WO2002072635A2 - Specific binding members - Google Patents

Abstract

The present invention relates to specific binding members which are able to bind to two or more different antigens at the same time. Such members comprise one or more pairings of variable domains consisting of an immunoglobulin VL and an immunoglobulin VH domain. The VH and VL domains in the pairing both contain a antigen-binding domain which allows the pairing to bind to two antigens simultaneously. Therapeutic applications of such molecules, along with methods and means for production are also described herein.

Description

Specific Binding Members

The present invention relates to specific binding members, in particular, specific binding members which are able to bind to two or more different antigens at the same time. Specific binding members, uses thereof and methods and means for production are also described herein.

Recombinant antibodies and their fragments represent over 30% (worth 10 billion $US per annum) of all biological proteins undergoing clinical trials for diagnosis and therapy. The focus on antibodies as the ideal cancer-targeting reagents recently culminated in approval by the Food and Drugs Administration for the first engineered therapeutic antibodies Rituxan, Bexxar (both targeting CD20; Leget, G.A. & Czuczman, M.S. Curr. Opin . Oncol . 10, 548-551 (1998)) and Herceptin (anti-Her2, Goldenberg M.M. Clin . Ther. 21, 309-318 (1999)).

Antibodies are the paradigm for the design of high-affinity, protein-based binding reagents and recombinant antibody construction has allowed a reduction in size, dissection into minimal binding fragments and rebuilding into multivalent, high avidity reagents (Hudson, P.J. Curr. Opin . Biotechnol . 9, 395-402 (1998); Dall'Acqua, W. and Carter, P. Curr. Opin . Struct . Biol . 8, 443-450 (1998)). Recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes, liposomes and biosensor surfaces for diagnostic and therapeutic purposes (Hudson, P.J. Curr. Opin . Immunol . 11, 548-557 (1999) ) .

The cross-reactivity of an auto-antibody to DNA and cardiolipin (i.e. the same auto-antibody recognising two different auto-antigens) has long been known in the art. This cross-reactivitity lies in the recognition by the auto- antibody of an epitope shared between DNA and cardiolipin, possibly with a constituent phosphate (Lafer, E.M. et al 1981. J. Exp . Med. 153: 897-909). The cross-reactivity of auto-antibodies is thus dependent on either the presence of a single binding domain which is capable of recognising an epitope which is common to different auto-antigens or the over-all charge (probably via arginine residues) of the auto- antibody, which allows interaction with negatively charged auto-antigens such as DNA.

Computer modelling studies have suggested differences in how an auto-antibody can interact with DNA. The auto-antibody may have a groove within which a complete turn of B-DNA could be • accommodated as shown for B3 antibody (Kalsi, J.K. et al (1996) Mol. Immunol . 33: 471-483.). It may, alternatively, have abundance of positively charged and aromatic residues known to participate in DNA recognition, on the surface of the binding site (Ravirajan, C.T. et al . 1998. Eur . J. Immunol . 28: 339-50.). Positively charged amino acids bind to the phosphate backbone of the DNA via electrostatic interactions, while aromatic residues may participate in base stacking interactions (Barry, M.M. et al. (1994). J. Biol . Chem . 269: 3623-32.).

Hybrid Fab molecules have been constructed using heavy and light chains from autoimmune antibody fragments BB (B3) , RR (33H11) and UU (UK4) , which react to both DNA and cardiolipin (Kumar et al, (2000) J. Biol. Chem. 275 45 35129-35136). A compatible binding partner is essential in order that the hybrid auto-antibody molecule achieves auto-antigen reactivity. The compatible partner chain allows the specificity bearing chain to assume the correct folding upon combining, allowing the formation of a pocket that allows binding to the self-antigen (s) . The compatible partner chain is either inert, non-inhibitory, or promotes binding to the auto-antigen by the specificity bearing chain.

Multivalency means the binding of a single molecule to more than one antigen via different binding sites and is distinct from cross-reactivity, in which only one site is involved. Multivalency is one of the hallmarks of antibodies, and allows enormous gains in^' functional affinity leading to improved performance in vivo including improved targeting and more selective localization, and in a variety of in vitro assays (Pluckthun, A. & Pack, P. Immunotechnology 3, 83-105 (1997)).

Bispecific antibodies and related fusion proteins have been produced for cancer immunotherapy, effectively enhancing the human immune response in anti-cancer vaccines and T cell recruitment strategies and the beneficial effects of bispecific antibodies (bsAbs) have been shown in a number of Phase I and II clinical trials (Segal, D.M. et al. Curr. Opin . Immunol . 11, 558-562 (1999)).

Traditional methods used for the construction of these bsAbs include fusion of two different hybridoma cell lines leading to the construction of a quadroma (or hybrid hybridoma)

(Milstein, C. & Cuello, A.C. Nature 305, 537-540 (1983)) and cross-linking two different f(ab') fragments by their hinge disulfides leading to the construction of a hetero-F(ab' ) ₂ (Deo, Y.M. et al . J. Immunol . 160, 1677-1686 (1998)).

Recent advances in genetic engineering allow the production of large amounts of clinical-grade antibody reagents and have increased the usage of Fv-based genetically engineered bsAbs. The joining of two scFvs originally produced via non-covalent association only of V-domains (V_H/V_L) , can produce bs-miniAbs, sc-bsAbs (Pluckthun and Pack (1997) supra), diabodies (Holliger, P. et al. Proc . Na tl . Acad. Sci . , USA 90, 6444-644E (1993) ) or trispecific triabodies and tetraspecific tetrabodies (Hudson (1999) supra) .

F(ab') constructs, which eliminate the need for proteolytic digestion of intact monoclonal Abs, have been shown by animal studies to be stable in serum and to clear at about the same rate as conventional F(ab')₂ fragments (Bakacs et al. (1995) Int. Immunol. 7 947-955) . Dimeric and trimeric Fabs can be constructed using adhesive domains or helices, and aleiimide cross-linking reagent, TFM, respectively.

Known methods for the construction of bsAbs therefore involve linking pairs of H/L chain variable domains derived from different antibodies with desired specificities to form a bi- or poly-specific antibody.

Methods of producing bispecific antigen binding domains as provided herein complement known methods for the construction of bi-/multispecific antibodies and provide more choice and flexibility for the inclusion of multiple specificities without increasing the molecular size of the resultant polyspecific bi-/multivalent antibody molecule. Any such increase may, for example, result in reduced tumour penetration.

In the work described herein, the antigen specificities of two distinct antibodies have been transferred via an appropriate combination of H and L chains from these antibodies to generate a hybrid molecule, which possesses antigen reactivities of both the parent antibodies. The method allows the construction of monovalent specific binding members such as Fabs and single chain Fvs which are bispecific.

One aspect of the present invention therefore provides a specific binding member which comprises a variable domain pairing which specifically binds a first and a second antigen.

The variable domain pairing may consist of a VL domain which comprises a binding domain for said first antigen and a VH region which comprises a binding domain for said second antigen.

The variable domain pairing may therefore be a hybrid variable domain pairing i.e. comprising VL and VH domains from different parental antibodies.

The variable domain pairing may therefore consist of a VL domain of a first antibody molecule which specifically binds a first antigen and a VH region of second antibody molecule which specifically binds a second antigen.

The hybrid variable domain pairing therefore contains two antigen binding domains and is bi-specific i.e. it may bind to both the first and the second antigen at the same time. The hybrid variable domain pairing may additionally specifically bind to a third antigen different from said first and said second antigen. The binding affinity of the hybrid variable domain pairing to the first and/or second antigen may be increased relative to the binding affinity of the parent antibody which binds specifically to said antigen. Preferably, the VL domain as described herein is of a parent antibody in which the VL domain provides a functional antigen binding domain, i.e. binding to an antigen occurs through the VL domain, and the VH domain as described herein is of a parent antibody in which the VH domain provides a functional antigen binding domain i.e. binding to an antigen occurs through the VH domain.

Light and heavy chains of the parent antibody molecules and comprising VL and VH domains as described herein may be associated to form hybrid antigen-binding domains as described herein.

Light and heavy chains of the two parent antibody molecules (whose specificities are to be combined/incorporated into a hybrid antibody molecule) may be cloned into known prokaryotic and eukaryotic expression vectors (see, for example, Kumar et al (2000) supra, McCafferty, Hoogenboom and Chiswell (eds) Antibody Engineering (1996) IRL Press at Oxford University Press, Oxford) in a PCR (polymerase chain reaction, Sambrook, Fritsch and Maniatis 1990. Molecular cloning, Cold Spring Harbor Laboratory Press, New York) or non-PCR (Kumar et al (2000) supra) manner. The heavy and light chains of the parent antibody molecules may then be shuffled as described herein.

The hybrid variable domain pairing may be evaluated for ability to bind to the two antigens i.e. the parental antigens. This may be performed sequentially by determining the binding of the variable domain pairing to the first antigen and then to the second antigen or simultaneously by determining the binding of the variable domain pairing to the first and second antigens at the same time. Other aspects of the present invention provide a variable domain pairing as described herein specifically binding to two antigens at the same time.

The binding of a specific binding domain of a variable domain pairing to an antigen may be determined using conventional techniques such as Enzyme Linked Immunosorbant Assay (ELISA) or affinity (in solution, Lisa et al. 1996. In Antibody Engineering, eds. McCafferty, Hoogenboom and Chiswell, pp. 77- 96, IRL Press at Oxford University Press, Oxford) or biosensors (Hefta et al 1996. In Antibody Engineering, eds. McCafferty, Hoogenboom and Chiswell, pp. 41-58, IRL Press at Oxford University Press, Oxford) .

The heavy and light chains of the parent antibody molecules may be shuffled with the heavy and light chains of antibody molecules with known antigen specificities and which are known or pre-determined to be negative (i.e. to show no reactivity or binding) to the antigens of interest. The screening _tof the hybrid constructs against the antigens of interest indicates on which chain the antigen specificity resides. Appropriate heavy and light chains may then be selected for a hybrid variable domain pairing as described herein.

Structural studies on such hybrid Fabs may also reveal the underlying requirements/principles for the compatibility of the two chains of an antibody. Such information could be used to predict and design compatible partner chains which form a bi-specific antigen binding domain without screening, for example, the VH and VL domains may, by modelling and antigen docking, be predicted to fold to allow binding to the antigens of interest. Analysis of ^'the. structure of the hybrid variable domain pairings and antigens followed by molecular modelling studies may therefore allow the design of hybrid binding domains specific for any particular antigen or combination of antigens.

Variable domain, amino acid sequence variants of a VH or a VL domain from a parent antibody molecule may be employed in accordance with the present invention. Particular variants may include one or more amino acid sequence alterations

(addition, deletion, substitution and/or insertion of an amino acid residue), maybe less than about 20 alterations, less than about 15 alterations, less than about 10 alterations or less than about 5 alterations, 4, 3, 2 or 1. Alterations may be made in one or more framework regions and/or one or more CDRs.

Parent antibodies used to generate hybrid variable domain pairings as described herein may specifically bind to any antigen of interest. The antigen of interest may be a extracellular target, for example a cell surface receptor such as pl85 or an intracellular target such as RNA, DNA, Rib.PO or other auto-antigen (see for example, Isenberg, D. A., and Horsfall, A. (1998) In Oxford Textbook of Rheumatology (Maddison, P.J., Isenberg, S.A., Woo,. P, and Glass, D. editors) Oxford University Press, Oxford, UK.), or any other molecule- which affects cell physiology.

In some embodiments, one or more of the parent antibody molecules are auto-antibody molecules which bind to self- antigens, preferably human antigens. In other embodiments, neither of the parent antibody molecules are auto-antibody molecules . A hybrid variable domain pairing as described may bind to an antigen which is not specifically bound by either parent antibody. Novel antigens which may be bound in this way include RibPO.

In addition to a hybrid variable domain pairing, the specific binding member may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to the ability to bind antigen. Specific binding members of the invention may carry a detectable label, or may be conjugated to a toxin or enzyme (e.g. via a peptidyl bond or linker) .

Different types of specific, binding member are well-known in the art and include antibody molecules such as scFv, Fab, F(ab)₂, diabody, multimeric antibody fragments and quadroma/IgG formats. Due to its small size, a scFv has better penetration into solid tumours than larger specific binding members and is cleared more rapidly from non-target tissues. ScFvs may therefore be preferred in some therapeutic applications.

Specific binding members may comprise a light chain from a first parent antibody molecule and a heavy chain from a second parent antibody molecule. The VL and VH domains of these light and heavy chains then form the hybrid variable domain pairing. A specific binding member may comprise one, two, three or four or more hybrid variable domain pairings as described herein.

Examples of a VL domain which may be employed in a hybrid variable domain pairing as described herein includes a VL domain of B3 (Ace No: L22483) (Ehrenstein et al (1994) J. Clin . Invest .93: 1737-1797), 33H11 (Deposited as RH-14; Ace No: X95739) (Winkler et al (1991) Clin. Exp. Immunol. 85: 379- 385) and 4D5 (Ace No: 442925).

Examples of a VH domain which may be employed in a hybrid variable domain pairing as described herein includes a VH domain of B3 (Ace No: Y18126) , UK4 (Ace No: X91130) (Menon et al. (1997) J. Autoimmun. 10: 43-57) or 4D5 (Ace No: 442926) (Carter et al (1992) Proc Natl Acad Sci USA 89:4285-4289).

For example, a hybrid variable domain pairing may comprise a heavy chain from one of B3, UK4, and 4D5 and a light chain from a different antibody molecule selected from B3, 4D5 and 33H11, in particular, a light chain from 4D5 and a heavy chain from UK4, a light chain from B3 and a heavy chain from 4D5, a light chain from 4D5 and a heavy chain from B3, a light chain from 33H11 and a heavy chain from B3, or a light chain from 33H11 and a heavy chain from 4D5.

4D5 heavy chain is shown herein to possess reactivity to pl85 immunodominant epitope derived from its extra-cellular domain, when combined with a compatible partner chain. An antibody molecule that has the specificity for this epitope also has the potential to bind the whole molecule (185kDa; Genosys catalogue antibody) which is, for example, derived from the SKBR-3 cells that over-express pl85. The heavy chain of 4D5 may, in principle, be combined with the light chain of a cytotoxic antibody (with its specificity lying on its light chain) to generate novel combinations of anti-pl85 reactivity and cytotoxicity in a hybrid variable domain pairing as described herein. Such a bi-specific antibody molecule is capable of targeting as well as killing the cancer cells. 4D5 heavy chain may therefore be used for targeting a gene/or a molecule of interest to pl85-over-expressing cells (such as cancerous cells) . The heavy chain may be assembled with a light chain to construct a bi-/multi-specific antibody molecule as described herein.

A suitable light chain may provide the hybrid molecule with a cytotoxic activity. For example, a light chain possessing specificity/reactivity to Rib.PO, which is absolutely required for the ribosomal activity and cell viability (Santos and Ballestea 1994. J. Biol. Chem. 269:15689-15696)

A further aspect of the present invention is therefore a specific binding member comprising a hybrid variable domain pairing which consists of the VH domain of the parent antibody molecule 4D5 and the VL domain from a different parent antibody molecule. A specific binding member comprising such a hybrid variable domain pairing may be used in a method of targeting a therapeutic activity to a target cell, for example a cancer cell.

Alternatively, a suitable light chain may allow the hybrid molecule to be used for gene targeting. This may be achieved if the light chain possesses an anti-DNA activity; for example, the light chain of the anti-DNA antibody B3 as described herein. The hybrid Fab BD possesses significant residual anti-pl85 activity following its binding to dsDNA.

The variable domain of the B3 light chain is shown herein to possess anti-DNA activity. This domain finds application in gene delivery. The B3 light chain comprising this domain may be assembled with a heavy chain to construct bi-/multi- specific antibody molecule useful in delivering a gene to the targeted cell via the specificity of the heavy chain. For example, a gene may be targeted to a pl85 over-expressing cell via 4D5 heavy chain, for example, by means of the antibody molecule BD described herein.

A further aspect of the present invention is therefore a hybrid variable domain pairing comprising the VL domain of the parent antibody molecule B3 and the VH domain from a different parent antibody molecule. A specific binding member comprising such a hybrid variable domain pairing may be used in a method of gene therapy for delivering a gene to a target cell.

Specific binding members of the invention may be labelled with a detectable or functional label. Detectable labels include radiolabels such as ¹³¹I or ⁹⁹Tc, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase.

Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

Specific binding members of the present invention may be used in methods of diagnosis or treatment in human or animal subjects, preferably human. For example, a bispecific antibody molecule may be targeted to a particular cell-type using one binding specificity (e.g. pl85) and produce a therapeutic effect by means of the other binding specificity. The therapeutic effect may, for example, be produced by the binding of the specific binding member to a cellular protein, such as Rib.Po. Such binding may impede the function of the cellular protein. In other embodiments, the therapeutic effect may be produced through the introduction to the cell of a nucleic acid molecule bound to the bispecific antibody molecule, for example in a method of gene therapy.

In particular, specific binding members of the present invention may be used for immunotherapy, for example in the treatment of cancer, malaria, auto-immune disorders and disorders involving over-expression and/or pathological involvement of a cell surface protein such as pl85 and/or an intracellular protein such as Rib.Po.

Accordingly, further aspects of the invention provide methods of treatment of a condition as described above comprising administration of a specific binding member as provided, pharmaceutical compositions comprising such a specific binding member, and use of such a specific binding member in the manufacture of a medicament for administration for the treatment of cancer, malaria or other condition, for example in a method of making a medicament or pharmaceutical composition comprising formulating the specific binding member with a pharmaceutically acceptable excipient.

Further aspects of the invention provide the use of a hybrid variable domain pairing which includes a VH or VL domain having a sequence described herein or a fragment thereof which has binding activity in the manufacture of a medicament for administration for the treatment of cancer, malaria or other condition.

In accordance with the present invention, specific binding members and compositions comprising such members may be administered to individuals. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibody are well known in the art; see Ledermann J.A. et al. (1991) Int J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Specific binding members of the present invention may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream or directly into the site to be treated e.g a tumour. The precise dose will depend upon a number of factors, including whether the specific binding member is for diagnosis or for treatment, the size and location of the area to be treated (e.g. tumour), the precise nature of the specific binding member (e.g. antibody molecule such as a whole antibody or an antibody fragment, including a diabody) , and the nature of any detectable label or other molecule attached to the specific binding member.

A typical dose will be in the range 0.5mg to lOOg for systemic applications, and lOμg to lmg for local applications. The specific binding member may be an antibody molecule such as a whole antibody, for example an IgG isotype in a quadroma format, or a fragment such as a Fab, (Fab)₂ or scFv. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.

Specific binding members as described herein which bind Rib.Po find particular application in the treatment of malaria.

Rib.PO has been shown to be a host-protective immunogen which provides a cross-species passive protection and may represent a universal target antigen against malarial infections. This is evidenced by rabbit/mouse anti-PfPO antibodies which protect mice against challenge with the lethal 17XL variant of the rodent malarial parasite P. yoelii (Chatterjee et al (2000) Mol. Biochem. Parasitol. 107:143-154).

Human and Plasmodium falciparum Rib.PO exhibit significant homology in different regions of the molecule and their C terminal regions are highly conserved. Antibodies to both the N and C termini of the molecule protect against P. falciparum . 10 per cent of sera from Systemic lupus erythematosus patients that recognise Rib.PO of human origin also bind to that of P. falciparum origin, while normal subjects show no reactivity to either human or P. falciparum PO. Further, Systemic lupus erythematosus (SLE) sera containing anti-Rib. PO IgG, specifically inhibit the growth of P. falciparum through the cross-reactive anti-PO antibodies.

Specific binding members which bind Rib.PO, such as those described herein, are therefore useful in the treatment of malaria, for example cerebral malaria. Active vaccination attempts with large PfPO constructs may have the risk of generating activated B cells that may produce host cross-reactive anti-PO antibodies. Passive immunisations, on the other hand, have been performed on malaria patients with preparations of IgG obtained from immune adults, and the recipients have shown no ill effects from such treatments (Cohen et al (1961) Nature 192:733-737, Edozien et al (1962) Lancet ii: 951-955, McGregor et al (1963) Trans. R. Soc. Trop. Med. Hyg. 57:170-175, Sabchareon et al (1991) Am. J. Trop.

Med. Hyg. 45:297-308). Further, mice that recovered completely following passive immunisation also lack SLE-like symptoms (Chatterjee et al 2000 Supra) .

Passive immunisation using a bispecific anti-Rib.Po antibody molecule may thus provide a non-toxic approach towards achieving host protection against malaria.

The therapeutic usage of bispecific anti-Rib. PO antibody molecules as described herein is particularly beneficial in acute cases of cerebral malaria, especially in regions of drug-resistant P. falciparum. Rib.PO is an essential protein, as established by the knockout studies performed in S. cerevisiae (Santos and Ballesta 1994. supra) as well as in vitro growth inhibition assays in P. falciparum culture

(Chatterjee et al. 2000. supra, Goswami et al (1997) J. Biol. Chem. 272: 12138-43). PfPO is therefore unlikely to undergo deletion or a significant degree of polymorphism under therapeutic pressure.

Malaria therapy involving the passive immunisation against PfPO using an antibody molecule provided herein reduces the probability of the parasite developing resistance. Small antibody molecules such as Fabs and scFvs find particular application because the absence of an Fc domain may reduce toxicity, and the reduced molecular weight increases the ability to access intracellular targets such as RibPo.

Further aspect of the present invention provides a method of treatment of malaria comprising administration of a specific binding member which binds Rib.Po as described herein, pharmaceutical compositions for use in treating malaria comprising such a specific binding member, and use of such a ^• specific binding member in the manufacture of a medicament for administration in the treatment of malaria.

Specific binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.

Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatine or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous administration, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Such administration may be by means of an injection. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Other treatments may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics.

A further aspect of the present invention provides a method comprising causing or allowing binding of a specific binding member as provided herein to a target antigen. As noted, such binding may take place in vivo, e.g. following administration of a specific binding member, or nucleic acid encoding a specific binding member, or it may take place in vitro, for example in ELISA, Western blotting, immunocytochemistry, immuno-precipitation or affinity chromatography. Binding may include the binding of a variable domain pairing to one antigen or to two different antigens at the same time.

The amount or extent of binding of specific binding member to a target antigen may be determined. Quantitation may be related to the amount of the antigen in a test sample, which may be of diagnostic interest.

The reactivities of specific binding members on a sample may be determined by any appropriate means. Radioimmunoassay (RIA) is one possibility. Radioactive labelled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the specific binding member. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the specific binding member determined. The more antigen there is in the test sample the less radioactive antigen will bind to the specific binding member. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochro es include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine .

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may also be employed.

The signals generated by individual specific binding member- reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant antibody binding in samples (normal and test) .

The present invention also provides the use of a specific binding member as above for measuring antigen levels in a competition assay, that is to say a method of measuring the level of antigen in a sample by employing a specific binding member as provided by the present invention in a competition assay. This may be where the physical separation of bound from unbound antigen is not required. Linking a reporter molecule to the specific binding member so that a physical or optical change occurs on binding is one possibility. The reporter molecule may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule. The present invention also provides for measuring levels of antigen directly, by employing a specific binding member according to the invention, for example in a biosensor system.

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

Specific binding members and antibody molecules comprising variable domain pairings according to the present invention may be provided, isolated and/or purified, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes other than the sequence encoding a polypeptide with the required function.

A further aspect of the present invention comprises a nucleic acid molecule comprising a sequence encoding a VL domain and a sequence encoding a VH domain which together form a variable domain pairing as described herein. The two sequences may be operably linked to one or more regulatory elements.

Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

Systems for the cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast baculovirus and plant systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. Cloned antibody molecules may also be expressed in plants (Tavladoraki et al. (1993) Nature 366: 469-472) such as tomato and tobacco using conventional techniques, for example, Agrobacterium or Biolistic techniques (Reviewed in Ma and Hein (1996) Ann. N.Y. Acad. Sci. 792:72-81). Expression in a transgenic plant may provide a cheap method of producing medicaments, for example anti-malarials such as anti-Rib. PO antibody molecules, suitable for developing countries.

A common, preferred bacterial host is E. coli .

The expression of specific binding members and antibody molecules in prokaryotic cells such as E. coli is well established in the art (For a review, see Plϋckthun, A. Bio/Technology 9: 545-551 (1991)). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding member, see for recent reviews, for example Ref, M.E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J.J. et al . (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual : 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

Thus, a further aspect of the present invention provides a host cell containing nucleic acid which encodes a hybrid variable domain pairing domain as described herein. Nucleic acid sequences encoding the VH and VL domains comprised in the hybrid variable domain pairing may be present on the same or different nucleic acid molecules in said cell. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE- Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include heat shock coupled with calcium chloride transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing. or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

A host cell containing nucleic acid according to the present invention, e.g. as a result of introduction of the nucleic acid into the cell or into an ancestor of the cell and/or genetic alteration of the sequence endogenous to the cell- or ancestor (which introduction or alteration may take place in vivo or ex vivo) , may be comprised (e.g. in the soma) within an organism which is an animal, in particular, a mosquito. Genetically modified or transgenic animals, in particular mosquitoes, comprising such a cell are also provided as further aspects of the present invention.

Anti-Pf.PO antibodies bind to gametocytic stages of Plasmodium falciparum (Goswami et al . Journal of Biological Chemistry 272: 12138-12143 (1997)). Gametocytic stages are infective to mosquitoes that act as a carrier of the malarial parasite.

The introduction of genes encoding anti-Rib. PO antibody molecules as described herein into the mosquito gut provides a novel approach to control the parasite growth in mosquito. The growth of the gametocytes ingested by a mosquito via blood meal may be inhibited by the anti-Rib. PO antibodies being expressed in the gut. Technology for the construction of transgenic mosquitoes expressing antibody molecules in their guts is well known to skilled workers in the art (Coates. Malaria. A mosquito transformed. Nature. 2000 405: 900-1).

Thus, in various further aspects, the present invention provides a mosquito with genes encoding VL and VH domains which form a hybrid antigen binding domain which binds Rib.PO as described herein within its genome. Preferably the genes are operably linked to a regulatory element for expression in the mosquito gut .

A number of monoclonal antibodies directed against a range of antigens of interest, have already been cloned using hybridoma technology. The variable domains of these known antibodies can be cloned into suitable vectors, for example those described herein, and subsequently expressed in a heterologous expression system such as E. coli, using methods well-known in the field of antibody technology (see McCafferty et al 1996. Antibody Engineering, IRL Press at Oxford University Press, Oxford) .

Alternatively, antibodies with novel binding specificities for the novel antigens of interest, can be selected using phage display libraries. Phage-display technology provides an efficient in vitro method for the selection of high-affinity recombinant antibody fragments and has been used extensively to isolate human Fab and scFv fragments against a wide range of cancer cell surface markers (Hoogenboom et al . 1998.

Immunotechnolog. 4, 1-20) and proteins that can be targeted for cancer therapy (Pini et al 1998 J. Biol . Chem . 273, 21769- 21776) .

Mono-specific antibody molecules, for example Fab fragments, reactive against antigen A (Fab AA) or B (Fab BB) selected following panning using phage display libraries, may be used to generate antibody Fabs with dual reactivity (AB and/or BA) simply by described shuffling of their H/L chains. The art of affinity maturation of antibodies using phage display is well documented- in the literature (Johnson and Hawkins 1996. In Antibody Engineering, eds. MsCafferty, Hoogenboom and Chiswell, pp. 41-58, IRL Press at Oxford University Press, Oxford; Jackson et al . 1992. In Protein Engineering, eds. Rickwood and Ha es, pp. 277-300, IRL Press at Oxford University Press, Oxford) .

Light and heavy chains of the two antibody molecules of interest (whose specificities are to be combined/incorporated into a hybrid antibody) generated/selected via phage display may be sequenced at DNA level to deduce/determine the amino acid sequences of their heavy and light chains. These sequences may then be cloned into the vectors described herein (light, pAGP2, V4; heavy, pAGPl, V5; Kumar et al 2000 supra) via recursive PCR (Prodromou and Pearl 1992. Prot . Eng. 5: 827-829) .

Alternatively, the genes encoding for the variable domains of antibody molecules of interest can be directly cloned in an appropriate vector, preferably a prokaryotic expression vector such as those described herein, via PCR. Heavy and light chains can then be shuffled as described. The hybrid antibodies may then be evaluated for their ability to bind to the two antigens in ELISA.

Alternatively, the heavy and light chains of these antibodies may be shuffled with the heavy and light chains of the antibodies with known antigen specificities and which are known or pre-determined to be negative to the antigens of interest. The screening of the hybrid constructs against the antigens of interest reveals on which chain the antigen specificity resides. An appropriate pairing or combination of the heavy and light chains is then made.

The above approach may be used to screen for antibody molecules in which reactivity to the antigens predominantly resides on one of the two chains. Such molecules may be used to generate specific binding members such as Fab (or scFv) molecules with dual reactivity as described herein.

For example, monospecific Fab molecules reactive against antigen A (Fab AA) or B (Fab BB) which are selected following panning using phage display libraries and whose reactivity resides predominantly on one of the chains, can be used to generate an antibody molecule (for example, an antibody fragment) with dual reactivity (AB and/or BA) simply by described shuffling of their H/L chains.

Suitable parent antibody molecules for use in accordance with the present invention may be identified by various methods, for example phage display as described above.

An antibody molecule may be tested to determine whether reactivity for an antigen resides predominantly on one of the chains, by determining the reactivity for the antigen of each individual chain (i.e. a heavy or light chain) when in combination (i.e. paired with a light or heavy chain to form a binding domain) with a chain which is not reactive for said antigen.

The reactivity of such an antibody molecule, which comprises a heavy or a light chain from a parent antibody molecule, may be compared to the reactivity of the parent antibody molecule.

Reactivity of an antigen may be determined by conventional methods known in the art as described herein such as ELISA.

A further aspect of the present invention provides a method of producing a specific binding member comprising a hybrid variable domain pairing as described herein, the method comprising, providing a first antibody molecule which specifically binds a first antigen, in which the reactivity for the first antigen resides on the light chain, providing a second antibody molecule which specifically binds a second antigen, in which the reactivity for the second antigen resides on the heavy chain, expressing the light chain from the first antibody molecule and the heavy chain from the second antibody molecule, and combining the chains to associate to produce a hybrid variable domain pairing.

The binding of the hybrid variable domain pairing to the first antigen and the second antigen may be determined. This may include determining either sequentially or simultaneously the^' binding affinity of the antigen binding domain for the first antigen and determining the binding affinity of the antigen binding domain for the second antigen.

The binding of the hybrid variable domain pairing to a third antigen different from the first and second antigens may also be determined.

Binding of the hybrid variable domain pairing may be determined by known methods, including ELISA and Surface Plasmon Resonance.

Antibody molecules which bind to the first or second antigen may be identified and/or obtained by screening a phage display library. Antibody molecules in which the antigen reactivity resides on a particular chain may be identified using methods described herein. A method of producing a specific binding member comprising a hybrid variable domain pairing as described herein, may therefore include, isolating from a phage display library a first population of antibody molecules which specifically bind a first antigen, selecting a first antibody molecule of said population in which reactivity for the first antigen resides on the light chain, isolating from a phage display library a second population of antibody molecules which specifically bind a second antigen, selecting a second antibody molecule of said second population in which reactivity for the second antigen resides on the heavy chain, expressing the light chain of the first antibody molecule and the heavy chain of the second antibody molecule, and combining the chains to produce a hybrid variable domain pairing.

Methods as described herein may include causing expression of a light and a heavy chain comprising a VL and VH domain respectively as described herein from encoding nucleic acid. Host cells may be cultured under appropriate conditions for production of the heavy and light^' chains or VH and VL domains.

The reactivity, for example, the binding affinity, of the hybrid variable domain pairing for the first and second antigen may be determined using conventional techniques.

Methods may include isolating and/or purifying specific binding member comprising the hybrid variable domain pairing. Methods of production may also comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Methods of generating, manipulating and screening phage display libraries are routine in the art and are described in Sambrook J. and Russell D. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2001) and references therein.

Aspects of the present invention will now be illustrated with reference to the accompanying figures described below and experimental .exemplification, by way of example and not limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art. All documents mentioned in this specification are hereby incorporated herein by reference.

Figure 1 shows binding of hybrid Fabs to antigens in ELISA. Quantitative comparative assessment is expressed as per cent binding of recombinant Fabs in their native or hybrid (a:33Hll/4D5, b: B3/4D5) H/L chain combinations, to cardiolipin, Smith (S ) or Sm / ribonucleoprotein (RNP) and a pl85₉₂₉_₉₄₇ peptide, in relation to total binding to anti-human λ or K monoclonal antibody. Letters B, R and D denote B3,

33H11 and 4D5 respectively; the first of the two-letters name of the Fab construct denotes the light chain and the second of the letters the heavy chain.

Figure 2 shows a diagram of the construction of bi- and multispecific antibodies. Figure 3 shows a quantitative comparative assessment as per cent binding in relation to total binding to anti-human λ or K monoclonal antibody, of the recombinant Fab proteins in ELISA, to an immuno-dominant peptide (pl85₃₇₈-₃₉₄) derived from the extra-cellular domain of human c-erbB-2. Binding of a, native H/L chain combinations and 4D5 related hybrids or b, 4D5 unrelated hybrids. Letters B, R, U and D denote B3, 33H11, UK4 and 4D5 respectively; the first of the two-letters name of the Fab construct denotes the light chain and the second of the letters the heavy chain.

TERMINOLOGY

Specific binding member This describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. An example of a type of specific binding pair is antigen-antibody and this application is concerned with such reactions.

Antibody Molecule

This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is substantially homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibody molecules are the immunoglobulin isotypes and their isotypic subclasses, fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb and Fd, and diabodies.

As antibody molecules can be modified in a number of ways, the term "antibody molecule" should be construed as covering any specific binding member or substance having an immunoglobulin binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or . equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) . Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996) . Minibodies comprising a scFv joined to a CH3 domain may also be made (S. Hu et al, Cancer Res.,^" 56, 3055-3061, 1996) .

Conventional bispecific antibodies may be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the immunogenicity (including the immune response following the binding of the antibody to the target antigen) of the molecule and the effects of anti-idiotypic reaction.

Bispecific diabodies, .as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli . Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries.

Hybrid variable domain pairings as described herein may be used to produce antibody molecules, including bivalent antibody fragments such as diabodies, in a ^λbispecific' format which have four reactivities. Variable Domain Pairing

A variable domain pairing as described herein comprises an antibody light chain variable region (VL) associated with an antibody heavy chain variable region (VH) . The pairing may comprise a single antigen binding domain formed by both the light chain variable region (VL) and an antibody heavy chain variable region (VH) or two antigen binding domains, one domain being formed by each of the heavy (VH) and light chain (VL) variable domains.

Antigen binding domain

This describes the part of an antibody molecule which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain usually comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) . In the molecules described herein, the antigen binding domain comprises either an antibody light chain variable region (VL) or an antibody heavy chain variable region (VH) .

Cross Reactivi ty This is used to describe the binding of more than one antigen by the same antigen binding domain of a molecule.

Bi specifici ty

This is used to describe the binding of two different antigens by two separate antigen binding domains of the same molecule. In the molecules described herein, a hybrid variable domain pairing comprises the two separate antigen binding domains. Specific

This may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner (s) . The term is also applicable where for example, an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

Comprise

This is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

Isola ted

This refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members, will be in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo . Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Specific binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells e.g. CHO or NSO (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E.A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services-, 1987) and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu).

Experimental General

The principle underlying the present methods involves H (V_H- C_H1) and L (V_L-C_L) chain shuffling of antibodies with differing specificities .

Combinations of H and L regions from three hypothetical antibodies with specificities for antigen A, B or C indicated by white (Fig.2a), black (Fig.2b) and hatching (Fig.2c) respectively, are shown in Figure 2. V_L and C_L refer to variable and constant domains of the light chain; V_H, C_H1, C_H2 and C_H3 refer to the variable and the three constant regions of the heavy chain.

A bi- or multi-specific antibody molecule may have particular application in the situations where more than one cell surface marker represents the cell type(s) in a disease. Assuming that the respective antigen specificities reside on the L chain of anti-A antibody and on the heavy chains of anti-B and anti-C antibodies, bispecific antibodies (chimeric monovalent Fabs) with reactivity against antigen A and B (H2-L1) or A and C (H3-L1) can be generated (Fig.2d) by H and L chain shuffling. In a hypothetical situation where antigens B and C represent cell-surface markers in a disease state and their antibody reactivities reside on the same chain (H or L) thus not allowing chimeric Fab construction, the monovalent Fabs H2-L1 and H3-L1 can be linked to generate tri-specific F(ab)2 (Fig.2e) exhibiting reactivities against both the antigens. Cell targeting and its killing both can be achieved in this situation if anti-A reactivity confers toxicity to the targeted cell.

Alternatively, all the three antigens A, B and C could represent mutually exclusive cell surface markers in a disease state and the F(ab)2 can target all the three cell types. The chimeric Fabs can be either genetically engineered in their monovalent (Fig.2d), F(ab)2 (Fig.2e) or quadroma (Fig.2f) format or could be chemically conjugated using for example the maleiimide cross-linking reagent, TFM (Fig.2g) .

Only three H/L chain combinations are shown in the Fab format; there are obviously alternative and additional combinations; such combinations can also be used in scFv format. Bispecific Hetero-F(ab) 2 (prepared by cross-linking two different F(ab') fragments by their hinge disulfides) and quadroma (hybrid hybridoma; resulting from the fusion of two different hybridoma cell lines) are currently being used in clinical trials .

Materials and Methods Construction of Expression Vector.

4D5 heavy and light chain variable domains cloned on the basis of their published sequence (Celltech Therapeutics, Slough, UK) were employed in the study described herein. B3, 33H11 and UK4 all possess lambda, whereas 4D5 possesses kappa light chain. The L (ompA-V_λ-C_λ, B3, 33H11 and UK4; ompA- V_κ-C_κ, 4D5) and subsequently the H (ompA-V_H-C_Hl) chain variable and constant domains along with the ompA signal sequence cloned previously (Kumar et al (2000) supra) , were inserted into pAWtac via pAGP2 (K) (4D5) or our new vectors pAGP2 (λ) or V4 (pAGP2 λ equivalent, Kumar et al 2000 supra) (B3, 33H11 and UK4) and pAGPl or V8 (pAGPl equivalent, Kumar et al 2000 supra) .

The L chains of the four antibodies (B3, 33H11, UK4 and 4D5) were singly inserted into pAWtac as Xho I - Eco RI fragments thus constructing the four intermediate vectors - one for each antibody L chain: pAWtac-L (B3) , pAWtac-L (33H11) , pAWtac-L (UK4) and pAWtac-L (4D5) . The four H chains were finally singly inserted in each of the three constructs as Eco RI fragments, thus allowing the construction of the antibody and related hybrid final expression vectors.

The sequence of the VL domain of B3 has a database accession number: L22483. The sequence of the VL domain of 33H11, which was originally deposited as RH-14, has a database accession number: X95739. The sequence of the VL domain of UK4 has a database accession number: X91134. The sequence of the VL domain of 4D5 has a database accession number: 442925. One mutation was present in the sequence used in the present application: p55 E->Y.

The sequence of the VH domain of B3 has a database accession number: Y18126. Two mutations were present in the sequence used in the present application: p24 S->T, p62 D->G. The sequence of the VH domain of UK4 has a database accession number :X91130. The sequence of the VH domain of 33H11 was deposited as RH-14 and has a database accession number: X95740. The sequence of the VH domain of 4D5 has a database accession number: 442926.

Protein Expression and Detection

The final expression vector pAWtac containing V_H-C1 and V_λ-C_λ or V_κ-C_κ was transferred to the E. coli . expression strain

W3110.

At least five single colonies per construct were picked from the transformants and 50 ml cultures prepared in 250 ml shake flasks (overnight, 30 or 25 °C) . Fresh 1.5 L cultures in 5 L flasks, were then set up using the overnight culture to inoculate the medium to an OD₆oo of 0.08. Cultures were induced with ImM isopropyl β-D-thiogalactoside (IPTG) at an ODeoo of 0.5 and allowed to grow for further 4 to 16 h. 2TY containing 30 μg/ml chloremphenicol was used to prepare cultures. Muslin cloth was used on the flasks to allow better aeration. OD₆oo was recorded at periodic intervals before and after induction, and of the uninduced control cultures. Cultures were spun (9,000 rpm; 30 min; 4 °C; sorvall, Du Pont, USA) following induction. The cell pellet was suspended in ice-cold water (30 ml dH₂0 per 1 L culture) , stirred for 30 min at 4 °C and spun as above described. Supernatant was filtered (0.22 μm filters; millipore, Bedford, USA) and stored as periplasmic fraction at 4 °C.

Samples of periplasmic preparations were applied on the nitrocellulose membrane (Millipore; Bedford, USA) for dot blot analysis. The membrane was then blocked (skimmed milk, 20 min) , probed with goat anti-human lambda or kappa antibody (α- HL) conjugated to alkaline phosphatase (lh) and developed using 5-bromo-4-chloro-4-indolyl phosphate/nitro blue tetrazolium (BCIP-NBT) substrate. Proteins were transferred to nitro-cellulose membrane for their Western blot analysis following SDS-PAGE (Bollag and Edelstein (1991) Protein Methods. Wiley-Liss, New York) and probed as described above for dot-blot analysis.

Comparative assessment of the expression levels of the Fabs of B3, 33H11, UK4, 4D5 and their hybrids: Protein concentration of the purified Fab was determined by measuring the absorbance at 280 nm using UV-2401 PC (UV-VIS recording spectrophotometer, Shimadzu Corporation, Japan) . The level of expression in individual periplasmic batches was assessed using an enzyme-linked immunosorbant assay (ELISA) using the purified Fab as a standard.

Comparative functional assessment of the Fabs. Anti-cardiolipin activity of the Fabs was determined by ELISA. Reactivity against Sm and Sm/RNP was measured using a standard solid-phase ELISA involving antigen (Shield Diagnostics; Dundee, UK) or monoclonal anti-human light chain antibody (Mo -HL) pre-coated plates, according to the manufacturer's instructions .

For the assay of the Fab activity to pl85₉₂₉-₉ or pl85₃₇₈__394, the peptides (SIGMA/GENOSYS, Cambridge, UK; catalogue peptides) derived from the intra-cellular (pl85₉₂9-9 ) or extra-cellular domains (immuno-dominant peptide pl85₃₇₈-₃₉) of human c-erbB-2 were synthesised (Alta Bioscience, Birmingham) with N-terminus biotin conjugation. The polystyrene plates were coated (4 °C, overnight) with streptavidin (5 μg/ml, half of the wells) and Moα-HL (1:1000; one-fourth of the wells). The biotinylated peptide was applied to the streptavidin coated wells (5 μg/ml, lh, 37 °C) . The plate was then blocked with 2% bovine serum albumin (BSA) in PBS (lh, 37 °C) and the periplasmic preparations for the Fab constructs with or without IPTG induction, applied (lh, 37 °C) .

Binding of the Fabs to the pl85 peptides, cardiolipin, Sm, Sm/RNP or Moα-HL (total Fab/IgG capture) was detected using goat α-HL conjugated to alkaline phosphatase and p-nitrophenyl phosphate as substrate. The antigen specific binding is expressed in relation to that for Moα-HL as per cent binding using the following equation:

% Binding = [ (OD_antigen ~ background) ÷ (ODMOC-HL - background)] x 100

Reactivity against nuclear antigens was detected using an ANA- Bioblot kit (Biocode Biotechnology, Sclessin, Belgium) according to the manufacturer's instructions. In brief, nuclear antigens extracted from human HeLa cells (ENA) were separated by SDS-PAGE and blotted onto nitrocellulose together with pre- stained molecular weight markers facilitating accurate interpretation of the results. These ready-to-use strips were , incubated (1 h) with the Fab preparations at the concentration of approximately 20 μg/ml diluted in Tris-buffered saline containing blocking proteins and Tween-20. The strips were probed with α-HL conjugated to alkaline phosphatase (lh) and developed using 5-bromo-4-chloro-4-indolyl phosphate/nitro blue tetrazoliu (BCIP-NBT) substrate. Results were interpreted with reference to the control strips provided showing the exact positions of Sm, RNP, Ro, La, Jo-1, Scl-70 and Ribosomal Po antigen bands.

The letters B, R, U and D denote B3, 33H11, UK4 and 4D5, respectively, in the text and the figures; the first of the two letters name of the Fab construct denotes the L chain and the second of the letters the H chain. All chemicals were purchased from Sigma Immunochemicals, St. Louis, MO, unless otherwise mentioned. Polystyrene plates (Immunolon type 1) were purchased from Dynatech Labs, VA, USA. Moα-HL was from Immunostics, London.

Results

Dot blot analysis demonstrated the presence of the recombinant Fabs in the periplasm, supernatant and cell extracts from IPTG-induced, but not in those from uninduced cells. The expression of the Fabs was confirmed by western blot analysis. Bands in approximately 45 kDa (unreduced, Fab) and 22 kDa (reduced, free H and λ/κ chains) regions on Western blots, demonstrated the Fabs to be of correct size and in assembled form. Expression levels of up to 8 mg Fab protein per litre of culture, were recorded using the quantitative ELISA.

Functional ELISAs revealed that appropriate H and L chain combinations possessed a combination of antigen reactivities originally possessed by the parent antibodies (Fig. la, b) . RR exhibited nearly 40% binding against cardiolipin compared to around 20% binding of DD, whereas the anti-pl85₉₂9-₉7 reactivity of DD was nearly two (BB) or three (RR) fold higher compared to the other two Fabs .

Interestingly, ^' RD possessed significantly high anti- cardiolipin reactivity and its anti-pl85₉₂9-₉₄₇ reactivity was almost comparable to that of DD. BB possessed significantly higher anti-Sm and anti-Sm/RNP activity compared to that of DD. This high anti-Sm and anti-Sm/RNP activity of BB was transferred to BD via its L chain. In addition, BD retained anti-pl85929-₉7 reactivity comparable to that of DD. The hybrid Fabs DR and DB possessed only weak reactivity against the antigens tested. RD and BD thus represent bi- and tri-specific antibodies.

The Fabs exhibited a range of reactivity against ENA on

Western blots. BB exhibited significant activity to a number of ENA including 38 kDa Rib.PO protein. However, BD and DU exhibited significant reactivity to 38 kDa Rib.PO protein. DB exhibited weak reactivity to PO, whereas BD exhibited additional significant reactivity to pl85-derived peptides.

The Fabs exhibited anti-pl85₃₇a-₃9 activities in the following decreasing order of their per cent binding to pl85: BD>RD>DD = BB>DU>RR = UU ≡. UD ≤ DB = DR (including 4D5 related hybrids, Fig.3a); RB>BU>BR>UR>RU>UB (4D5 unrelated hybrids, Fig. 3b). The anti-pl85₃₇8-₃9 reactivity of 4D5 unrelated hybrid Fabs containing BB L chain, was either comparable to (BU) , or was a little less (BR) than, that of BB. RB exhibited highest anti- pl85₃7₈-₃₉₄ reactivity. RR and UU however, exhibited only insignificant anti-pl85₃g_9₄ reactivity.

The hybrid Fabs exhibited a range of reactivity against ENA on Western blots. BB exhibited significant activity to a number of ENA including 38 kDa Rib.Po protein. However, BD and DU exhibited significant reactivity for 38 kDa Rib.Po protein.

DU presents an interesting Fab construct exhibiting high reactivity/specificity predominantly against 38 kDa Rib.PO on Western blots. BD however, possesses significantly high reactivity to both 38 kDa Rib.PO protein and pl85₃₇₈-₃₉ peptide . The principle of the construction of bispecific monovalent Fab fragments described herein (Fig. 2d) could have various applications, for example in the field of cancer since more than one marker often identifies tumours (Scott, A.M. &• Welt, S. Curr. Opin . Immunol . 9, 717-722 (1997)). Such Fabs would be of use in the simultaneous recognition of at least one of the two cell surface markers followed by rapid tumour penetration due to their small size, and in in vi tro studies for instance, on cell-targeting, and cell-cell interactions where their fast systemic clearance due to their small size, is not a consideratio .

Since antigen specificity of a bispecific Fab or scFv, for the two antigens resides on separate polypeptide chains, the antibody reactivity with one antigen may not be inhibitory to its reactivity to the other antigen. Such antibody fragments may also have application as chelating antibodies binding to adjacent epitopes on the same antigen (Neri, D. et al. J. Mol . Biol . 246: 367-373 (1995)). Minimal-size antigen-binding protein domains have further application in studies on function, in vivo diagnosis and therapy and in diagnostic histopathology (Spooner, R.A. et al . Hum . Pathol . 25, 606-614 (1994)). The bispecific Fab or scFv would thus be useful for the simultaneous detection of one of the two antigens of interest.

An antibody fragment however, with more antigen binding arms and the size range of 60-120 kDa is ideal as it would allow rapid tumour penetration without fast clearance in the kidney

(Casey, J.L. et al . Br. J. Cancer. 81, 972-980 (1999), Humphreys, D.P. et al . J. Immunol . Methods . 217, 1-10

(1998)), in addition to retaining high avidity due to its higher valency (Muller, K.M. et al. Anal . Biochem . 261, 149- 158 (1998)).

The construction of for instance, a diabody with constituent bispecific scFvs, would present an ideal molecule possessing all of the described characteristics in addition to being multispecific - tetraspecific, in this instance. A bispecific diabody with constituent bispecific scFvs of the same species, will have the advantage of possessing high avidity to both the target antigens, simultaneously. The specificities can be recruited in a diabody for up to four different antigens, as per requirement. In addition, construction of the following formats is also possible: Fab dimer (Fig. 2e) , tri er (Fig. 2g) or chimeric Fab in quadroma format (Fig. 2f) .

A number of cell-specific antibodies are being evaluated as delivery agents to direct a cytotoxic component to a tumour site (Reiter, Y. & Pastan, I. Trends Biotechnol . 16, 513-520 (1998)). Since both Fab and scFv fragments provide effective and highly specific in vivo targeting reagents (Hudson P.

(1999) Curr. Opin. Immunol. 11, 548-557), the ability of the construction of monovalent bispecific Fab or scFv molecules using the described technology would complement traditional and currently available methods of the construction of bi- /multivalent antibodies.

The technology described herein provides more choice and flexibility of the inclusion of multiple specificities without the need to increase the molecular size of the resultant polyreactive bi-/multivalent antibody species which may result in reduced tumour penetration. The specific binding members DU, BD and DB are useful as anti- malarial compounds via passive immunization. They exhibit significant anti-Rib. PO activity, and possess at least one chain (DU, BD and DB; the other chain, D, is humanised) of human origin, thus they are less immunogenic in humans. DU and BD appear to be the best candidates as they both exhibit high anti-Rib. PO activity and recognise this antigen relatively specifically on the .Western blots. Further, the cross- reactivity of BD to pl85₃₇₈-₃g peptide is unlikely to be toxic to the host. P185 has been proposed to provide useful target for serotherapy (Goldenberg (1999) supra; Bast et al (1993) Cancer 71:1597-1601) and the anti-pl85 antibody 4D5, is currently under clinical trials (Ye et al (1999) Oncogene 18: 731-738) against the neoplastic phenotype among the patients over-expressing c-erbB-2.

BB exhibited significant anti-pl85₃₇₈_₃₉ cross-reactivity comparable to that observed for 4D5 (fig 3a) . Two hybrid Fabs possessed significantly high (RB) or comparable (RD) anti- pl85_{3 8}-₃₉₄ reactivity relative to 4D5. BD presented an interesting Fab- construct exhibiting significantly high reactivity to both pl85₃7₈-₃₉ and 38 kDa Rib.Po protein. DU however, possessed high reactivity/specificity predominantly against 38 kDa Rib.Po.

Interestingly, the hybrid Fab RB with constituent H/L chains derived from two distinct ^' anti-DNA antibodies, without any 4D5 H/L chain contribution, exhibited highest anti-pl85₃₇₈_₃₉₄ reactivity (Fig 3b) . RR H chain in a previous study (Kumar et al (2000) supra) appeared to be either inert or inhibitory to the RR auto-antigen reactivity probably in part, because the length of its H3 region (5 residues) was much smaller than the L3 region (8, B3; 9, RR; 6, UK4; 8, 4D5) of the antibodies currently employed. Effective antigen binding may be favoured when B cells express immunoglobulin molecules with CDR3s of more similar lengths (Brezinschek et al (1998) J. Immunol.160^': 4762-4767) . Other H3 regions (10, B3; 10, UK4 ; 9, 4D5) could thus offer better H/L chain combinations and may provide a partial explanation for the better antigen reactivity of some of the H/L chain combinations like RD and RB.

pl85 is encoded by a normal cellular gene c-erbB2 present on chromosome 17 (Fukushige et al (1986) Mol. Cell. Biol. 6: 955- 958) . In normal breast cells, c-erbB2 is present as a single gene copy, whereas its amplification and consequent over- expression have been found in 25-30% (van de Vijver et al (1988) Eur. J. Surg. Oncol. 14: 111-114; Sla on et al (1987) Science 235:177-182) of primary human breast cancer. Different studies (Natali et al (1990) Int J. Cancer .45 : 457-461) have demonstrated that when over-expressed, the c-erbB2 gene product represents a tumour-restricted marker.

Anti-receptor antibodies have shown potential utility in the down modulation of these cell-surface proteins (Ohnishi et al (1995) Br. J. Cancer.71: 969-973) and suppression of the malignant phenotype (Mountain and Adair (1992) Biotechnl. Genet. Eng. Rev. 10:1-142). pl85 has been proposed to provide useful target for serotherapy (Bast et al (1993) supra) . The molecules described herein with high anti-pl85 epitope activity with or without additional anti-Rib.Po reactivity, are useful in efficient cell targeting/therapy in a neoplastic condition.

Because pl85 is over-expressed in cells from a number of tumour types, hybrid Fabs exhibiting either anti-pl85₃₇₈-₃₉₄ peptide reactivity or reactivity to both pl85₃₇8-394 peptide and 38 kDa Rib.PO protein are useful as therapeutic and/or drug targeting molecules for use in the treatment of the tumours which over-express pl85. For example, anti-Rib. PO activity, which resides on BB L' chain, is retained in the hybrid BD (Fig. 2) , which also possesses anti-pl85 activity and is therefore useful as a therapeutic agent.

Rib.PO occurs both in the nucleus^' and cytoplasm and in addition to its role in protein translation, has been shown to be involved in DNA repair (Yacoub et al 1996. Nucleic Acids Research 24: 4298-303) and to over-express in some carcinoma (Barnard et al 1992. Cancer Research 52: 3067-72). DNA repair activity is known to be increased in cancer.

Cross-reactivity to other cellular antigens is often undesirable in a therapeutic antibody molecule. Cross- reactivity in the parent antibody molecule may be eliminated by the novel H/L chain combinations produced by shuffling as described herein.

The additional presence of anti-Rib.Po activity on an anti- pl85 antibody molecule allows the antibody molecule to interfere with protein translation and DNA repair machinery after the cell has taken up the antibody through the anti-pl85 activity of the antibody molecule.

Heavy and/or light chains of an auto-antibody may possess specificity/reactivity to an auto-antigen. A number of such examples are now known. Such auto-antigens may be crucial for cell physiology and the binding of an antibody molecule to such an antibody may therefore be cytotoxic. Such a binding specificity may be employed in a therapeutic/targeting antibody molecule to deliver the cytotoxic reactivity to a target cell.

There is an acute need of reagents that can be used to efficiently target tumour specific epitopes in cancer. Such targeting is required for various purposes for instance, for chelation of cell surface molecules or for the delivery of toxic molecules to .the cancer cells. Better recognition of, or reactivity to, the target molecules by an antibody should allow efficient achievement of such objectives.

The Fabs RB, BD and RD all possess higher reactivity to pl85_37g- ₃₉₄ compared to 4D5 (Fig 3) . RB, BD and RD are interesting candidates for their application as therapeutic or drug targeting molecules on two counts: first, they exhibit significantly high anti-pl85_{3 8}-₃₉₄ reactivity thus offering the possibility of better pl85 recognition in vivo, and second, they possess either both (RB) or at least one chain (BD and RD; D is humanised) of human origin thus being less immunogenic in humans.

The antibody molecule BD is of particular interest as it also possesses significant anti-Rib.Po activity. The presence of anti-Rib.Po activity in association with anti-pl85 activity on an antibody molecule, will have further application as Rib.Po protein has recently been shown to be a multifunctional protein with possible role in DNA repair beyond its known participation in protein translation (Yacoub et al (1996) supra) and to be absolutely required for the ribosomal activity and cell viability in yeast (Santos and Ballesta (1994) supra) . The presence of anti-Rib.Po activity on BD has direct therapeutic application, as described above. Its dual reactivity allows BD to efficiently target cancer cells via its high anti-pl85 activity and then to cause toxicity following its uptake by the cell, by its interference with protein translation and DNA repair machinery of the cell via its anti-Rib.Po activity. Both of these functions are provided by a single antibody molecule species, avoiding the need of expensive process of the conjugation of the targeting antibody with a toxic molecule to achieve cancer cell killing.

If the anti-Rib.Po activity of BD is inhibited following its exposure to DNA and/or pl85, BD could be linked in a (Fab')₂ or diabody format to DU, which possesses high anti-Rib.Po activity (Fig. 2). This (Fab')₂ should overcome the described inhibition.

Although many of these constructs also possess reactivity to DNA, BD is of particular interest in this regard. The inhibition experiments show that although dsDNA inhibits

(<50%) the reactivity of BD (with constituent B3 light chain known to possess specificity to DNA) against-pl85₃₇₈-₃9₄ the residual anti-pl85₃₇₈-₃9₄ activity was still fairly high. This indicates the application of this antibody in gene delivery. For example, BD would efficiently bind a gene (to be delivered, naked DNA) via its anti-DNA activity and following administration would target the complex to pl85 over- expressing cells thus efficiently delivering the gene following the uptake of the complex by these cells. Given the Fab fragments lack the effector function determined by the Fc region, anti-DNA activity of BD should exhibit minimal pathogenicity due to its possible non-specific ^• reactivity to some cells in vivo via its anti-DNA activity. Such pathogenicity (if any) would be further minimised by the blocking of anti-DNA reactivity of the Fab fragment by the DNA of the gene to be delivered. Further, although 4D5 also exhibited anti-DNA activity, it does not seem to exhibit pathogenicity, as it is now in clinical trials. However, anti- pl85₃₇₈-₃₉ activity of 4D5 was significantly inhibited following its exposure to dsDNA.

Reactivity to DNA may therefore be conferred to a hybrid antibody with specificity to a cell surface molecule and this characteristic can be directly used to deliver a gene (DNA) to the cell displaying the said surface marker. Such antibody molecules may therefore be useful in a method of gene therapy.

Claims

CLAIMS :

1. A specific binding member comprising a hybrid immunoglobulin ^• variable domain pairing which specifically binds a first and a second antigen.

2. A specific binding member according to claim 1 wherein said pairing consists of a VL domain which comprises a binding domain for said first antigen and a VH domain which comprises a binding domain for said second antigen.

3. A specific binding member according to any one of the preceding claims wherein said hybrid variable domain pairing additionally binds specifically to a third antigen different from said first and said second antigen.

4. A specific binding member according to any one of the preceding claims wherein said pairing consists of a VL domain of a first parent antibody molecule which specifically binds a first antigen and a VH region of second parent antibody molecule which specifically binds a second antigen.

5. A specific binding member according to claim 4 wherein the binding affinity of the hybrid variable domain pairing to the first and/or second antigen is increased relative to^' the binding affinity of the parent antibody which binds specifically to said antigen.

6. A specific binding member according to any one of the preceding claims wherein said pairing specifically binds to said first and second antigens at the same time.

7. A specific binding member according to any one of the preceding claims comprising a detectable label.

8. A specific binding member according to any one of the preceding claims comprising a light chain from a first parent antibody molecule and a heavy chain from a second parent antibody molecule, wherein said hybrid variable domain pairing is formed by the VL and VH domains of said light and heavy chains

,

9. A specific binding member according to any one of the preceding claims comprising two or more hybrid variable domain pairings .

10. A specific binding member according to any one of the preceding claims which is a scFv, F(ab)₂, F(ab'), bs-miniAb, sc-bsAb, diabody, triabody or tetrabody.

11. A specific binding member according to any one of the preceding claims wherein the VH domain is selected from the group consisting of a VH domain of B3 (Ace No: Y18126) , UK4 (Ace No: X91130) or 4D5 (Ace No: 442926) .

12. A specific binding member according to claim 11 comprising a hybrid variable domain pairing which consists of the VH domain of the parent antibody molecule 4D5 and the VL domain from a different parent antibody molecule.

13. A specific binding member according to claim 12 wherein said VL domain is cytotoxic.

14. A specific binding member according to claim 12 wherein said VL domain binds DNA.

15. A specific binding member according to claim 14 wherein said VL domain is a B3 VL domain.

16. A specific binding member according to any one of claims

1 to 11 comprising a hybrid variable domain pairing comprising the VL domain of the parent antibody molecule B3 and the VH domain from a different parent antibody molecule.

17. A specific binding member according to claim 16 wherein said VH domain binds to a cell surface receptor.

18. A specific binding member according to claim 17 wherein the cell surface receptor is pl85.

19. A specific binding member according to claim 11 comprising a hybrid variable domain pairing selected from the group consisting of a light chain from 4D5 and a heavy chain from UK4, a light chain from 4D5 and a heavy chain from B3, a light chain from 33H11 and a heavy chain from B3, or a light chain from 33H11 and a heavy chain from 4D5.

20. A specific binding member according to any one of the preceding claims which specifically binds Rib.PO.

21. A specific binding member according to any one of claims 1 to 12 which specifically binds a cell surface receptor

22. A specific binding member according to any one of claims 1 to 12 which specifically binds RNA or DNA.

23. A specific binding member according to any one of the preceding claims for use in the treatment of the human or animal body.

24. A method of treatment comprising administration of a specific binding member according to any one of claims 1 to 22 to an individual in need thereof.

25. A pharmaceutical composition comprising a specific binding member according to any one of claims 1 to 22.

26. Use of a specific binding member according to any one of claims 1 to 22 in the manufacture of a medicament for use in a method of immunotherapy.

27. A method of making a pharmaceutical composition comprising formulating a specific binding member according to any one of claims 1 to 22 with a pharmaceutically acceptable excipient .

28. Use of a specific binding member according to claim 20 in the manufacture of a medicament for the treatment of malaria.

29. A method of treatment of malaria comprising administration of a specific binding member according to claim 20 to an individual in need thereof.

30. A nucleic acid molecule comprising a sequence encoding a VL domain and a sequence encoding a VH domain which together form a hybrid immunoglobulin variable domain pairing which specifically binds a first and a second antigen.

31. A host cell containing nucleic acid which encodes a specific binding member according to any one of claims 1 to 22.

32. A transgenic animal comprising a cell according to claim 31.

33. A transgenic animal according to claim 32 which is a mosquito.

34. A method of determining the level of target antigen in a sample comprising causing or allowing binding of a specific binding member according to any one of claims 1 to 22 to the target antigen.

35. A method of producing a specific binding member comprising a hybrid variable domain pairing, the method comprising, providing a first antibody molecule which specifically binds a first antigen, in which the reactivity for the first antigen resides on the light chain, providing a second antibody molecule which specifically binds a second antigen, in which the reactivity for the second antigen resides on the heavy chain, expressing the light chain- from the first antibody molecule and the heavy chain from the second antibody molecule, and; combining the chains to produce a hybrid variable domain pairing.

36. A method according to claim 35 wherein said first antibody molecule is obtained by isolating from a phage display library a first population of antibody molecules which specifically bind the first antigen, and; selecting from said population, a first antibody molecule in which reactivity for the first antigen resides on the light chain.

37. A method according to claim 36 wherein said second antibody is obtained by isolating from a phage display library a second population of antibody molecules which specifically bind the second antigen, and; selecting from said second population, a second antibody molecule in which reactivity for the second antigen resides on the heavy chain.

38. A method of producing a specific binding member comprising a hybrid variable domain pairing, comprising; isolating from a phage display library a first population of antibody molecules which specifically bind a first antigen, selecting a first antibody molecule of said population in which reactivity for the first antigen resides on the light chain, isolating from a phage display library a second population of antibody molecules which specifically bind a second antigen, selecting a second antibody molecule of said second population in which reactivity for the second antigen resides on the heavy chain, expressing the light chain of the first antibody molecule and the heavy chain of the second antibody molecule, and combining the chains to produce a hybrid variable domain pairing.

39. A method according to any one of claims 35 to 38 comprising determining the binding of the hybrid variable domain pairing to the first antigen and the second antigen.

40. A method according to any one of claims 35 to 39 comprising determining the binding of the hybrid variable domain pairing to a third antigen different from the first and second antigens.

41. A method according to any one of claims 35 to 40 including isolating and/or purifying a specific binding member comprising said hybrid variable domain pairing.

42. A method according to claim 41 comprising formulating the specific binding member into a composition including a pharmaceutically acceptable excipient.