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WO1996018105A1 - Recepteur de lymphocyte t monocatenaire - Google Patents

Recepteur de lymphocyte t monocatenaire Download PDF

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Publication number
WO1996018105A1
WO1996018105A1 PCT/US1995/015696 US9515696W WO9618105A1 WO 1996018105 A1 WO1996018105 A1 WO 1996018105A1 US 9515696 W US9515696 W US 9515696W WO 9618105 A1 WO9618105 A1 WO 9618105A1
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WIPO (PCT)
Prior art keywords
cell receptor
cell
domain
single chain
chain
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PCT/US1995/015696
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English (en)
Inventor
Jack L. Strominger
Shan Chung
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The President And Fellows Of Harvard College
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Publication of WO1996018105A1 publication Critical patent/WO1996018105A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • T-cell receptors are recognition molecules present on the surface of T lymphocytes. Although closely related to antibody molecules, T-cell receptors have numerous properties which distinguish them from antibodies. One fundamental difference between T-cell receptors and antibody molecules is that antibody molecules exist in two alternative forms: cell surface molecules or soluble secreted molecules. Native T-cell receptors exists, however, only as cell surface molecules.
  • the T-cell receptors which are found most commonly on the surface of T-cells are comprised of two glycoprotein subunits which are referred to as the ⁇ and ⁇ chains. Both chains have a molecular weight of about 40 kDa and possess a variable and a constant domain.
  • the genes which encode the ⁇ and ⁇ chains are organized in a manner which is similar to antibody gene organization in that there are libraries of V, D and J regions from which the genes are formed by genetic rearrangement.
  • TCRs recognize antigen which is presented by an antigen presenting cell as a part of a complex with a specific self-molecule encoded by a histocompatibility gene.
  • the most potent histocompatibility genes are known as the major histocompatibility complex (MHC) .
  • MHC major histocompatibility complex
  • T-cell receptor which retains specificity for an antigenic peptide presented by an antigen presenting cell as a component of a complex including the MHC is useful in a variety of contexts which are discussed in greater detail below.
  • Several approaches have been employed to produce soluble, recombinant T-cell receptors.
  • the transmembrane/cytoplasmic regions of the ⁇ and ⁇ chains were replaced with sequences from lipid-linked proteins (GPI anchor) , the CD3-f chain or with immunoglobulin (Ig) heavy or light chains. Soluble T-cell receptors were recovered either as secreted proteins (Ig constructs) or obtained by enzymatic cleavage of the
  • GPI-anchor or the CD3-f chain All of these approaches rely, however, on the assembly of the heterodimer which is inefficient. In addition, high level expression of the human T-cell receptor ⁇ chain in transfected eukaryotic cells is not stable.
  • the present invention relates in one aspect to a single chain T-cell receptor which binds specifically to an MHC/peptide ligand.
  • the single chain T-cell receptor of the present invention has been demonstrated to be soluble in aqueous media.
  • the single chain T-cell receptor is a 3-domain single chain T-cell receptor comprising an ⁇ chain variable domain (V ⁇ ) , a ⁇ chain variable domain (V ⁇ ) and a constant domain.
  • a single chain T-cell receptor wherein the V ⁇ -J ⁇ domain of the ⁇ chain variable domain is linked to the N-terminus of the ⁇ chain variable domain by a flexible amino acid linker and the C-terminal region of the ⁇ chain variable domain is linked to the ⁇ chain constant domain (C ⁇ ) .
  • the invention in another aspect, relates to a self- signaling single chain T-cell receptor which binds specifically to an MHC/peptide ligand.
  • the self- signaling T-cell receptor is preferably comprised of: 1) a soluble T-cell receptor domain comprising an ⁇ chain variable domain, a ⁇ chain variable domain and a constant domain; and 2) a transmembrane and intracellular signaling domain from a transmembrane receptor.
  • the self-signaling T-cell receptor When displayed on the surface of a T-cell, the self-signaling T-cell receptor has the ability to transduce signal following specific binding to MHC/peptide ligand.
  • the invention also relates to nucleic acid molecules which encode the single chain T-cell receptors described above, expression vectors containing such nucleic acid molecules, and cells which carry expressible copies of such nucleic acid molecules.
  • the invention relates to methods for diagnosing viral infection in a patient. Disclosed are methods which employ both the single chain T-cell receptor, and the self-signaling embodiment of the single chain T-cell receptor.
  • FIG. 1 is a diagrammatic representation of T-cell receptor ⁇ - and /3-chain genes and various chimeric constructs which employs the following designations:
  • S—S disulfide bond
  • L leader
  • V variable segment
  • J joining segment
  • C constant region
  • TM transmembrane region
  • Cy cytoplasmic region
  • ATG start codon
  • Li 15-residue peptide linker containing three repeats of GGGGS
  • PI GPI domain of human placental alkaline phosphatase with the sequence --APPAGTTDAAHPGRSV ⁇ -ALLPLLAGTLLLL (SEQ ID NO. 3).
  • the ⁇ region contains transmembrane and cytoplasmic domains of the marine CD3 f chain starting at position 31 (Engel et al., Science 256 : 1318 (1992)).
  • the present invention is based on experiments which resulted in the synthesis of a single chain T-cell receptor which binds specifically to a predetermined MHC/peptide ligand which is presented by an antigen presenting cell (e.g, a macrophage) .
  • the MHC component of the MHC/peptide ligand can be Class I or Class II.
  • the term "single chain" is used to describe an amino acid copolymer having a single N-terminus and a single C-terminus. This single chain T-cell receptor is to be contrasted with the native T-cell receptor which is comprised of two glycoprotein subunits which are referred to as the ⁇ and ⁇ chains.
  • the single chain T-cell receptor of the present invention is soluble in an aqueous medium.
  • aqueous medium includes, for example, physiologically compatible buffered solutions. It has been determined, for example, that the single chain T- cell receptor of the present invention is soluble in phosphate buffered saline up to a concentration of 1 mg/ml.
  • the native T-cell receptor is comprised of two glycoprotein subunits which are referred to as the and ⁇ chains.
  • Each of the glycoprotein subunit chains comprise two domains, one variable and one constant.
  • the glycoprotein subunits are encoded by genes which are formed by rearrangement of segments (V, D and J segments ( ⁇ chain) , and V and J segments ( ⁇ chain) ) from libraries found in discrete chromosomal regions.
  • the single chain T-cell receptor is a 3-domain single chain T-cell receptor comprising a leader sequence and an ⁇ chain variable domain, a ⁇ chain variable domain and a constant domain.
  • Such a molecule can be prepared in a variety of ways. For example, conventional synthetic techniques may be employed to produce the desired amino acid copolymer from its component amino acids (see e.g., Houghton et al., Proc. Natl . Acad. Sci . USA 82 : 5135 (1985)).
  • DNA encoding the single chain T-cell receptor is generated at the genetic level by combining elements from isolated T-cell receptor genes by recombinant DNA techniques (see e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). In order to practice such methods it is first necessary to isolate nucleic acid which encodes the ⁇ and ⁇ chains of the T-cell receptor having the desired specificity.
  • DNA encoding the ⁇ and ⁇ chains which comprise a T-cell receptor which binds specifically with a predetermined epitope of the human myelin basic protein (MBP) when presented in association with an MHC Class II protein, is described in detail in the Examples which follow.
  • MBP myelin basic protein
  • the procedures specified in connection with the isolation of DNA encoding a T-cell receptor specific for an epitope of the human MBP are applicable to the isolation of T-cell receptor genes which encode a T-cell receptor which binds specifically to any predetermined MHC/peptide ligand.
  • an initial step in obtaining DNA encoding T-cell receptor genes which encode a T-cell receptor which binds specifically to any predetermined MHC/peptide ligand is to isolate an antigen specific T- cell clone from a mammal (preferably a human) which is known to possess the antigen of interest in its tissue.
  • a mammal preferably a human
  • the predetermined MHC/peptide ligand contains a peptide representing a T-cell epitope from an HIV virus, an individual infected with the virus would be selected.
  • a peripheral blood cell preparation is obtained from the selected individual by conventional techniques (Wucherpfennig et al., Science 248 : 1018 (1990); Wucherpfennig et al., J . Immunol . 152 : 5581 (1989)).
  • the peripheral blood cell population is stimulated by contacting the cells with the predetermined antigen using conventional techniques intended to expand the population of T-cells which bind specifically to the antigen of interest.
  • stimulation is carried out with a purified peptide known to contain a T-cell epitope, or with an enzymatically degraded protein preparation (e.g., a partial trypsin digest) comprising a mixture of peptides.
  • This stimulation procedure enriches the T-cell population for those T-cells which specifically bind to an epitope of the antigen of interest.
  • a clonal population of cells encoding a single T-cell receptor specific for the antigen of interest is isolated.
  • DNA encoding the ⁇ and ⁇ chains which comprise a T- cell receptor is then prepared from the clonal T-cell population using spliced mRNA as template.
  • spliced mRNA One way in which this can be accomplished is through the construction of a cDNA library from a clonal cell culture of the type described above.
  • the isolation of mRNA and the synthesis and cloning of double stranded cDNA molecules encoded by the mRNA is a routine procedure (see e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) ) .
  • the cDNA library is then screened using probes specific for the constant region of the T-cell receptor ⁇ or ⁇ chains (see e.g., Davis et al., Nature 334 : 395 (1988)).
  • a variety of alternative approaches can also be used to prepare DNA encoding the ⁇ or ⁇ chains from mRNA isolated from a cell culture demonstrating the desired binding specificity.
  • Such methods include the use of a standard PCR protocol using first-strand cDNA template prepared from the isolated mRNA. The latter method is, in fact, the method which was used to generate DNA encoding the ⁇ chain in the experiments described in detail in Example 1 which follows.
  • Yet another alternative is the anchored PCR technique described by (Loh et al., Science 243 : 217 (1989)). Using standard recombinant DNA techniques, a 3-part single chain T-cell receptor of the type described herein can be assembled from domains isolated from the ⁇ and ⁇ chain DNA.
  • a leader sequence must be included for proper intracellular processing of the single chain T-cell receptor.
  • a leader sequence In genomic DNA, a leader sequence is always linked to the DNA encoding the ⁇ chain variable domain such that in the encoded product, the leader sequence is located at the N-terminus. Therefore, in the T-cell receptor of the present invention, a leader sequence is located at the N-terminus of the molecule and is considered to be an element of the V ⁇ domain. It will be recognized, however, that although a specific leader sequence is linked to a specific V ⁇ domain in genomic DNA, leader sequences can be exchanged using recombinant DNA techniques without a detrimental affect on the intracellular processing of the encoded fusion.
  • the V ⁇ - J ⁇ domain of the ⁇ chain variable domain is linked to C- terminus of a leader sequence.
  • the V ⁇ -J ⁇ domain of the o chain variable domain is also linked to the N-terminus of the ⁇ chain variable domain by a flexible amino acid linker and the C- terminal region of the ⁇ chain variable domain is linked to the ⁇ chain constant domain.
  • these domains which must be present at a minimum, it may be possible to add additional segments without interfering with the essential properties of the encoded molecule.
  • the identity of individual amino acids in the amino acid linker, and the number of amino acids which comprise the amino acid linker are variable.
  • the purpose of this linker is to provide sufficient flexibility within the molecule to enhance the binding characteristics of the soluble T-cell receptor.
  • a linker comprised of about 10 to 30 amino acid residues would be considered sufficient.
  • glycine is preferred in light of the fact that a glycine polymer exhibits increased flexibility relative to other mono-amino acid polymers due to the fact that it lacks j8-carbons.
  • amino acids which tend to increase solubility in an aqueous solution are preferred (e.g. , serine, glutamine, aspartic acid, arginine, etc.).
  • T-cell receptor variable region i.e., the portion of the variable region other than the CDRs
  • CDR complimentarity determining region
  • the scope of the present invention is intended to encompass derivatives of the T-cell receptor of the invention wherein the addition of the derivatizing group would not be expected to substantially alter the binding characteristics of the soluble T-cell receptor.
  • modifications include, for example, the addition of a radiolabel group or the conjugation of a toxin molecule.
  • conservative substitution refers to the substitution of a second amino acid residue for a first amino acid residue, both residues having an R group exhibiting substantially similar chemical properties. Standard biochemistry textbooks provide groupings of amino acid residues according to substantial similarity in R group chemistry.
  • DNA encoding the single chain T-cell receptor produced in the manner described above, can be expressed in eukaryotic or prokaryotic cells by placing the DNA under the control of regulatory sequences appropriate for expression in the cell type of interest, and introducing the construct into the cell type of interest. Regulatory sequences appropriate for expression can exert their influence at many levels including, for example, the transcriptional, translational and post-translational levels.
  • the DNA encoding the single chain T-cell receptor, together with appropriate regulatory sequences are typically carried within a larger DNA molecule (often referred to as an expression construct) which facilitates the introduction and expression of the expressible nucleic acid encoding the single chain T-cell receptor.
  • An extrachromosomal plasmid is an example of an expression vector which could be used to facilitate prokaryotic expression.
  • Modified eukaryotic viral DNA capable of integrating into the chromosome of a host-cell is an example of an expression vector which could be used to facilitate eukaryotic expression.
  • the selection of expression vectors and appropriate regulatory signals is highly dependent upon the particular application. Given a particular experimental goal, the choice of an appropriate vector and appropriate regulatory sequences would be clear to one of skill in the art based on fundamental and well established principles.
  • the present invention relates to a self-signaling single chain T-cell receptor which binds specifically to an MHC/peptide ligand when presented by an antigen presenting cell.
  • the native T- cell receptor is not a self-signaling molecule. Rather, the signaling function of the native T-cell receptor is mediated by a number of ancillary proteins. Briefly, there are two major classes of T-cells: cytotoxic T lymphocytes (CTLs) and helper T (T H ) lymphocytes. CTLs have the ability to lyse cells which display a foreign antigen on their cell surface (in association with an MHC component) .
  • CTLs cytotoxic T lymphocytes
  • T H helper T lymphocytes
  • T H lymphocytes recognize the degradation products of foreign antigens and, in response, secrete stimulatory factors which induce response by other immune system components.
  • CTLs can be distinguished from T H lymphocytes based on the presence of certain surface proteins. More specifically, CTLs typically have the CD8 protein on their cell surface while T H lymphocytes typically have CD4 protein on their cell surface.
  • the interaction between the antigenic peptide/MHC complex on an antigen presenting cell, and the T-cell receptor on the T-cell, is thought to involve either a class I MHC-CD8 interaction or a class II MHC-CD4 interaction.
  • a variety of other proteins also participate in the effector signaling process which results ultimately in the secretion of protein factors by both classes of T-cells (the secreted protein factors include, for example, factors which induce cell lysis in the case of CTLs, and lymphokines in the case of T H lymphocytes) .
  • T-cell receptor proteins which participate in the signaling process
  • proteins which participate in the signaling process include, for example, the CD3 complex of proteins which are tightly bound to the T-cell receptor, as well as a tyrosine kinase protein encoded by the lck gene which is bound to both CD4 and CD8.
  • CD3 complex of proteins which are tightly bound to the T-cell receptor as well as a tyrosine kinase protein encoded by the lck gene which is bound to both CD4 and CD8.
  • tyrosine kinase protein encoded by the lck gene which is bound to both CD4 and CD8.
  • the self-signaling T-cell receptor of the present invention differs from the native form in that a signaling domain is incorporated as a component of the fusion protein.
  • the self-signaling T-cell receptor of the present invention includes all of the elements of the single chain T-cell receptor described above (i.e., an a chain variable domain, a ⁇ chain variable domain and a constant domain) as well as a transmembrane and intracellular signaling domain from a transmembrane receptor.
  • a leader sequence is essential for intracellular processing and is located at the N-terminus of the molecule.
  • the C-terminus of the leader sequence is linked to the N-terminus of the V ⁇ -J ⁇ domain of the ⁇ chain variable domain. This, in turn is linked at its C-terminus to the N-terminus of the ⁇ chain variable domain by a flexible amino acid linker.
  • the C-terminal region of the ⁇ chain variable domain is linked to the ⁇ chain constant domain and the C-terminal region of the ⁇ chain constant region is linked to the N-terminal region of the transmembrane and intracellular signaling domain from a transmembrane receptor.
  • construction of the self-signaling single chain T-cell receptor is preferably carried out at the genetic level although alternative methods are well known in the art.
  • DNA encoding the transmembrane/signaling domain of the transmembrane receptor is also isolated according to well established experimental steps protocols including, for example, standard PCR.
  • the identity of the transmembrane receptor from which the transmembrane and intracellular signaling domain is derived depends upon the nature of the signal which is preferred. For example, if it is desired that self-signaling T-cell receptor is to be displayed on the surface of a T-cell with the ultimate goal being the stimulation of a response which mimics the T-cell response in vivo (e.g., secretion of protein factors such as lymphokines (T H lymphocytes) or factors which induce cell lysis (CTLs) ) , the preferred transmembrane and intracellular domains are derived from the CD3 f chain.
  • T H lymphocytes lymphokines
  • CTLs cell lysis
  • T-cell clone having: 1) a predetermined MHC/peptide ligand specificity; and 2) the ability to mimic activity of cytotoxic T lymphocyte following binding to the MHC/peptide ligand offers the opportunity for a new method of therapeutic intervention which is discussed in greater detail below.
  • transmembrane/signaling domains can be used. Based on previous work with chimeric transmembrane receptors, it would be predicted with a high degree of certainty that the fusion of the extracellular domain of the single chain T-cell receptor of this invention, with the transmembrane/signaling domain of virtually any transmembrane receptor, would result in the production of a chimeric single chain T- cell receptor in which the signaling function would be operable following specific binding of the extracellular domain of the single chain T-cell receptor to the MHC/peptide ligand for which it is specific.
  • tyrosine kinase receptor the intracellular and transmembrane domains of a tyrosine kinase receptor can be used.
  • Many transmembrane tyrosine kinase receptors have been identified (for reviews see, e.g.. Hanks, Current Opinion in Structural Biology 1 : 369 (1991) and Pawson and Bernstein, Trends in Genetics 6 : 350 (1990)).
  • Such a receptor when stimulated, functions to phosphorylate intracellular protein targets.
  • an increase in intracellular phosphorylation could be detected using specific antibodies (i.e., anti- phosphotyrosine antibodies such as antibody 4G10 which is available commercially from Upstate Biotechnology Incorporated) thereby providing a positive indication of specific binding of the self-signaling T-cell receptor to its MHC/peptide ligand binding partner.
  • specific antibodies i.e., anti- phosphotyrosine antibodies such as antibody 4G10 which is available commercially from Upstate Biotechnology Incorporated
  • Such a detection method would be useful, for example, in connection with a diagnostic embodiment of the present invention which is described below.
  • the self-signaling single chain T-cell receptor is expressed in a T lymphocyte which is then clonally expanded.
  • any T lymphocyte can be employed as a host-cell for expression of the self- signaling T-cell receptor.
  • expression in a cytotoxic T lymphocyte is preferred.
  • T lymphocytes to be used as host-cells can be purified from the peripheral blood cells of an individual.
  • established T lymphocyte tumor cell lines can be employed. Many such tumor cell lines have been reported in the literature. Expression is accomplished by the introduction of an expressible genetic construct carrying DNA encoding the self- signaling single chain T-cell receptor into the T lymphocyte. As discussed previously, the ability to design and synthesize such an expressible genetic construct is a matter of routine experimentation given the current state of the art.
  • the single chain T- cell receptor and related compositions are useful in a method for diagnosing viral infection in a patient.
  • a single chain T-cell receptor which binds specifically to an antigenic peptide from the virus when presented by an antigen presenting cell in association with an MHC is produced as described previously.
  • the soluble T-cell receptor may be labeled with a reporter group (e.g., a radiolabeling group) .
  • a peripheral blood cell preparation from the individual to be tested for viral infection is then incubated with the soluble single chain T-cell receptor under conditions appropriate for binding of the soluble single chain T-cell receptor to the MHC/peptide ligand for which it is specific.
  • Such conditions include, for example, incubation in a physiologically compatible buffer at a temperature of about 4° C. Following an appropriate incubation period (e.g., 30 minutes) the cells are pelleted and resuspended in a wash solution to remove labeled single chain T-cell receptor which is not specifically complexed with an MHC/peptide ligand on the surface of antigen presenting cells.
  • the amount of radiolabel associated with the cellular fraction is determined. This determination can be made using a variety of standard techniques depending upon the identity of the label employed. Values determined in this manner are then compared with values determined in an otherwise identical incubation using a peripheral blood cell preparation from an individual known to be noninfected with the virus of interest. Viral infection is indicated by the detection of substantially greater signal (i.e., at least about 2- fold) from the peripheral blood cell preparation from the individual to be tested for viral infection when compared with the signal detected using the peripheral blood cell preparation from the individual known to be noninfected.
  • the method of diagnosing viral infection discussed above can also be modified to employ a self-signaling T- cell receptor of the type described above.
  • this method of diagnosis it is not necessary that the signal produced by receptor binding to the MHC/peptide ligand result in a T-cell response which mimics the T-cell response n vivo (i.e., secretion of protein factors). Rather, all that is required is that specific binding of the self-signaling single chain T-cell receptor to the MHC/peptide ligand result in the generation of a specific and detectable signal.
  • the transmembrane/signaling domain of the self-signaling T- cell receptor can be selected from virtually any transmembrane receptor possessing both a transmembrane and a signaling domain. It will be recognized, of course, that the transmembrane/signaling domain from a transmembrane receptor known to participate in a signaling process which stimulates a T-cell response which mimics the T-cell response in vivo can be used.
  • An example of such a transmembrane receptor is the CD3 f-chain.
  • DNA encoding the self-signaling single chain T-cell receptor is introduced in expressible form into a mammalian T-cell. When incubated under conditions appropriate for metabolic activity, the self-signaling single chain T-cell receptor is expressed and displayed on the surface of the host-cell.
  • a peripheral blood cell preparation from an individual to be tested for infection is provided.
  • the peripheral blood cell preparation is subsequently fixed in solution using a fixing agent (e.g. , paraformaldehyde) .
  • a fixing agent e.g. , paraformaldehyde
  • fixing the antigen presenting cell ensures that any cytokine secretion subsequently detected is secreted from the T-cell.
  • the fixed cells and the cells bearing the self-signaling single chain T-cell receptor are incubated together under conditions appropriate for binding of a T-cell receptor to the MHC/peptide target for which it is specific.
  • the self-signaling single chain T-cell receptor to the MHC/peptide ligand for which it is specific results in a triggering of the signaling domain of the self-signaling receptor.
  • Signal is then detected by conventional techniques. For example, if the signaling domain is from a tyrosine kinase receptor, signal is detected using anti-phosphotyrosine antibodies. If, on the other hand the signaling domain is from a transmembrane receptor having the properties CD3 f-chain, signal is detected for example by the detection of secreted lymphokine (e.g., IL-2) using appropriate immunological reagents.
  • secreted lymphokine e.g., IL-2
  • the level of signal determined is then compared with levels determined in an otherwise identical experiment using a peripheral blood cell preparation from an individual known to be noninfected.
  • the soluble single chain T-cell receptor of the present invention (and the self- signaling counterpart) can be used in a therapeutic context. Soluble T-cell receptors with proper specificity, either alone or with another functional group (such as toxins) can be used to block specific immune response or target malignant or virus-infected cells.
  • the self-signaling single chain T-cell receptor when expressed in a suitable host-cell, can be applied to regulate specific immune response or to target malignant or virus-infected cells.
  • the single chain design of the T-cell receptors of this invention will also facilitate the construction of T-cell receptor phage libraries similar to those made for single chain antibodies (Clackson et al, Nature 352 : 624 (1992)).
  • the success in producing a bacterially- expressed three-domain single chain design supports this application.
  • Such libraries are powerful tools for the isolation of T-cell receptors with defined specificity and/or high affinity.
  • the present example discloses studies in which different single chain T-cell receptor designs were evaluated in transfected eukaryotic cells with respect to surface expression of T-cell receptor molecules, proper folding and recognition of the appropriate MHC/peptide ligand.
  • a single-chain three-domain construct (V ⁇ -linker-VjS-CS) was stably expressed on the cell surface when linked to a glycosyl- phosphatidylinostol (GPI) anchor and recognized by a conformation dependent Ab specific for the VS17 segment.
  • GPI glycosyl- phosphatidylinostol
  • T-cell receptor ⁇ and ⁇ chains were prepared from mRNA of 2H9 cells with Superscript reverse transcriptase (BRL) and an oligo(dT) primer (Sigma) and were amplified by PCR using Vent DNA polymerase (New England Biolabs) and primers 5'- GCTCGAGGCGGCGATGGAAACTCTCCTGGGAGT-3' (A5) (SEQ ID NO.
  • a Ban I site was engineered after the fifth amino acid residue beyond the last cysteine by oligonucleotide-directed utagenesis. The region 3' of the Ban I site was then replaced with a Ban 1-Not I fragment encoding the GPI signal domain from the human placental alkaline phosphatase.
  • variable domains of the 2H9 T- cell receptor ⁇ and ⁇ chains were prepared by PCR using primers A5 and 5'-CAGAGCTCACGGATGAACAATAAGGCTGGT-3' (SEQ ID NO.
  • 5'GTGGGAGATCTCTGCTTCTGATGGCTCAAAC (SEQ ID NO. 11) and B5 for the V ⁇ domain in AB-PI-2
  • 5'- CACGGATCCCCGTCTGCTCTACCCCAGGC (SEQ ID NO. 12) and B5 for the V ⁇ and C ⁇ domains in ABC-PI
  • CACGGATCCCCGTCTGCTCTACCCCAGGC-3' (SEQ ID NO. 13) and B5 for the V ⁇ and C ⁇ domains in ABC-f.
  • the cDNA encoding the transmembrane and cytoplasmic domains of murine CD3 f chain (Engel et al., Science 256 : 1318 (1992)) was employed. Convenient restriction sites were engineered at the end of each fragment to aid in the assembly of the construct.
  • the linker was a 15-amino acid motif of GGGGS repeated three times (Huston et al., Int . Rev. Immunol . 10 : 195 (1992)) with Sac I at the 5' end and -EcoRV at the 3*" end.
  • ⁇ -PI was cloned into pBJ-neo, which carries the G418-resistance gene.
  • ⁇ -PI was cloned into pCEP-4 (Invitrogen) , which bears the hygromycin-resistance gene. All constructs were verified by multiple restriction digests and by sequencing with the Sequenase kit (United States Biochemical) .
  • Recombinant T-cell receptor molecules were generated by using the T-cell receptor ⁇ - and 3-chain sequences of the human myelin basic protein (MBP) specific T-cell clone Hy.2H9 (Wucherpfennig et al..
  • This clone T-cell receptor is composed of the V ⁇ 3.1 and V317.1 segments and is specific for the immunodominant MBP peptide MBP-(85-99) in the context of HLA-DR2 (DRA, DRB1*1602) (Wucherpfennig et al., J. Immunol . 152 : 5581 (1994)).
  • Usage of the V017.1 segment allowed the proper folding of recombinant T-cell receptors to be probed using the superantigen SEB (Choi et al., Proc . Natl . Acad . Sci .
  • ⁇ Fl and 0F1 recognize nonconformational epitopes located in the C region of the T-cell receptor ⁇ and ⁇ chains, respectively.
  • Surface expression of the T-cell receptor ⁇ chain is independent of heterodimer formation and assembly of the CD3 complex (Lin et al., Science 249 : 677 (1990)).
  • high-level expression of ⁇ -PI was confirmed by staining with 3F1.
  • V3l7-specific Cl epitope is conformational and dependent on the proper pairing of T-cell receptor ⁇ and ⁇ chains and can therefore be used to assess the proper folding of recombinant T-cell receptors bearing a V017 sequence.
  • C ⁇ domain was added to the sc construct.
  • a complete C ⁇ domain should provide enough distance for the Va-V ⁇ domains to be expressed on the cell surface and, more importantly, should allow surface expression to be monitored with another antibody, jSFl (Brenner et al., J. Immunol . 138 : 1502 (1987)).
  • This three-domain sc T-cell receptor was constructed by extending the T- cell receptor S-chain sequences to the residue right before the last cysteine (the sixth cysteine) , which was then fused to the GPI domain. The last cysteine was deleted to prevent dimerization between C ⁇ domains.
  • the molecule could be efficiently cleaved from the cell surface with PI-PLC.
  • Soluble three-domain single chain T-cell receptor was purified from transfectants after PI-PLC cleavage followed by affinity chromatography using the 3F1 antibody. More specifically, after transfection and G418 selection (Engel et al. Science 256 : 1318 (1992)), cells expressing a high level of GPI-linked three-domain single chain T-cell receptor (ABC-PI) were isolated by three rounds of sorting. The resulting cells were grown in spinner culture to a density of 10 6 per ml and harvested by centrifugation.
  • the pellet was washed twice with phosphate-buffered saline (PBS) and resuspended in PBS containing 2 mM Pefabloc protease inhibitor (•••) (Centerchen, Inc., Stamford, CT) to a density of 5 X 10 7 per ml with PI-PLC (Sigma) added at 1 unit/ml. Cells were incubated at 37°C for 1 hr with constant rocking. The supernatant was collected by centrifugation and by passage through a 0.45- ⁇ m filter and applied to a column of Acti-gel (•••) (Sterogen, Inc., Arcadia, CA) with immobilized 0F1.
  • the column was washed with 10 volumes of PBS and the soluble T-cell receptor was eluted with 0.15 M glycine (pH 2.8). Fractions were immediately neutralized with 0.1 volume of saturated Tris. The soluble T-cell receptor was then dialyzed against > 100 volumes of PBS at 4°C with at least four changes and concentrated to 0.5 mg/ml by vacuum dialysis against PBS. Five micrograms of purified soluble three-domain single chain T-cell receptor was analyzed by SDS/PAGE under reducing conditions. The purified three-domain single chain T-cell receptor appeared as multiple bands at 50-70 kDa after SDS/PAGE.
  • the observed heterogeneity of single chain T- cell receptor is probably the result of variable glycosylation; its polypeptide size calculated from amino acid composition is 40 kDa.
  • the structural integrity of the three-domain single chain T-cell receptor was verified by a two-antibody ELISA. The molecules were first captured by the 0F1 antibody immobilized to the plate and then assessed for reactivity with the Cl antibody.
  • the single chain T-cell receptor from the eukaryotic system gave 10-20 times higher Cl reactivity.
  • the purified three-domain single chain T-cell receptor was stable and could be stored in PBS at 4°C for months without significant loss of Cl reactivity.
  • a self- signaling single chain T-cell receptor was produced by replacing the GPI domain with the transmembrane and cytoplasmic domains of the CD3 ⁇ chain. These regions have been shown to be sufficient for signal transduction when its extracellular fusion partner is crosslinked by an antibody or by the proper ligand (Engel et al. Science 256 : 1318 (1992); Irving et al. , Cell 64 : 891 (1991); Romeo et al., Cell 64 : 1037 (1991)).
  • a linker containing a thrombin cleavage site was inserted into the junction of three-domain single chain T-cell receptor and the f domain.
  • the construct (ABC-f) was transfected into BW " cells and the rat basophilic leukemia cell line RBL-2H3 (RBL) (Engel et al. Science 256 : 1318 (1992)), and the populations displaying high-level expression of three- domain single chain T-cell receptor were isolated by three rounds of cytofluorometric sorting using the antibody ⁇ Tl .
  • the ABC-f-transfected cells were first stimulated with various antibodies to confirm the self- signaling nature of this recombinant molecule.
  • ABC-f-transfected BW5147 / 8 ⁇ ' jS " cells (5 X 10 4 per well) were cultured in a 96-well round-bottom plate to which various antibodies had been immobilized (1 ⁇ g per well) .
  • the supematants were collected after 24 hr and interleukin 2 (IL-2) production was assessed in a bioassay using an IL-2- dependent-cell line (CTLL) and the CellTiter-96 nonradioactive proliferation assay (Promega) .
  • IL-2 interleukin 2
  • ABC-f transfectants displayed a concentration- dependent response toward SEB when the superantigen was presented by transformed B-cell lines with high-level expression of DR1 (DRA, DRB1*0101; cell line LG2) or DR2 (DRA, DRB1*1602; cell line 9016).
  • DR1 DRB1*0101; cell line LG2
  • DR2 DRB1*1602; cell line 9016
  • DRB1*1601 and DRB1*1602 differ only at position 67 in the D-R/Sl domain; this T-cell receptor contact-residue substitution does, however, abolish recognition of the peptide by the parent T-cell clone (Wucherpfennig, et al, J. Immunol . 152 : 5581 (1994)).
  • the ABC-f transfected BW " cells secreted IL-2 in response to peptide-pulsed 9016 cells, but not to peptide-pulsed 9009 cells. Similar results were obtained with RBL transfectants, as serotonin release was dependent on the concentration of the MBP peptide used to pulse 9016 cells. The signal appeared to be weak when compared with antibody stimulation.
  • T-cell receptor could be obtained from the ABC-f transfectants by thrombin cleavage and affinity purification.
  • T-cell receptor ⁇ and ⁇ chain sequences of the human yelin basic protein (MBP) specific T-cell clone Hy.2H9 as described in Example 1.
  • MBP human yelin basic protein
  • This T-cell receptor is composed of the V ⁇ 3.l and Vj8l7.l segments and is specific for the immunodominant MBP(85- 99) peptide in the context of HLA-DR2 (DRA, DRB1*1602) .
  • cDNAs for the T-cell receptor ⁇ and ⁇ chains of the Hy.2H9 cells were prepared as described in Example 1.
  • DNA fragments corresponding to the variable or constant domains of the ⁇ and ⁇ chains were prepared by PCR using cDNA template with convenient restriction sites engineered at the ends of each fragment.
  • the ⁇ and ⁇ - chain domains were connected by a 15-residue peptide linker with the primary sequence of GGGGS GGGGS GGGGS (SEQ ID NO. 14) (Huston et al., Proc . Natl . Acad . Sci . USA 85: 5879 (1988)).
  • the assembled single chain T-cell receptor was cloned in frame into a bacterial expression vector with PelB signal sequence and an isopropyl j8-D- thiogalactopyronoside (IPTG) inducible Tac promoter.
  • IPTG isopropyl j8-D- thiogalactopyronoside
  • E. coli strain BMH transformed with the single chain T-cell receptor plasmid was grown in Luria broth containing 15 ⁇ g/ml tetracycline and 2% glucose (w/v) at 30°C to a density at A ⁇ of 0.5 to 1.0. Cells were then pelleted by centrifugation and washed once with Luria broth. The cells were resuspended in fresh Luria broth containing 1 mM IPTG, grown for a further 8- 12 hours at 37°C before being harvested by centrifugation.
  • the cell pellet was resuspended in 10 ml/3g cell paste of 50 mM Tris-HCl (pH 8.0)/l mM EDTA/lysozyme (1 mg/ml)/l mM phenylmethylsulfonyl fluoride (PMSF) and incubated on ice for 30 min.
  • DNase and MgCl 2 were added to final concentrations of 10 ⁇ g/ml and 10 mM respectively and the suspension was incubated for 30 min at room temperature.
  • EDTA was added to a concentration of 20 mM and the suspension was subjected to 2 cycles of freeze/thaw. An additional 20 mM MgCl 2 was added to the suspension and incubated at room temperature for a further 15 min.
  • the cell suspension was centrifuged at 10,000 xg for 30 min at 4°C. The supernatant was removed and the cell pellet containing inclusion bodies was washed twice with 0.5% Triton x-100/50 mM Tris-HCl (pH 8.0)/l00 mM NaCl/0.1% sodium azide. After a final wash in 50 mM Tris-HCl (pH 8.0)/100 mM NaCl, the pellet containing inclusion bodies was solubilized in 8M urea/100 mM NaHPO ⁇ /lO mM Tris-HCl (urea buffer) (pH 8.0).
  • the suspension was centrifuged at 10,000 xg for 30 min, and the supernatant was loaded onto a Ni-NTA column (Qiagen) and washed with 10 volumes of urea buffer (pH 8.0) and then 5 volumes of urea buffer (pH 6.3). Protein was eluted in fractions from the column in 2 volumes of urea buffer (pH 4.5). Protein content was determined by a Coomassie blue protein assay, and fractions containing proteins were pooled and diluted in refolding buffer to a protein concentration of 50 ⁇ g/ l.
  • the final refolding buffer contained 3M urea/50 mM Tris-HCl (pH 8.0)/l mM EDTA/0.3 mM oxidized glutathione/3 mM reduced glutathione/1 mM PMSF.
  • the refolding solution was incubated at 4°C with gentle mixing for 48 hours and then dialyzed against 100 volume of PBS at 4°C with at least 4 changes.
  • Refolded protein was first concentrated in an Amicon stir cell with a YM10 membrane to a volume of 25-50 ml, then further concentrated by vacuum dialysis against PBS.
  • Binding was detected with streptavidin- conjugated alkaline phosphatase (Sigma) after the addition of substrate (Bio-rad) . Absorbence at 410 nm was determined using a 96-well plate reader. For the direct-binding ELISA, soluble T-cell receptors in serial dilution were immobilized on the plate. After the blocking step, Cl antibody was added to the wells followed by an alkaline phosphatase conjugated anti- mouse immunoglobulin antibody. The binding was measured after the addition of the substrate as described above.
  • the present example relates to the expression of a three-domain single chain T-cell receptor in bacteria.
  • the final construct for bacterial expression bears a three-domain design similar to its eukaryotic counterpart described in Example 1, with a few modifications. More specifically, the N-terminal endogenous ⁇ -chain leader sequence was deleted, a histidine tag was added to the C-Terminus for convenient purification with Ni-NTA resin and additional amino acid residues created convenient restriction sites at these junction regions.
  • This three-domain single chain T-cell receptor was cloned into a bacterial expression vector in frame with a N-terminal PelB signal sequence.
  • the presence of the PelB sequence was intended to allow recombinant proteins to be expressed as secretory proteins and to be isolated from either the growth medium or the periplasmic space. However, it was determined that the yield from these preparations was extremely low and the majority of the proteins were accumulated in bacteria as inclusion bodies. Based on these observations, the three-domain single chain T-cell receptors were isolated from inclusion bodies. The recombinant proteins were subsequently purified with a one-step Ni-NTA chromatography procedure through the binding of histidine tag engineered at its C-terminus. SDS-PAGE analysis of the induction of the three-domain single chain T-cell receptor, the inclusion body preparation, the flowthrough of the NI-NTA column and purified proteins eluted from the column was performed.
  • the recombinant protein could be purified from each liter of bacteria culture.
  • the purified protein which was reduced and denatured following the NI-NTA column chromatography step was subsequently refolded with glutathione.
  • the efficiency of refolding was evaluated with a two- antibody ELISA where the fraction of Cl reactive protein was measured.
  • Cl had been demonstrated to recognize a Vj8 17 specific epitope. Further, it had been shown and that the formation of this epitope is dependent upon the proper folding and pairing of the T- cell receptor ⁇ and ⁇ chains.
  • the eukaryotically expressed three-domain single chain T-cell receptor was used as a control in the two-antibody ELISA.
  • the eukaryotically produced single chain T-cell receptor was purified from the cell surface after the PI-PLC cleavage and likely consists of T-cell receptors completely folded in their native conformation. Based on a determination of Cl reactivity between the two preparations, 5-10% of the bacterially-expressed three- domain single chain T-cell receptor was properly folded. To confirm the importance of the C ⁇ domain, a two- domain single chain T-cell receptor based on the design of scFv was constructed and purified in the manner described above in connection with the three-domain single chain T-cell receptor. The folding of the two- domain T-cell receptor was assayed by direct-binding
  • the Cl reactive fraction of the bacterially expressed three-domain single chain T-cell receptor can be further separated from the refolded protein solution using a Cl-affinity column. Consistent with the result from the two-antibody ELISA, 5-10% of the protein was recovered from the Cl column. Additionally, the Cl purified three-domain single chain T-cell receptor appeared as a single band in a non-reduced SDS-PAGE, in contrast to the multiple bands observed from the refolded protein solution. Furthermore, in a two- antibody ELISA, the Cl purified protein displayed comparable Cl reactivity to the eukaryotically expressed three-domain single chain T-cell receptor preparation.
  • CAG AAA GGA GAT ATA GCT GAA GGG TAC AGC GTC TCT CGG GAG AAG AAG 672 Gin Lys Gly Asp lie Ala Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys 210 215 220
  • CAC ACC CAA AAG GCC ACA CTG GTG TGC CTG GCC ACA GGT ATC TTC CCT 864 Hie Thr Gin Lys Ala Thr Leu Val Cys Leu Ala Thr Gly lie Phe Pro 275 280 285
  • ATC CAT CAC CAT CAC CAT CAC TAA 1176 lie His His His His His His His 385 390
  • Lys Thr Ser lie Asn Asn Leu Gin Trp Tyr Arg Gin Asn Ser Gly Arg 50 55 60
  • MOLECULE TYPE cDNA

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Abstract

L'invention a pour objet un récepteur de lymphocyte T monocaténaire qui se lie de manière spécifique à un ligand de peptide système HL.A. Le récepteur de lymphocyte T monocaténaire est un produit de recombinaison à trois domaines, comprenant un domaine variable à chaîne α; un domaine variable à chaîne β et un domaine constant. L'invention a également pour objet un récepteur de lymphocyte T monocaténaire d'auto-signalisation qui se lie, de manière spécifique, à un ligand de système HL.A peptide et assure la médiation de la transduction des signaux.
PCT/US1995/015696 1994-12-06 1995-12-04 Recepteur de lymphocyte t monocatenaire WO1996018105A1 (fr)

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EP1019439A4 (fr) * 1997-10-02 2002-11-06 Sunol Molecular Corp Proteines solubles du recepteur des lymphocytes t a chaine unique
US6534633B1 (en) * 1998-10-21 2003-03-18 Altor Bioscience Corporation Polyspecific binding molecules and uses thereof
WO2004033685A1 (fr) * 2002-10-09 2004-04-22 Avidex Ltd Recepteurs de lymphocytes t de recombinaison a chaine unique
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EP1159457A4 (fr) * 1999-02-23 2004-11-17 Baylor College Medicine Sequence vbeta-dbeta-jbeta du recepteur de leucocytes t et procedes de sa detection
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