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WO2004049794A2 - Anticorps simple chaine - Google Patents

Anticorps simple chaine Download PDF

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Publication number
WO2004049794A2
WO2004049794A2 PCT/GB2003/005274 GB0305274W WO2004049794A2 WO 2004049794 A2 WO2004049794 A2 WO 2004049794A2 GB 0305274 W GB0305274 W GB 0305274W WO 2004049794 A2 WO2004049794 A2 WO 2004049794A2
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single chain
genes
antibody
transgenic mouse
heavy
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PCT/GB2003/005274
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WO2004049794A3 (fr
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Marianne BRÜGGEMANN
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The Babraham Institute
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Publication of WO2004049794A3 publication Critical patent/WO2004049794A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present invention relates to single chain antibodies.
  • the invention includes a transgenic mouse capable of expressing single chain antibodies and single chain antibodies produced by such a transgenic mouse.
  • H and L chains Classical antibodies (Abs), and particularly immunoglobulins (Igs), are present in plasma of mammalian species as units of paired heavy (H) and light (L) chains.
  • Each H chain consists of an N-terminal variable (V H ) domain of -110 amino acids linked to 3 or 4 constant (C) domains of similar size (C H 1-C H 3 or C H 1-C H 4), while light chains comprise a variable (V L ) domain and a single C domain, C -
  • V H N-terminal variable domain of -110 amino acids linked to 3 or 4 constant (C) domains of similar size (C H 1-C H 3 or C H 1-C H 4), while light chains comprise a variable (V L ) domain and a single C domain, C -
  • the pairing of H and L chains creates the conventional antigen combining site through the association of V H and V , each domain contributing 3 sets of hypervariable or complementarity determining regions (CDRs) via which contact is made to antigen.
  • H chain isotypes There are 5 major H chain isotypes ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), which characterise the 5 Ig classes (M, G, A, E and D) and vary in the number of C domains (3 for ⁇ , , ⁇ or 4 for ⁇ , ⁇ ).
  • Ig classes M, G, A, E and D
  • K, ⁇ L chain isotypes
  • the Ig classes differ in the number of H 2 L 2 units they contain; thus IgG, IgD and IgE are single H 2 L 2 units, while IgA is a dimer (2 x H 2 L 2 ) and IgM a pentamer (5 x H 2 L 2 ).
  • BCR B cell receptor
  • the genes encoding the H-chain contain the CHI exon, which is spliced out during mRNA maturation as a result of a point mutation at the canonical splicing donor site (Nguyen et al, 1999, Mol.Immunol. 36: 515).
  • Single chain HCAbs are absent in other mammals except in pathological cases (i.e. heavy chain diseases), where various parts of the V H domain and the C H I are eliminated due to DNA rearrangements.
  • V H H genes (Nguyen et al, 2001, Adv Immunol. 79: 261), which differ from VH genes used in the generation of conventional Abs as they carry changes in some of the codons that encode the residues normally contacting the V L domain.
  • V H / V L interface mutations e.g. G44E, L45R and W47G
  • a repertoire of such single V H domains was expressed on phage and selected against haptens and proteins, which led successfully to single V H binding domains of moderate affinity (Riechmann and Muyldermans, 1999, J. Immunol. Methods 231_: 25; Davies and Riechmann, 1995, Biotechnology 13: 475).
  • Riechmann and Muyldermans 1999, J. Immunol. Methods 231_: 25; Davies and Riechmann, 1995, Biotechnology 13: 475.
  • there will be several advantages in utilising in vivo methods for selection of human HCAbs particularly in the use of transgenic mice.
  • B cells rearrange the human V genes in normal fashion (i.e. V H DJ H and V L J L ) and express fully human Ig's, responding to immunisation by production of fully human antibodies.
  • V H DJ H and V L J L the expression of endogenous mouse Ig is suppressed by knockout of the mouse H and L chain loci.
  • knockout strategies such as silencing of H chain expression by deletion of the ⁇ chain membrane domain (Kitamura et al, 1991, Nature 350: 423) and inactivation of the L chain locus by deletion of J L segments.
  • human monoclonal antibodies can be obtained by the hybridoma method, and such mice are now used for the production of therapeutic human antibodies (for review see Bruggemann and Taussig, 1997, Curr Opin Biotechnol 8: 455).
  • transgenic mouse capable of expressing a single chain antibody, in which expression includes either extracellular display or secretion or both.
  • Transgenic mice of the invention have the capacity to express, on their B cells and/or in plasma, single chain antibodies.
  • An advantage in the use of animals for the production of classical antibodies lies in the natural process of selection and affinity maturation which occurs in B cells of the immunised animal in vivo, resulting in a potentially quicker and easier derivation of desired antibodies.
  • HCAbs were observed exclusively in camelids and not in other mammalian species it was thought that HCAb production may involve camelid-specific accompanying specific factors to process, assemble and express HCAb genes into functional antigen-binding entities. For these reasons, the ability to utilise a noncamelid species to produce bona fide HCAbs, as demonstrated here for the first time, is an unexpected result.
  • the term 'single chain antibody' designates in a first embodiment an Ig H chain, unpartnered by L chain.
  • the H chain may carry a V region linked to C H domains and capable of binding to antigen ligand via a single V domain.
  • a single chain antibody may designate a combination of the domains of an Ig H and Ig L chain to form a chimeric single chain antibody.
  • a 'single domain antibody', also covered by the present invention, is defined as an unpaired V region capable of binding to an antigen ligand.
  • the single chain antibody of the transgenic mouse may thus be a heavy chain antibody.
  • the single chain antibody may in a further embodiment comprise a heavy chain domain linked to a light chain domain.
  • the transgenic mouse may carry immunoglobulin heavy and/or chain locus genes of a heterologous species for single chain antibody expression.
  • the heterologous heavy and/or light chain locus genes may include V L and/or V H , and or D and/or J H and/or C genes.
  • a heterologous heavy chain locus gene may be modified (for example by mutation and/or deletion) to allow expression and/or antigen binding of the single chain antibody.
  • a C region exon for example, the C H I exon
  • the V H and/or complementary determining regions CDRs, i.e. hydrophobic residues located in the conventional VH VL interface and which take part in domain-domain interaction
  • CDRs complementary determining regions
  • heterologous heavy and or light chain locus genes in the transgenic mouse are not rearranged and comprise a repertoire of V H , D and J H segments, or V L and J L segments, and a constant region gene.
  • the transgenic mouse comprises a repertoire of genes encoding rearranged or germline variable V H or V L domains of a heterologous species, modified for single chain antibody expression and optionally antigen binding by modification of variable-region framework and/or CDR residues and/or constant-region genes.
  • any or all of the endogenous mouse heavy chain genes and or light chain genes may be modified, functionally silenced and/or deleted. This can, for example, allow for expression of specific heterologous antibodies only.
  • the single chain antibody may be in monomeric or multimeric (for example, dimeric) configuration.
  • the transgenic mouse may express a repertoire of single chain antibodies.
  • the heterologous heavy and/or light chain locus genes or the repertoire of genes may undergo rearrangement and expression in mouse B cells.
  • Immunoglobulin gene segments can be rearranged in B cells in vivo and the mouse may thus respond to immunisation by production of a variety of single chain Abs, for example, of the heterologous species.
  • different V H genes may be rearranged with different D and J segments.
  • Mixed loci may be used for the creation of single (e.g. heavy) chain antibody repertoires. It may be advantageous to mix genes or segments of intervening sequences from different species to improve single chain antibody expression.
  • the heterologous species may be mammalian, for example a noncamelid such as a human.
  • a method for the production of a transgenic mouse capable of expressing a single chain antibody having a heavy chain or a heavy chain linked to a light chain, comprising the steps of inserting immunoglobulin heavy or light chain locus genes of a heterologous species into the mouse, allowing expression of the genes to form the single chain antibody which is expressed either by extracellular display or by secretion or both.
  • a single chain antibody produced by the transgenic mouse as defined above.
  • the single chain antibody may be in the form of a monomer or multimers in which identical chains are associated.
  • the single chain antibody may be in dimeric or multimeric form such that variable domains of the antibody bind antigen independently.
  • Also provided according to the invention is a monoclonal or polyclonal heavy chain only antibody, or a heavy-light chain chimeric antibody, which has a structure including a single domain antigen-combining V H or V L region and which is made upon immunisation of a mouse as defined above.
  • a method for producing a single chain antibody of the invention comprising the step of immunising the mouse as defined above with an antigen to elicit an immune response, the immune response comprising antigen-specific antibody production.
  • Another aspect of the invention is an antibody display library derived from the transgenic mouse as defined above, in which the display library comprises immunoglobulin heavy and/or light chain genes which are transcribed and translated in vitro to encode a population of single chain antibodies.
  • the library may comprise lymphocyte DNA isolated from a mouse as defined above.
  • Also provided according to the invention is a method for the production of the display library as defined above, comprising the step of introducing the immunoglobulin heavy andor light chain genes into a bacterial, yeast, phage or ribosome display system.
  • a further aspect of the invention is an isolated non-camelid immunoglobulin locus comprising heavy chain genes modified to encode a single chain antibody capable of being expressed in vivo.
  • the locus may further comprise light chain genes.
  • the heavy and light chain genes may comprise variable genes which are modified at their V K /V interface.
  • the invention also provides a hybridoma obtainable from a transgenic mouse as defined herein by fusion of a splenocyte from the transgenic mouse with a B-cell tumour line cell (for example an NSO myeloma cell), and subsequent selection of single clones.
  • a single chain antibody preferably human, obtainable from this hybridoma.
  • human genes encoding single chain antibodies may be recovered from the immune B cells of transgenic mice by PCR for further genetic manipulation.
  • the antigen-combining site of a conventional antibody is provided by the association of V H and V L domains
  • that of single chain antibodies may include a V H H domain only and consequently be much smaller in size than that of classical antibodies.
  • This provides single chain antibodies certain advantageous properties, such as the ability to recognise smaller antigens, for example the binding site clefts on the surfaces of enzymes where active sites are located or canyons on the surfaces of viruses, both regions which may be inaccessible to the combining sites of conventional Abs. In camelids, this may be assisted by the CDR3 region of the HCAb, which may be particularly long and include a disulphide bridge.
  • single domain combining sites may have advantages as therapeutic and diagnostic agents in their easier penetration into target tissues such as tumours and more rapid clearance from the human body.
  • a further aspect of the invention therefore covers use of single chain antibodies according to the invention as a therapeutic and/or diagnostic agent.
  • Single chain antibodies of the invention may be used in the manufacture of a medicament for the treatment of a disease, for example cancer.
  • a method for producing a repertoire of single chain antibodies in a mouse may use the mouse as defined above.
  • the method may encompass the method for producing the antibody as defined above.
  • the repertoire may comprise single chain antibodies of the invention as defined above.
  • Also provided according to the invention is a repertoire of single chain antibodies produced by the method.
  • Figure 1 depicts constructs of V ⁇ H- ⁇ 2a with ("VaH-C ⁇ aTM”; top figure) or without ("V H H-0)2a"; bottom figure) transmembrane (TM) exons;
  • Figure 2 shows ELISA results detecting hen egg-white lysosyme (HEL)-specif ⁇ c dromedary Ig produced by transfected NSO mouse myeloma cells carrying the V H H- Q)2aTM, V H H-C ⁇ a or parental pSV2 vector transcripts.
  • OD optical density (values at 405 nm);
  • Figure 3 shows an SDS-PAGE of camel HCAbs from transfected NSO mouse myeloma cells carrying the VsH-C ⁇ aTM, VnH-C ⁇ a or parental pSV2 vector transcripts, stained with Coomassie brilliant blue (panels A and B) or revealed by Western blotting (panels C and D);
  • Figure 4 illustrates the predicted structure of two mRNA products, with (panel A, top) and without (panel A, bottom) C H I, that could be obtained from a rearranged camel Ig H-gene.
  • Panels B to D show PCR identification of transcription products from transfected NSO mouse myeloma cells carrying the V H H-Q) aTM, V H H-Oy2a or parental pSV2 vector transcripts;
  • Figure 5 shows flow cytometry of NSO mouse myeloma cells transfected with VHH- C ⁇ 2aTM (A, D), V H H-C ⁇ 2a (B) or parental pSV2 vector only (C).
  • Panel (D) shows cells stained with biotinylated rabbit anti-dromedary Ig and with FITC labeled streptavidine prior to (shaded histogram) or after (open profile) incubation with phosphatidyl-inositol- specific phospholipase C (PI-PLC) to reveal BCR configuration;
  • PI-PLC phosphatidyl-inositol- specific phospholipase C
  • Figure 6 shows PCR analysis of transgenic mice containing the camel V H H-C ⁇ 2a transgene
  • Figure 7 shows ELISA results detecting camel single chain antibodies detection in transgenic mice containing the V ⁇ H-C ⁇ 2a transgene.
  • the left panel shows HEL-specific camel Ig in serum from transgenic mice while the right panel shows HEL-specific camel Ig in serum from normal mouse;
  • Figure 8 shows FACS analysis of B cells in normal mice (left), transgenic mice carrying the V E iH-C ⁇ 2a construct in a ⁇ MT " ⁇ heavy chain knockout background (centre), and control ⁇ MT " mice (right);
  • Figure 9 shows flow cytometry analysis of B cells from bone marrow (left three rows) and spleen (right three rows) from normal FI mice, ⁇ MT mice and two separately generated camel heavy chain mice bred to homozygosity with ⁇ MT animals, viz. camlgH(l) ⁇ MT and camIgH(2) ⁇ MT;
  • Figure 10 depicts a IgH YAC construct (prior art) from Nicholson, I.C. et al. (1999) J. Immunol. 163: 6898-6906; and
  • Figure 11 depicts a IgH * YAC construct which is modified from the IgH YAC construct of Figure 10 by truncation and removal of the C gene.
  • Example 2 we demonstrate the presence of camelid HCAb in the plasma of transgenic mice carrying the HCAb V ⁇ H- ⁇ 2a gene.
  • Examples 3 and 4 we demonstrate and analyse the rescue of B cell development in heavy chain knockout mice by the HCAb transgene.
  • Examples 5 to 7 relate to expression of appropriately modified human V H or V genes as single chain antibodies in transgenic mice.
  • Example 1 A camel heavy-chain antibody secreted and displayed by mouse myeloma cells
  • Antigen- binding HCAbs were displayed on the surface of the mouse cell as GPI-linked B cell receptors (BCR) when the transmembrane exons were present in the recombinant gene.
  • BCR GPI-linked B cell receptors
  • DNA manipulations were carried out using standard PCR and DNA subcloning techniques (Sambrook J, et al. 1989, In: A laboratory manual. Cold Spring Harbor Laboratory Press, especially pp. 2.64-2.68; 6.3-6.15; 6.16-6.22; 9.14-9.23).
  • intermediate cloning steps recombinant plasmids (pBluescript) were propagated in E.coli DH5a cells, and DNA was prepared using a Qiagen-mini® prep kit (Qiagen, Westburg, Leusden, The Netherlands).
  • the cloning strategy was as follows: the Ig promoter region derived from the germline V H H clone cvhhpll (Nguyen et al, 2000, EMBO J.
  • the cAb-Lys3 is a V H H coding for a HEL-specific single domain antibody fragment.
  • the arrow at the left in Fig. 1 denotes the Ig promoter starting with the conserved Ig octamer sequence.
  • the other gene components are boxed: the rearranged V H HDJ region encoding the cAb-Lys3 sequence, the J H 6 (not used in the D-J recombination), the enhancer (E), matrix attachment region (MAR) and switch region and the different exons (C for constant domains and M for transmembrane regions) of the dromedary C ⁇ 2a are indicated.
  • the position of the Notl, BstEII, EcoRI, Sail and Kpnl restriction enzyme sites used during cloning and screening are indicated as well as the position of the stopcodons ($).
  • the star denotes the non-canonical splicing site at the 3' end of the CHI of the camel C ⁇ 2a gene.
  • the VEiH-C ⁇ 2aTM construct (Fig. 1, top) of 11.7 kb is the part comprised between the unique Notl and Sail sites, and the V H H-C ⁇ 2a construct (Fig. 1, bottom) of 7.4 kb, comprised between the Notl and Kpnl sites, lacks the membrane bound exons. (The space between E-MAR and switch region is not to scale.)
  • V ⁇ H- ⁇ 2aTM or the V ⁇ H- ⁇ 2a gene constructs or the parental pSV2-Neo cloning vector used as control were electroporated separately into NSO myeloma cells (Desmyter A. et al, 1996, supra) by 2 pulses using a BIORAD Gene Pulser set at 230 V and 500 ⁇ F.
  • the transfected NSO cells were maintained at 37°C and 5% C0 2 in RPMI 1640 medium containing 10% FCS. After 24 hrs of growth, G418 (Invitrogen, Paisley, UK) was added to a final concentration of 400 ⁇ g/ml. At least 5 antibiotic resistant clones were chosen for each construct and grown to a density of 2-3xl0 5 cells/ml for further studies.
  • HEL (10 ⁇ g/ml in PBS) was coated overnight at 4°C onto 96-well-plates (Nunc- MaxisorbTM, Life Technologies, Invitrogen, Merelbeke, Belgium). Residual protein binding sites were blocked with PBS-1% casein for 2 hrs at room temperature. Serial fivefold dilutions (100 ⁇ l) of cell-free supernatants from different clones of the transfected NSO cells were added to the wells and incubated at room temperature for 1 hr. The " retention of recombinant HEL binding Abs (anti-HEL-IgG2a) was detected with rabbit anti-camel IgG (1/1000 anti-dromedary rabbit serum R17, provided by T.
  • the positive control contains camel anti HEL antiserum diluted 1/5000.
  • Clones 1 and 3 for constructs V ⁇ H- ⁇ 2aTM and V H H- ⁇ 2a respectively gave ELISA signals similar to those of the positive control and were inhibited by free HEL (not shown).
  • the camel HCAbs secreted by the NSO cells were characterized by SDS-PAGE and Western blotting, after absorption and elution from HEL-coupled Sepharose, or visualized by Western blot using rabbit anti-camel IgG ( Figure 3).
  • Recombinant HCAb was enriched on immuno-adsorbent obtained by coupling HEL to CNBr-activated Sepharose (Amersham Pharmacia, Little Chalfont, UK) (3 mg HEL per ml resin) in 0.5 M NaCl and 0.1 M NaHC0 3 pH 8.3 following the instructions provided by the manufacturer.
  • HEL- coupled Sepharose 50 ⁇ l wet gel was incubated with 0.5 ml culture supernatant of the different NSO transfectants for 1 hr at room temperature and washed repeatedly with PBS.
  • the captured proteins were solubilized by adding 100 ⁇ l SDS-sample buffer, boiled and 4 ⁇ l applied to a 10% polyacrylamide gel. SDS-PAGE was carried out under reducing (0.5% DTT in the loading buffer) and non-reducing conditions.
  • Fractionated IgGl Abs and IgG2 HCAbs isolated from dromedary serum were applied in adjacent lanes as references. To visualize the proteins after electrophoresis, gels were stained using Coomassie Brilliant Blue.
  • proteins separated on SDS-PAGE were transferred onto nitrocellulose membranes (Amersham Pharmacia), using a Mini Trans-Blot Cell (BioRad, Nazareth EKE, Belgium) and following standard protocols (Galfre et al, 1982, Immunology 45: 125-128).
  • the recombinant IgG2a enriched by adsorption was detected with rabbit anti-dromedary serum as first Ab, alkaline phosphatase-conjugated goat anti-rabbit IgG as second Ab (the same reagents as in ELISA), and 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium (BCIP/NBT, Sigma-Aldrich) as substrate.
  • Culture supernatant was applied from NSO cells transfected with V ⁇ H-C ⁇ 2a-TM (Fig. 3, lanes 1), VHH- C ⁇ 2a (Fig. 3, lanes 2) and parental pSV2 vector only (Fig. 3, lanes 3).
  • the isotypes fractionated form dromedary serum were loaded in adjacent lanes: IgGl, the conventional H-L containing Abs (Fig. 3, lanes 5) and the HCAb IgG2 (Fig. 3, lanes 6). All proteins were separated under non-reducing (Fig. 3, panels A and C) or under reducing conditions (Fig. 3, panel B and D).
  • the molecular weight marker is a prestained protein ladder, and the sizes (in kDa) are given along the bands. The amount of material applied in the Coomassie blue stained gels was ten times higher than in gels revealed by Western blotting.
  • the QuickPrep micro mRNA purification kit (Amersham Pharmacia) was used for the preparation of mRNA from 10 7 transfected NSO cells and first strand cDNA was synthesized using the 'Ready-to-Go' kit (Amersham Pharmacia). PCR conditions using 0.5 ⁇ l cDNA were: 30 cycles of 45 s at 94°C, 30 s at 52°C and 45 s at 72°C.
  • V3FR1B (5' GAGGTGCAGCTGGTGGCGTCTGGAGGAGG 3'; SEQ LO NO: 1), derived from the sequence of the V H H/V H -FR1 region and G2AHIF (5' GGGACACGTGCATTCTGGTTCA 3 ' ; SEQ ID NO: 2), a sequence annealing at the long hinge region of dromedary C ⁇ 2a;
  • V3FR1B and CH1290F (5' CTCTTGTCGACCTTGGTGCTGCTG 3'; SEQ ID NO: 3), representing a conserved sequence of the first constant exon of all camelid C ⁇ genes; and
  • CH1242B (5' GCATCTAGACCGGMAAGACCTTCAYCT 3'; SEQ ID NO: 4), a consensus sequence of the first constant exon of all camelid C ⁇ , and the long hinge-specific G2AHIF primer.
  • Fig. 4(A) illustrates the predicted structure of two mRNA products, with and without C H I, that could possibly be obtained from the rearranged camel Ig H-gene.
  • the theoretically possible PCR amplification fragments of these mRNAs are indicated with the expected sizes in kb.
  • the fourth lane shows the 123 bp size ladder (position of 123 bp monomer is indicated in panel B).
  • oligonucleotides V3FR1B and G2AHIF With oligonucleotides V3FR1B and G2AHIF, the mRNA from the transfected NSO cells as template resulted in a single fragment of 0.5 kb, identifying the transgenic mRNA product without C H I (Fig. 4B, lanes 1 and 2). No band was discerned with a size of 0.8 kb that would result from the mRNA in which the CHI exon sequences would have been retained. The absence of the C H I exon in the V ⁇ H- ⁇ 2a spliced products was further shown by RT-PCR using C H I -specific oligonucleotides which failed to amplify any products (Fig. 4C & 4D, lanes 1 and 2).
  • HEL and affinity-purified rabbit anti-camel IgG were biotinylated using biotin-X-sulfo-NHS (Calbiochem, Nottingham, UK).
  • Clones from the NSO transfectants (10 cells) were resuspended in 200 ⁇ l RPMI medium and incubated with 1 mg biotinylated HEL at 4°C for 45 min. Unbound antigen was removed by two washings with PBS containing 0.002% TritonX-100 and 0.01%) sodium azide, and FITC-labeled streptavidin (PharMingen, San Diego, CA) was added to detect cells that captured HEL.
  • PI-PLC phosphatidyl-inositol-specific phospholipase C
  • Figure 5 shows cells transfected with V ⁇ H-C ⁇ 2aTM (A, D), V ⁇ H-C ⁇ 2a (B) and vector only (C) that were incubated with biotinylated HEL and stained with FITC-labeled streptavidin. The background signal obtained when the biotinylated HEL was omitted is superimposed (shaded histograms).
  • panel D cells transfected with V ⁇ H-C ⁇ 2a TM were stained with biotinylated rabbit anti-dromedary Ig and with FITC labeled streptavidine prior to (shaded histogram) or after (open profile) incubation with PI-PLC. Each histogram shows the analysis of approximately 10 4 cells.
  • Example 1 confirm removal of the CHI domain by mouse B cells, which allows expression of camel HCAb.
  • the HCAbs are also found on the B cell surface. This has major implications for the formation of antibody repertoires because the B cell receptor is essential to allow cell survival and progression of B-cell development.
  • the successful expression of HCAbs by mouse cells suggests that developmental regulation of HCAbs in a transgenic mouse and in vivo HCAb expression and maturation is feasible.
  • mice and all other healthy non-camelidae mammals studied, do not express single chain antibodies and there are no reports that B-cell development can progress without expression of Ig H and L (light) association on the cell surface.
  • camel-type H chains could be expressed in a transgenic mouse, retaining their specificity and the effect on B-cell development and B-cell receptor (BCR) configuration, transgenic mice were constructed.
  • V ⁇ H-C ⁇ 2a TM construct (see Example 1) was introduced into the germline of FI CBA x C57BL/6 mice by conventional DNA injection into fertilised mouse eggs (Hogan, B. et al. 1994, Production of transgenic mice, In: Manipulating the mouse mouse embryo, a laboratory manual, Cold Spring Harbour Laboratory Press, pp 217-251).
  • mice Following reimplantation into pseudopregnant FI females, several mice were obtained carrying the camel heavy chain gene in multiple copies in their genome. Integration of the camel H chain gene was verified by PCR using mouse tail DNA prepared according to Matise et al. (2000, Gene Targeting: A practical approach, Joyner, Ed., Oxford University Press, p.122). PCR was performed using CamlgH Primer 1 (5'- GCATCTAGACCGGAAAGACCTTCATCT-3'; SEQ ID NO: 5) and CamlgH Primer 2 (5'-GGGACACGTGCATTCTGGTTCA-3'; SEQ ID NO: 2). PCR conditions were: 94°C for 3 min and then 94°C, 55 s; 52°C, 40 s and 72°C, 55 s for 30 cycles with a final extension of 10 min. The expected product size is 486 bp.
  • Figure 6 shows the PCR results for the following lanes: 1) control no DNA; 2) normal mouse DNA; and 3-6) transgenic camel antibody mice.
  • a ⁇ -based ladder flanks the PCR lanes.
  • lanes 3-6 containing amplification products from transgenic camel antibody mice, but not the control and normal mouse DNA lanes a band of about 486 bp was generated (compare with strong 600 bp band of ⁇ -based ladder).
  • the results in Figure 6 confirm that camel-type H chains can be expressed in transgenic mice.
  • V H chain is expressed as a single chain antibody in dimeric H2 form with specificity for HEL.
  • ELISA assays for camel Ig HEL binding activity were carried out using serum obtained from the tail vein blood from each of the 3 separately derived transgenic founder mice.
  • HEL-specific single chain antibodies in serum of transgenic mice carrying the V ⁇ H-C ⁇ 2a transgene were detected using 96-well Dynatech microtiter plates coated with 50 ⁇ l of 10 ⁇ g/ml hen egg lysozyme and blocked with 3% BSA overnight.
  • Serum (50 ⁇ l) at various dilutions (1:10, 1:100, 1:1000 or 1:10000) was added and incubated for 2 hours at room temperature. Normal mouse serum was used as control.
  • the transgenic camel HCAb mice of Example 2 above carrying the V ⁇ H-C ⁇ 2a construct were crossed with mice in which the endogenous H chain locus was silenced by gene targeting ( ⁇ MT mice, Kitamura et al, 1991, supra).
  • Flow cytometry analysis was carried out using spleen cells. Mice were sacrificed, spleens removed and cell suspensions prepared. Cells were stained using anti-mouse B220 antibodies (01125A, 01129A, PharMingen, San Diego, CA) and for FACS analysis lymphocytes were gated according to their expected size.
  • Results are shown in Figure 8 for normal mice (left), transgenic mice carrying the V H H- C ⁇ 2a construct in the ⁇ MT _ " heavy chain knockout background (centre), and control ⁇ MT " /_ mice (right). The percentage of B cells in each spleen preparation are indicated. FACS analysis revealed that B cells were rescued in the transgenic mice in the ⁇ MT " " background, with good recovery of B-cell development. Indeed from the results it is clear that a substantial number of B-cells leave the bone marrow and settle in the spleen. Since the ⁇ MT _ " mouse is normally devoid of B cells (see controls in Figure 8), it is clear that the camel HCAb transgene rescues B cell development. We conclude from these experiments that a single chain antibody of camel origin is successfully processed and expressed in mouse B cells, both when transfected in cell culture and when incorporated into the mouse germline as a transgene.
  • mice carrying the rearranged camel heavy chain gene were set up for breeding with mice in which the heavy chain locus and, separately, the kappa and lambda light chain locus had been silenced by gene targeting (see Kitamura, D. et al, 1991, supra, and International patent application No: PCT/GB02/002867 published as WO03/000737). Homozygous animals were obtained.
  • Homozygous animals are analysed by flow cytometry to establish the state of B-cell development and IgH expression.
  • Example 6 Design of a human heavy chain locus that allows expression of camel- type single chain antibodies in mice
  • a human IgH YAC with V, D, J and C genes has been described (Nicholson et al, 1999, supra; see Figure 10).
  • the human IgH YAC is modified by truncation and removal of the C gene (C ⁇ and C ⁇ regions).
  • C ⁇ and C ⁇ regions C ⁇ and C ⁇ regions.
  • a human C gamma ( ⁇ ) gene (Flanagan and Rabbitts, 1982, Nature 300: 709-713; Briiggemann et al, 1987, J. Exp. Med. 166: 1351-1361) without the CHI exon, obtained by restriction digest or PCR, is subcloned into a YAC arm vector (Burke et al, 1987, Science 236: 806-812).
  • a region 5' of Cmu (C ⁇ ) is added to the human IgH YAC construct. This allows in yeast transfection the replacement of the region comprising the mu ( ⁇ ) and delta ( ⁇ ) C genes to form a IgH , YAC construct ( Figure 11).
  • a loxP sequence can be added to permit future modification of the IgH , YAC construct.
  • Yeast methodologies are described in Guthrie and Fink, 1991, Methods in Enzymology, Vol 194, Academic Press). Suitable techniques for YAC modification are described in Popov et al, 1996, Gene 177: 195-205 and in International patent application publication No: Example 7: Introduction of a repertoire of unrearranged genes into mice
  • Protoplast fusion is used for YAC integration of the IgH ⁇ 4 YAC construct (see Example 6 and Figure 11) into ES cells.
  • Chimeric mice are produced by blastocyst transfer and breeding established germline transmission. Further breeding with H and L KO mice allows analysis of B-cell development and expression of (diversified) single chain Ig from this modified IgH YAC.
  • YAC can be enforced.
  • transgenic mice carrying this YAC are bred with animals not expressing any immunoglobulin genes (see knockout mice in Example 4 above). This establishes a human heavy chain-only antibody repertoire and after immunisation specific single chain antibodies are obtainable.
  • mice or mice cells may also be desirable to introduce specific human single chain antibody gene configurations into mice or mice cells so that no rearrangement takes place and "monoclonal" antibodies are produced. In situ somatic mutation of these configurations may allow for some optimisation of binding to antigen.

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Abstract

La présente invention concerne des anticorps simple chaîne. L'invention concerne également une souris transgénique et des anticorps simple chaîne produits par cette souris transgénique. Selon un mode de réalisation décrit dans cette invention, la souris transgénique peut exprimer un anticorps simple chaîne, tel qu'un anticorps à chaîne lourde uniquement; l'expression comprend, soit la sécrétion ou la diffusion extracellulaire, soit les deux.
PCT/GB2003/005274 2002-12-03 2003-12-03 Anticorps simple chaine WO2004049794A2 (fr)

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