequence heavy chain at a first time point,
A transgenic mouse comprising B cells that transswitch to produce a chimeric heavy chain consisting of a human variable region and a mouse constant region at a second time point. 16. A chimeric antibody comprising a chimeric heavy chain comprising a human sequence heavy chain variable region, a mouse sequence heavy chain constant region and a mouse sequence heavy chain constant region is produced, and further, a human sequence heavy chain variable region and human sequence. A transgenic mouse containing B cells that produce an antibody containing a human heavy chain containing a heavy chain constant region. 17. The transgenic mouse of claim 16, wherein the chimeric antibody comprises a human sequence light chain. 18. The chimeric antibody is at least about 1 × 10 ^7.
18. The transgenic mouse of claim 17, which binds to a predetermined antigen (eg, immunogen) with an affinity of M- ¹ . 19. The transgenic mouse according to claim 18, wherein the predetermined antigen is human CD4 or human CEA. 20. Two human V _H gene segments, 8
One human D gene segment, six human J _H gene segments, one human J-μ enhancer, one human μ switch region, one fully human μ C _H gene, one human sterile promoter, one human γ switch Region, one fully human γC _H gene and a genome comprising a human heavy chain transgene containing a heavy chain 3 ′ enhancer, wherein the unplaced human heavy chain transgene has a mouse V _H
A gene segment, a mouse D gene segment, a mouse J _H gene segment, a mouse C _H gene, a mouse switch region, and a mouse heavy chain enhancer are absent, and the B lymphocytes of this mouse are unassigned by VDJ joining. Human heavy chain transgene is rearranged to produce the fully human μ or fully human γ _{C H} on the transgene.
Producing an in-frame joined VDJ gene encoding a heavy chain variable region that is expressed in a polypeptide bond to a constant region encoded by the gene,
Transgenic mouse. 21. The human heavy chain transgene comprises a 5.3 kb Hind III fragment of the human heavy chain locus containing the first exon of the preswitch sterile and the γ1 switch region, and the B lymphocytes are B of this transgenic mouse rearranged with the human heavy chain transgene.
In-frame joined VD- encoding heavy chain variable region expressed as human μ or human γ chain in lymphocytes
The transgenic mouse according to claim 20, which forms the J gene. 22. The transgene further comprises a 0.7 kb Xba I / Hind III fragment of the human heavy chain locus, the 0.7 kb Xba I / Hind III fragment being essentially immediately upstream of the 5.3 kb γ1 fragment. It is composed of a sequence flanking this fragment, and further upstream of the human heavy chain locus flanks 3.1 kb of Xba.
22. The transgenic mouse of claim 21, which comprises an I fragment. 23. The human heavy chain transgene comprises a human γ1 constant region containing a switch region associated with the exon and an exon associated with the sterile transcription, and a flanking sequence of about 4 kb upstream from the sterile transcription initiation site, And 5'CAG GAT
CCA GAT ATCAGT ACC TGA AAC AGG GCT TGC31 51GAG CAT
23. The transgenic mouse of claim 22, comprising a rat heavy chain 3'enhancer that can be PCR amplified with the oligonucleotide primer GCA CAG GAC CTG GAG CAC ACA CAGCCT TCC 3 '. 24. The human heavy chain transgene contains pHCl.
24. The transgenic mouse of claim 23, which comprises the NotI insert of 25. The transgenic mouse of claim 24, which comprises an intact germline copy of one of the NotI inserts of pHCl and expresses both human μ and human γ1 chains in serum. 26. The human heavy chain transgene undergoes an isotype switch and thus the frame-joined VDJ gene is first expressed within a peptide bond to the human mu constant region and then the transgenic mouse. 26. The transgenic mouse of claim 25, which is to encode a human heavy chain variable region expressed in a peptide bond to a human gamma constant region in B lymphocytes of E. coli. 27. A human V _H gene segment that expresses human μ and human γ1 chains in serum, and is joined in-frame to each human μ or human γ1 chain as a VDJ gene,
PHCl or pIGM1 No containing a variable region basically composed of a polypeptide sequence encoded by a human D gene segment and a human J _H gene segment
Transgenic mice containing an intact, integrated germline copy of the tI insert. 28. The transgenic mouse of claim 20, further comprising a functionally disrupted endogenous heavy chain locus lacking the mouse J _H gene segment. 29. An intact human heavy chain transgene which expresses in its serum an immunoglobulin chain which comprises a human or human γ1 constant region and is encoded by said human heavy chain transgene and which basically consists of a NotI insert of pHCl. Transgenic mouse having an integrated germline copy of. 30. A method for producing an antibody containing human immunoglobulin in the serum of a transgenic mouse, the step of immunizing the transgenic mouse according to claim 1 or 20 with a predetermined antigen and a body fluid. A method comprising collecting serum from this animal after an appropriate time for a sexual immune response. 31. A B cell of a transgenic mouse according to claim 1 or 20 comprising this B cell immunized with a predetermined antigen fused with a second cell capable of immortalizing the B cell. A hybridoma in which a monoclonal antibody containing a human heavy chain is regenerated in the hybridoma and the monoclonal antibody binds to the predetermined antigen. 32. The hybridoma of claim 31, wherein the predetermined antigen is a human antigen. 33. The hybridoma according to claim 32, wherein the human antigen is CEA, CD4 or NCA-2. 34. With an affinity of at least 1 × 10 ⁷ M ⁻¹ ,
32. The hybridoma of claim 31, wherein the monoclonal antibody binds a human antigen. 35. The hybridoma comprises a functionally disrupted mouse immunoglobulin allele.
The hybridoma according to 31. 36. The hybridoma of claim 34, wherein the monoclonal antibody binds human CD4 with an affinity of about 8 × 10 ⁷ M ⁻¹ . 37. A human monoclonal antibody regenerated by the hybridoma according to claim 31. 38. The human antigen is CEA, CD4 or NCA-.
38. The human monoclonal antibody of claim 37, which is 2. 39. The hybridoma of claim 31, wherein the predetermined antigen is human CD antigen. 40. The predetermined antigen is a human non-CD antigen,
The hybridoma according to claim 31. 41. The hybridoma according to claim 31, wherein the predetermined antigen is a human pathogen or an antigen of a human pathogen.