WO2012122512A1

WO2012122512A1 - Recombinant production of mixtures of single chain antibodies

Info

Publication number: WO2012122512A1
Application number: PCT/US2012/028583
Authority: WO
Inventors: Roland Buelow
Original assignee: Hco Antibody, Inc.
Priority date: 2011-03-10
Filing date: 2012-03-09
Publication date: 2012-09-13

Abstract

The invention relates to the recombinant production of mixtures of single chain antibodies.

Description

RECOMBINANT PRODUCTION OF MIXTURES OF SINGLE CHAIN

ANTIBODIES

Field of the Invention

The invention is from the field of recombinant production of polyclonal antibodies, in particular single chain antibodies.

Background of the Invention

Therapeutic Antibodies

Monoclonal antibodies have become increasingly important not only for biomedical research but also as clinical therapeutics in the treatment of various diseases, such as cancer. The list of therapeutic antibodies in clinical use is steadily increasing and includes, for example, HERCEPTIN® (trastuzumab), REOPRO™ (abciximab).

MYLOTARG™ (gemtuzumab), RITUXAN® (rituximab), SIMULECT™ (basiliximab), REMICADE™ (infliximab), SYNAGIS™ (palivizumab), ZENAPAX™ (daclizumab), and CAMPATH™ (alemtuzumab). Despite these successes, however, there is still room for new antibody products and for improvement in the properties and efficacy of existing antibody products.

For example, cancer cells are genetically unstable. Consequently, subpopulations of cancer cells that no longer express the target antigen (so called "antigen-loss tumor variants") can arise and may escape destruction by antibodies targeting the antigen, thereby making monoclonal antibody treatment less effective. For example, loss of CD20 expression has been reported in patients with B-cell non-Hodgkin's lymphoma following treatment with the anti-CD20 antibody, rituximab, leading to relapse of the lymphoma (Foran et al., Br. J. Hematol, 2001, 114(4):711-3; Massengale et al., 2002, J. Am. Acad. Dermatol. 46:441-443). Methods described for increasing the potency of antibodies include, for example, fusions with toxic compounds, such as radionuclides, toxins, e.g. maytansonoids, and the like. Each of these approaches, however, has its limitations, including technological and production problems and/or high systemic toxicity.

Polyclonal antibodies can also be used for therapeutic applications, for instance, for passive vaccination or for active immunotherapy, and currently are usually derived from pooled serum from immunized animals or from humans who recovered from the disease. The pooled serum is purified into the proteinaceous or gamma globulin fraction, so named because it contains predominantly IgG molecules. Polyclonal antibodies that are currently used for treatment include anti-rhesus polyclonal antibodies, gamma globulin for passive immunization, anti-snake venom polyclonal (CroFab),

THYMOGLOBULIN™ for allograft rejection, anti-digoxin to neutralize the heart drug digoxin, and anti-rabies polyclonal antibodies. In currently marketed therapeutic antibodies, an example of the higher efficacy of polyclonal antibodies compared to monoclonal antibodies can be found in the treatment of acute transplant rejection with anti-T-cell antibodies. While monoclonal antibodies are more specific than traditional polyclonal antibodies, often the gain in specificity comes at the cost of loss of efficacy. In vivo, antibody responses are polyclonal in nature, i.e., a mixture of antibodies is produced because various B-cells respond to the antigen, resulting in antibodies with various specificities being present in the polyclonal antibody mixture. In addition, the monoclonal antibodies on the market (anti-CD25 basiliximab) have been reported to be less efficacious than a rabbit polyclonal antibody against thymocytes (thymoglobulin). The use of pooled human sera, however, potentially carries the risk of infections with viruses such as HIV or hepatitis, with toxins such as lipopolysaccharide, with proteinaceous infectious agents such as prions, and with unknown infectious agents. Furthermore, the supply is limited and insufficient for widespread human treatments. Problems associated with the current application of polyclonal antibodies derived from animal sera in the clinic include a strong immune response of the human immune system against such foreign antibodies. Therefore, such polyclonals are not suitable for repeated treatment or for treatment of individuals that were injected previously with other serum preparations from the same animal species.

The art describes the idea of the generation of animals with a human

immunoglobulin repertoire, which can subsequently be used for immunization with an antigen to obtain polyclonal antibodies against this antigen from the transgenic animals (see, e.g. WO 01/19394, the disclosure of which is incorporated herein by reference in its entirety). However, many technological hurdles still will have to be overcome before such a system is a practical reality in larger animals than mice and it will take years of development before such systems can provide the polyclonal antibodies in a safe and consistent manner in sufficient quantities. Moreover, antibodies produced from pooled sera, whether being from human or animal origin, will always comprise a high amount of unrelated and undesired specificities, as only a small percentage of the antibodies present in a given serum will be directed against the antigen used for immunization. It is, for instance, known that in normal, i.e., non- trans genie, animals, about 1% to 10% of the circulating immunoglobulin fraction is directed against the antigen used for hyper- immunization; hence, the vast majority of circulating immunoglobulins is not specific.

Thus, while the need for mixtures of antibodies, including monoclonal antibodies, may have been recognized in the art, no acceptable solutions exist to economically make mixtures of antibodies in a pharmaceutically acceptable way. Single Chain Antibodies

Creation of single chain antibodies was first described by Bird, R.E. et al. (1988) Science Oct; 242:423-6 and Huston, J.S. et al., 1988, PNAS Aug; 85:5879-8, and is also disclosed in numerous patent documents, such as, for example,in US Patent Nos.

4,946,778; 5,260,203; 5,455,030; 5,518,889; 5,534,621 ; 5,869,620; 6,103,889;

6,025,165; 6,121,424; 6,027,725; and 6,515,110. In one example, single chain antibodies are camelid immunoglobulins composed solely of heavy (H) chains (Heavy-chain Abs or HCAbs) without light (L) chains (Hamers-Casterman et al., 1993, Nature 363: 446). In such single chain antibodies, the combining site is formed by a single VH domain, termed VHH. In HCAbs, the H chains occur as disulphide bridged dimers- and lack the first constant domain (CHI) (Muyldermans et at, 1994, Protein Engineering 1: 1129). In the dromedary, the CHI exons from HC genes that give rise to HCAbs are spliced out during mRNA maturation as a result of a point mutation at the canonical splicing donor site (Nguyen et ah, 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 VH domain and the CHI are eliminated due to DNA rearrangements. Single chain antibodies comprising light chain gene elements, i.e. VL and/or Jl genes) also have been described (see, e.g. WO 2004/049794). Recombinant production of mixtures of antibodies, including single chain antibodies, is described, for example, in WO

2004/009618 and US Patent No. 7,429,486.

To date, an in vitro molecular approach to construction of single domain combining sites has been used in which VH/VL interface mutations are introduced (e.g. G44E, L45R and W47G) in order to prevent the formation of a domain pair. A repertoire of such single VH domains was expressed on phage and selected against haptens and proteins, which led successfully to single VH binding domains of moderate affinity (Riechmann and Muyldermans, 1999, J. Immunol. Methods 231: 25; Davies and

Riechmann, 1995, Biotechnology U: 475). However, there will be several advantages in utilising in vivo methods for selection of human single chain antibodies (HCAbs), particularly in the use of transgenic mice. Production of Human Immunoglobulins in Transgenic Mice

Because of their clinical therapeutic advantages for treatment of diseases including cancers and infections, several procedures have been explored for production of human antibodies, avoiding immunization of humans. They include molecular techniques of 'humanization', in which CDRs from mouse antibodies replace the human CDRs in human V region frameworks, selection of binders from genetic libraries of human antibody combining sites by phage display in vitro, and expression of human Ig genes in transgenic animals, including mice. In the last of these, elements of the human H and L genetic loci, including VHs, Ds, JHs and VLs and h segments with C region genes all in germline configuration, are cloned into yeast artificial chromosomes and introduced into mouse embryonic stem cells. In the transgenic animals thus obtained, B cells rearrange the human V genes in normal fashion (i.e. VHDJH and VLh) and express fully human immunoglobulins (Igs), responding to immunisation by production of fully human antibodies. Optimally, the expression of endogenous mouse Ig is suppressed by knockout of the mouse H and L chain loci. A number of different knockout strategies have been employed, such as silencing of H chain expression by deletion of the f .L chain membrane domain (Kitamura et al., 1991, Nature 350: 423) and inactivation of the L chain locus by deletion of h segments. After immunisation of such multifeature transgenic knockout mice, 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 Briiggemann and Taussig, 1997, Curr. Opin. Biotechnol. 8: 455-458).

Brief Description of the Drawings Fig. 1 Schematic representation of an antibody and single chain antibodies. Fig. 2 Schematic representation of a bi-specific monoclonal antibody and a bispecific single chain antibody.

Summary of the Invention

The present invention is based, at least in part, on the recognition that a mixture of single chain antibodies specifically binding to a target antigen can be efficiently produced in a single recombinant host cell.

In one aspect, the invention concerns a method for production of a mixture of single chain antibodies, the method comprising

(a) immunizing an animal capable of expressing a diversified repertoire of single chain antibodies with one or more target antigens to generate a repertoire of antigen- specific single chain antibodies;

(b) isolating nucleic acid molecules encoding at least three non-identical antigen-specific single chain antibodies from said transgenic animal; (c) introducing the nucleic acid molecules in a single recombinant host cell and

(d) producing a mixture of single chain antibodies.

In one embodiment, the animal is naturally capable of expressing a diversified repertoire of single chain antibodies. Such animals include camelids, such as camel, lama, and alpaca, as well as sharks.

In another embodiment, the animal is genetically engineered to express a diversified repertoire of single chain antibodies. Examples of such animals include rodents, such as mice and rats, rabbits, birds, goats, cattle and sheep. In yet another embodiment, the transgenic animal is a transgenic mouse or rat carrying artificial heavy chain immunoglobulin loci in unrearranged configuration.

In a further embodiment, the transgenic animal has been engineered to produce heavy chain only antibodies of various isotypes.

In a still further embodiment, the single chain antibodies are heavy chain only antibodies.

In an additional embodiment, the single chain antibodies are selected from the group consisting of scFv, VHH antibodies and fragments thereof, and domain antibodies (dAbs) and fragments thereof.

In another embodiment, in step (b) of the process described above, nucleic acid encoding three non-identical antigen- specifics single chain antibodies is isolated.

In yet another embodiment, in step (c) the recombinant host cell is an eukaryotic or prokaryotic cell.

In a further embodiment, the recombinant host cell expresses a first, a second and a third non-identical antigen specific single chain antibody, where the first, second and third non-identical antibodies may, for example, have differing specificities for the same target antigen and/or differing affinities for the same target epitope. In another embodiment, the first, second and third single chain antibodies bind to at least two different antigens.

In a still further embodiment, the mixture of single chain antibodies produced in step (d) of the above method comprises homodimers and heterodimers of said single chain antibodies.

In another embodiment, the mixture of single chain antibodies produced in step (c) is composed predominantly of homodimers of said single shain antibodies.

In yet another embodiment, the mixture of single chain antibodies produced in step (c) is composed predominantly of heterodimers of said single chain antibodies.

In a further aspect, the invention concerns a recombinant host cell comprising nucleic acid encoding at least three non-identical antigen- specific single chain antibodies.

In one embodiment, the recombinant host cell comprises nucleic acid encoding three non- identical antigen- specific single-chain antibodies. In another embodiment, the nucleic acid comprised in the recombinant host cell encodes heavy chain only antibodies.

In yet another embodiment, the single chain antibodies are selected from the group consisting of scFv, VHH antibodies and fragments thereof, and domain antibodies (dAbs) and fragments thereof. The recombinant host cell may be eukaryotic or prokaryotic, including

mammalian cells and plant cells.

In a further aspect, the inventionc concerns a composition comprising a mixture of at least three non-identical single chain antibodies.

In one embodiment, the composition is a pharmaceutical composition. In another embodiment, the composition is a diagnostic composition.

In a further embodiment, the composition is a pharmaceutical compsition, wherein the composition has a biological activity exceeding the biological activity of each single chain antibody present in the composition. In a further aspect, the invention concerns a transgenic non-human animal comprising a nucleic acid sequence or nucleic acid sequnces encoding at least three different single chain antibodies.

It is noted that two or more of the various embodiments listed above or otherwise disclosed herein can be used in any combination, and any and all of such combinations are within the scope of the present invention.

It is further noted that various embodiments described in connection with one aspect of the invention are also contemplated with respect to other aspects of the invention.

Detailed Description of the Invention

I. Definitions

Unless otherwise defined, all terms of art, notations and other scientific

terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Also, for example, Current Protocols in Molecular Biology, Supplement 93, January 2011, John Wiley & Sons, Inc. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise.

Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. When trade names are used herein, applicants intend to independently include the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification. Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

The term "antigen" refers to an entity or fragment thereof which can bind to an antibody or trigger a cellular immune response. An immunogen refers to an antigen which can elicit an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term antigen includes regions known as antigenic determinants or epitopes. As used herein, the term "immunogenic" refers to substances which elicit the production of antibodies, and/or activate T-cells and/or other reactive immune cells directed against an antigen of the immunogen.

An immune response occurs when an individual produces sufficient antibodies, T- cells and other reactive immune cells in response to administered immunogenic compositions of the present invention to moderate or alleviate the disorder to be treated.

The term immunogenicity as used herein refers to a measure of the ability of an antigen to elicit an immune response (humoral or cellular) when administered to a recipient. The present invention is concerned with approaches that reduce the immunogenicity of the subject human chimeric or humanized antibodies..

Antibodies, also referred to as immunoglobulins, generally comprise two identical heavy chains and two identical light chains. Each heavy and light chain comprises an amino terminal domain that is variable and a carboxy terminal end that is constant. The variable domain from one heavy chain (V_H) and the variable domain from one light chain (V_L) together form an antigen binding site of an antibody. Accordingly, a native antibody generally has two antigen binding sites. A schematic depiction a native full- length antibody structure is provided in Fig. 1A. As shown in Fig.lA, the two heavy chains are covalently bound to each other by disulphide bonds at the constant region (C_H), and each heavy chain is covalently bound to the constant region of one of the light chains (C_L). The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour, of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, ,ηοη-human primate, murine, rat, rabbit or chicken origin. The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a .beta.-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the .beta.-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see for example: US Patent Nos. 4,816,567 and 5,807,715). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597; for example.

The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US Patent 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen- binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences. An "intact antibody" herein is one comprising a V_L and V_H domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more "effector functions" which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR. Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes." There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005/0048572; US 2004/0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequences of their constant domains.

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and or those residues from a "hypervariable loop" (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

"Fv" is the rriinimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Humanized" forms of non-human (e.g., rodent) antibodies, including single chain antibodies, are chimeric antibodies (including single chain antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanization is a method to transfer the murine antigen binding information to a non-immunogenic human antibody acceptor, and has resulted in many therapeutically useful drugs. The method of humanization generally begins by transferring all six murine complementarity determining regions (CDRs) onto a human antibody framework (Jones et al, (1986) Nature 321:522-525). These CDR- grafted antibodies generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired. Besides the CDRs, select non-human antibody framework residues must also be incorporated to maintain proper CDR conformation (Chothia et al (1989) Nature 342:877). The transfer of key mouse framework residues to the human acceptor in order to support the structural conformation of the grafted CDRs has been shown to restore antigen binding and affinity (Riechmann et al (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al (1993) J. Immunol. 151, 2623-2632; Werther et al (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001) Thromb. Haemost. 85:379- 389). For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see US Patent 6,407,213; Jones et al (1986) Nature, 321:522-525; Riechmann et al (1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596. A "functional Fc region" possesses an "effector function" of a native-sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc-receptor binding; ADCC; phagocytosis; down-regulation of cell-surface receptors (e.g., B-cell receptor), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody-variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein. A "native-sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native- sequence human Fc regions include a native- sequence human IgGl Fc region (non-A and A allotypes); native- sequence human IgG2 Fc region; native- sequence human IgG3 Fc region; and native- sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native- sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native- sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native- sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

"Homology" between two sequences is determined by sequence identity. If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, Wis. 53711). Bestfit utilizes the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489, in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. When using Bestfit, the so-called optional parameters are preferably left at their preset ("default") values. The deviations appearing in the comparison between a given sequence and the above-described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program "fasta20u66" (version 2.0u66, September 1998 by William R. Pearson and the University of Virginia; see also W. R. Pearson (1990), Methods in Enzymology 183, 63-98, appended examples and http://workbench.sdsc.edu/). For this purpose, the "default" parameter settings may be used.

The term "Fc-region-comprising antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region according to this invention can comprise an antibody with K447, with all K447 removed, or a mixture of antibodies with and without the K447 residue.

The term "single chain antibody" as used herein means a single polypeptide chain containing one or more antigen binding domains that bind an epitope of an antigen, where such domains are derived from or have sequence identity with the variable region of an antibody heavy or light chain. Parts of such variable region may be encoded by V_H or V_L gene segments, D and J_H gene segments, or J_L gene segments. The variable region may be encoded by rearranged V_HDJ_H, V_LDJ_H, V_HJ_L> or V_LJ_L gene segments. V-, D- and J-gene segments may be derived from humans and various animals including birds, fish, sharks, mammals, rodents, non-human primates, camels, lamas, rabbits and the like.

Heavy chain antibodies constitute about one fourth of the IgG antibodies produced by the camelids, e.g. camels and llamas (Hamers-Casterman C, et al. Nature. 363, 446- 448 (1993)). These antibodies are formed by two heavy chains but are devoid of light chains. As a consequence, the variable antigen binding part is referred to as the VHH domain and it represents the smallest naturally occurring, intact, antigen-binding site, being only around 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies termed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

The structure of a specific single chain antibody, an scFv, is schematically represented in Fig.lB. Examples of single antibodies according to the invention include full length heavy chain only antibodies, bispecific heavy chain only antibodies, antibody fragments like scFv and the like, and immunoconjugates, and the like. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

Further included within this definition are domain antibodies (dAb) (see, e.g. Holt et al., Trends in Biotechnology (2003) Vol. 21, No. 11: 484-490; U.S. Patent Publication Nos. 20090148434, 20100081792). dAbs comprise only the VH or VL domain of an antibody and are consequently smaller than, for example, Fab and scFv. DAbs are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. They are highly expressed in microbial cell culture. Each dAb contains three of the six naturally occurring complementarity determining regions (CDRs) from an antibody.

A single chain antibody according to the present invention may be isotype IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM, and the like, or a derivative of these. Single chain antibodies according to the present invention may form monomers, dimers (homodimers and heterodimers) or multimers. Single chain antibodies according to the invention can be of any origin, including animal derived, of more than one origin, i.e. chimeric, humanized, or fully human antibodies. Immunoconjugates comprise antigen binding domains and a non-antibody part such as a toxin, a radiolabel, an enzyme, and the like. The single chain antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific to different epitopes of a single molecule or may be specific to epitopes on different molecules.

"Different single chain antibodies" according to the present invention may differ in the variable region and have the same constant region. In other embodiments, where it is clear from the context, they may have the same variable region and differ in the constant region, e.g. be of a different isotype. The use of a mixture of antibodies having different constant regions, such as the Fc-portion, may be advantageous if different arms of the immune system are to be mobilized in the treatment of the human or animal body. In other embodiments, the "different single chain antibodies"differ both in their variable domain and in their constant region sequences. The "different single chain antibodies" differ at at least one amino acid position in their variable domain and/or constant region sequences. In various embodiments, the "different- single chain antibodies" share at least about 99%, or at least about 95%, or at least about 90%, or at least about 85%, or at least about 80%, or at least about 75%, or at least about 70% amino acid sequence identity in their variable domain and/or constant region sequences.

A "mixture of single chain antibodies" according to the invention comprises at least three non-identical (different) single chain antibodies, but may comprise 4, 5, 6, 7, 8, 9, 10, or more different single chain antibodies, and may resemble a polyclonal single chain antibody mixture with regard to complexity and number of functional antigen binding molecules.

An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

An antibody "which binds" an antigen of interest, is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targetting a cell expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a non- targeted antigen will be less than about 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). "Complement dependent cytotoxicity" and "CDC" refer to the lysing of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.

"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or FcRn receptor). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low- affinity antibodies bind antigen (or FcRn receptor) weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen (or FcRn receptor) more tightly and remain bound longer.

A "functional" or "biologically active" antibody (including single chain antibodies) is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signaling transduction or enzymatic activity. A functional antibody may also block ligand activation of a receptor or act as an agonist antibody. The capability of an antibody to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

The term "host cell" (or "recombinant host cell"), as used herein, is intended to refer to a cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Specifically included within the definition are rodents, such as mice and rats and animals creating antibody diversity by gene conversion. II. Detailed Description

It is an object of the present invention to provide a method for producing a mixture of single chain antibodies to at least one target in a single host cell. In particular, in the methods of the present invention at least three different single chain antibodies are expressed in a single host cell. According to the present invention, animals capable of expressing a diversified repertoire of polyclonal single chain antibodies are immunized with one or more target antigens to generate a repertoire of antigen-specific polyclonal single chain antibodies.

The animals might be naturally expressing single chain antibodies, such as single heavy chains or fragment thereof, in the absence of light chains, e.g. VHH antibodies. Such animals include camelids, such as camel, lama, and alpaca, as well as sharks.

Of particular interest, are genetically engineered animals with a repertoire of human or humanized single chain antibodies, or single chain antibodies with fully human idiotypes. Subsequent to immunization, nucleic acid encoding at least three different single chain antibodies specific to the target antigen(s) is detected in and isolated from the animals and expressed in a single recombinant host cell. In specific embodiments, the recombinant host cell is provided with nucleic acid sequences encoding for 3, 4, 5, 6, 7, 8, 9, 10, or more single chain antibodies capable of pairing with at least one light chain (in case of singe heavy chain antibodies) or heavy chain (in case of single light chain antibodies) to increase the complexity of the produced mixture of single chain antibodies. The obtained single chain antibodies will be functional and bind to the target antigen(s).

Methods for the production of substantially human antibodies to a specific antigen in transgenic animals are known in the art.

For example, WO 01/19394 describes the production of substantially human antibodies in transgenic domestic birds comprising genetically altered light and heavy chain immunoglobulin loci and at least a portion of human light and heavy chain immunoglobulin loci. The method employs stepwise modification of a domestic bird in which the antibody repertoir is diversified predominantly by gene conversion (including but not limited to chicken, turkey, ducks, goose, and quail). Antibody preparations can be obtained by fractionating egg yolk from genetically engineered birds expressing human sequence immunoglobulins. Alternatively, antibodies may be purified from serum.

Production of antibodies from transgenic animals, especially members of the rodent family, such as mice and rats, is also described in US Patent Nos. 5,814,318; 5,545,806; 5,545,807; 5,661,016; 5,789,650; and 5,569,825. Homologous recombination for chimeric mammalian hosts is exemplified in US Patent 5,416,260. A method for introducing DNA into an embryo is described in US Patent 5,567,607. Maintenance and expansion of embryonic stem cells is described in US Patent 5,453,357.

Immunization of animals, isolation of cells and cDNAs Antigen- specific polyclonal single chain antibodies can be raised in animals

(mammals) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues),

glutaraldehyde, succinic anhydride, SOCl₂.

Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with protein, peptide or conjugate in Freund's incomplete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.lt is also possible to immunize animals with DNA encoding expression cassettes of antigens. Injection of DNA with or without adjuvants induces an antibody response against the antigen encoded by the expression construct. Alternatively, animals may be immunized with whole cells, virus particles, bacteria, toxins, carbohydrates, etc. Expression of three or many single chain antibodies

Expression of several different single chain antibodies in a single host cell results in the expression of a mixture comprising monospecific and bispecific single chain antibodies. The structure of a bispecfic heavy chain only antibody is schematically represented in Fig.2.

If desired, formation of monospecific antibodies in the mixture can be favored over the formation of bispecific antibodies. If desired, formation of bispecific antibodies in the mixture can be favored over the formation of monospecific antibodies.

When single chain antibodies according to the present invention are expressed in a host cell at equal levels, the theoretical percentage of bispecific single chain antibodies produced by the method according to the invention is (l-l/n)*100 . The total number of different antibodies in the mixture produced by the method according to the invention is theoretically n+{ (n -n)/2}, of which (n -n/2) are bispecific antibodies. Distortion of the ratio of expression levels of the different single chain antibodies may lead to values deviating from the theoretical values. The amount of bispecific single chain antibodies can also be decreased, compared to these theoretical values, if not all single chain antibodies pair with equal efficiency. It is for instance possible to engineer the heavy chain antibodies, e.g. by introducing specific and complementary interaction surfaces between selected heavy chains, to promote homodimer pairing over heterodimer pairing. Heavy chain antibodies may also be selected so as to minimize heterodimer formation in the mixture.

A special form of this embodiment involves heavy chain antibodies of two or more different isotypes (e.g. IgGl, IgG3, IgA). Nucleic acid encoding such heavy chain antibodies can, for example, be isolated from transgenic animals carrying artificial heavy chain immunoglobulin loci comprising constant region genes encoding IgGl, IgG3, IgA isotypes, essentially as described in Example 1. When heavy chain antibodies of different isotype are expressed in the same host cell in accordance with the present invention the amount of bispecific heavy chain antibodies will be reduced, possibly to very low or even to undetectable levels. Thus, when bispecific heavy chain antibodies are less desirable, it is possible to produce a mixture of antibodies according to the invention, wherein nucleic acid sequences encoding at least three different heavy chain antibodies with different variable regions are expressed in a recombinant host, and wherein such heavy chain antibodies further differ in their constant regions sufficiently to reduce or prevent pairing between the different heavy chain antibodies.

Alternatively, when monospecific heavy chain antibodies are less desirable, it is possible to produce a mixture of antibodies according to the invention, wherein nucleic acid sequences encoding at least three different heavy chain antibodies with different variable regions are expressed in a recombinant host, and wherein said heavy chain antibodies further differ in their constant regions sufficiently to reduce or prevent pairing between the identical heavy chain antibodies.

In one embodiment, at least two single chain antibodies from the mixture produced according to the invention comprise a heavy-heavy chain dimer having different specificities and/or affinities. The specificity determines which antigen or epitope thereof is bound by the single chain antibody. The affinity is a measure for the strength of binding to a particular antigen or epitope. Specific binding is defined as binding with an affinity (Ka) of at least 5 *10E4 liter/mole, more preferably 5 * 10E5, more preferably more than 5 * 10E6, still more preferably 5 *10E7, or more. The mixture of single chain antibodies produced according to the present invention may contain at least two single chain antibodies that bind to different epitopes on the same antigen molecule and/or may contain at least two single chain antibodies that bind to different antigen molecules present in one antigen comprising mixture. Such an antigen comprising mixture may be a mixture of partially or wholly purified antigens such as toxins, membrane components and proteins, viral envelope proteins, or it may be a healthy cell, a diseased cell, a mixture of cells, a tissue or mixture of tissues, a tumor, an organ, a complete human or animal subject, a fungus or yeast, a bacteria or bacterial culture, a virus or virus stock, combinations of these, and the like. Unlike monoclonal antibodies that are able to bind to a single antigen or epitope only, the mixture of single chain antibodies according to the present invention may therefore have many of the advantages of a polyclonal or oligoclonal antibody mixture. Expression Constructs

For the isolation of cDNAs encoding antigen specific antibodies, B-cells from antibody producing animals can be isolated from blood, spleen, bone marrow, lymph nodes and the like. The isolation of B cells is well known to those skilled in the art. B cells may be isolated using fluorescence activated cell sorting or with magnetic beads and the like.

Reverse transcription of RNA isolated from B cells results in the generation of cDNA. Antibody encoding sequences are generated from cDNA by PCR. PCR products can be used to determine the nucleotide sequence of antibody encoding cDNA and the generation of expression constructs.

The nucleic acid sequences encoding the sinlge chain antibodies obtained may be converted to encode any desired antibody format such as IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, kappa, lambda before introducing them into a host cell, using standard molecular cloning methods and means known to the person skilled in the art (e.g.

described in Boel et al., Immunol. Methods 239:153-166, 2000).

For recombinant production of the single chain antibody mixture herein, the nucleic acid encoding the components of the mixture is isolated and inserted into a repiicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the desired single chain antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody variant). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, .alpha, factor leader (including Saccharomyces and Kluyveromyces a- factor leaders), or acid phosphatase leader, the C. Albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody .

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein coriferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding antibody, wild- type DHFR protein, and another selectable marker such as aminoglycoside 3'- phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See US Patent 4,965,199.

A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2- deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 .mu.m circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8: 135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991). Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody nucleic acid. Promoters suitable for use with prokaryotic hosts include the phoA promoter , beta-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (tip) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.

Antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in US Patent 4,419,446. A modification of this system is described in US Patent 4,601,978. Alternatively, the rous sarcoma virus long terminal repeat can be used as the promoter.

Transcription of DNA encoding the antibodies of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5 Or 3' to the antibody-encoding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.

Host Cells

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 4 IP disclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. . thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234), Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

Pharmaceutical and therapeutic antibodies synthesized in plants can be produced in a variety of ways. Since the first report of antibody production in N. tabacum plants (Hiatt et al., 1989, Nature, 342:76-78), antibodies have been expressed in moss (for review, see Decker and Reski, 2008, Bioprocess Biosyst. Eng., 31, 3-9), algae (for review, see Franklin and Mayfield, 2005, Expert Opin. Biol. Ther., 5, 225-235) and various dicot and monocot species, such as tobaco, rice. For review see, for example, De Muynck et al., 2010, Plant Biotechnology Journal, 8(5):529-563. Transgenic plants or plant cells producing antibodies have also been described (Hiatt et al., 1989, Nature, 342:76-78), and useful plants for this purpose include corn, maize, tobacco, soybean, alfalfa, rice, and the like. Constitutive promoters that can for instance be used in plant cells are the CaMV 35S and 19S promoters, Agrobacterium promoters nos and ocs. Other useful promoters are light inducible promoters such as rbcS. Tissue-specific promoters can for instance be seedspecific, such as promoters from zein, napin, betaphaseolin, ubiquitin, or tuber-specific, leaf-specific (e.g. useful in tobacco), root- specific, and the like. It is also possible to transform the plastid organelle by homologous recombination, to express proteins in plants. Methods and means for expression of proteins in recombinant plants or parts thereof, or recombinant plant cell culture, are known to the person skilled in the art and have been for instance been described in (Giddings et al, 2000; WO 01/64929; WO 97/42313; US Patent Nos. 5,888,789, 6,080,560; See for practical guidelines: Methods In Molecular Biology vol. 49 "Plant Gene Transfer And Expression Protocols", Jones H, 1995).

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN.TM.drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

It is an aspect of the present invention to provide a recombinant host cell comprising a nucleic acid sequence a nucleic acid sequence or nucleic acid sequences encoding at least three different single chain antibodies. The single chain antibodies expressed by the host cell are able to bind the target antigen or target antigens. The host cells according to the invention are useful in the method according to the invention. They can be used to produce mixtures of single chain antibodies according to the invention.

High level of expression In a preferred embodiment, the host cell according to the method of the invention is capable of high-level expression of human immunoglobulin, i.e. at least 1 pg/cell/day, preferably at least 10 pg/cell/day and even more preferably at least 20 pg/cell/day or more without the need for amplification of the nucleic acid molecules encoding the sinele chains in said host cell, Preferably, host cells according to the invention contain in their genome between

1 and 10 copies of each recombinant nucleic acid to be expressed. In the art, amplification of the copy number of the nucleic acid sequences encoding a protein of interest in e.g. CHO cells can be used to increase expression levels of the recombinant protein by the cells (see e.g. Bendig, 1988; Cockett et al, 1990; U.S.Patent No. 4,399,216). This is currently a widely used method. However, a significant time- consuming effort is required before a clone with a desired high copy number and high expression levels has been established, and moreover clones harbouring very high copy numbers (up to hundreds) of the expression cassette often are unstable (e.g. Kim et al., 1998). It is therefore a preferred embodiment of the present invention to use host cells that do not require such amplification strategies for high-level expression of the antibodies of interest. This allows fast generation of stable clones of host cells that express the mixture of single chain antibodies according to the invention in a consistent manner. We provide evidence that host cells according to the invention can be obtained, subcloned and further propagated for at least around 30 cell divisions (population doublings) while expressing the mixture of single chain antibodies according to the invention in a stable manner, in the absence of selection pressure. Therefore, in certain aspects the methods of the invention include culturing the cells for at least 20, preferably 25, more preferably 30 population doublings, and in other aspects the host cells according to the invention have undergone at least 20, preferably 25, more preferably 30 population doublings and are still capable of expressing a mixture of single chain antibodies according to the invention.

Purification

The antibodies are expressed by the cells according to the invention, and may be recovered from the cells or preferably from the cell culture medium, by methods generally known to persons skilled in the art. Such methods may include precipitation, centrifugation, filtration, size-exclusion chromatography, affinity chromatography, cation- and/or anion-exchange chromatography, hydrophobic interaction chromatography, and the like. For a mixture of antibodies comprising IgG molecules, protein A or protein G affinity chromatography can be suitably used (see e.g. US Patent Nos. 4,801,687 and 5,151,504).

Mixture of antibodies

It is yet another aspect of the present invention to provide a mixture of single chain antibodies that is obtainable by the method according to the invention, described above. Such mixtures can be used for a variety of purposes, such as in treatment or diagnosis of disease, and may replace, or be used in addition to, monoclonal or polyclonal antibodies. Such mixtures of single chain antibodies are expected to be more effective than the sole components it comprises, in analogy to polyclonal antibodies usually being more effective than monoclonal antibodies to the same target. Such mixtures can be prepared against a variety of target antigens or epitopes.

Method of identifying at least one host cell clone

It is another aspect of the present invention to provide a method for identifying at 5 least one host cell clone that produces a mixture of antibodies, wherein the mixture of antibodies has a desired effect according to a functional assay, the method comprising the steps of: (i) providing a host cell comprising a nucleic acid sequence or sequences encoding at least three different single chain antibodies; (ii) culturing at least one clone of the host cell under conditions conducive to expression of said nucleic acid sequences; 10 (iii) screening said at least one clone of the host cell for production of a mixture of single chain antibodies having the desired effect by a functional assay; and (iv) identifying at least one clone that produces a mixture of single chain antibodies having the desired effect.

In specific embodiments said culturing in step (ii) and said screening in step (iii) 15 of the method is performed with at least two clones. The method may optionally include an assay for measuring the expression levels of the single chain antibodies that are produced, which assay may be during or after step (ii) according to the method, or later in the procedure. Such assays are well known to the person skilled in the art, and include protein concentration assays, immunoglobulin specific assays such as ELISA, RIA, '.0 DELFIA, and the like. In particular embodiments of said method according to the invention, the host cell comprises nucleic acid sequence or sequences encoding at least 3, 4, 5, 6, 7, 8, 9, 10, or more single chain antibodies. Functional assays useful for the method according to the invention may be assays for apoptosis, ADCC, CDC, cell killing, inhibition of proliferation, virus neutralization, bacterial opsonization, '.5 receptormediated signaling, cell signaling, bactericidal activity, and the like. Useful screening assays for anti-cancer antibodies have for instance been described in US Patent 6,180,357. Such assays may also be used to identify a clone according to the method of the present invention. It is for instance possible to use enzyme linked immunosorbent assays (ELISAs) for the testing of antibody binding to their target. Using such assays, it is possible to screen for single chain antibody mixtures that most avidly bind the target antigen (or mixture of target antigens against which the mixture of single chain antibodies is to be tested). Another possibility that can be explored is to directly screen for cytotoxicity or cytostatic effects. It is possible that upon such a different screen, other or the same clones producing mixtures of antibodies will be chosen than with the ELISA mentioned above. The screening for cell killing or cessation of growth of cancerous cells may be suitably used according to the invention. Cell death can be measured by various endpoints, including the absence of metabolism or the denaturation of enzymes. In one possible embodiment of the present invention, the assay is conducted by focusing on cytotoxic activity toward cancerous cells as an endpoint. For this assay, a live/dead assay kit, for example the LIVE/DEAD® Viability/Cytotoxicity Assay Kit (L-3224) by Molecular Probes, can suitably be used. Other methods of assessing cell viability, such as trypan blue exclusion, 51Cr release, Calcein-AM, Alamar Blue™, LDH activity, and similar methods can also be used. The assays may also include screening of the mixture of single chain antibodies for specificity to the desired antigen comprising tissue. The single chain antibodies according to the invention may have a limited tissue distribution. It is possible to include testing the mixtures of antibodies against a variety of cells, cell types, or tissues, to screen for mixtures of antibodies that preferably bind to cells, cell types or tissues of interest.

Irrespective of a functional assay as described above, the present invention also teaches ways to determine the identity of the single chain antibodies expressed by a clone, using methods such as iso-electric focusing (IEF), mass-spectrometry (MS), and the like. It is therefore an aspect of the invention to provide use of MS and/or IEF in selecting a clone that expresses a mixture of antibodies according to the invention.

When monoclonal single chain antibodies are produced by recombinant host cells, a screening step is usually performed to assess expression levels of the individual clones that were generated. The addition of more heavy chains to produce mixtures adds a level of complexity to the production of antibodies. When host cells are transfected with nucleic acid molecules encoding single chain antibodies that will form the mixture of single chain antibodies desired, independent clones may arise containing the same genetic information, but nevertheless differing in expression levels, thereby producing different ratios of the encoded single chain antibodies, giving rise to different mixtures of single chain antibodies from the same genetic repertoire. The method according to the invention is useful for identifying a clone that produces an optimal mixture for a certain purpose.

The culturing and/or screening according to steps (ii) and (iii) respectively, may be suitably performed using high-throughput procedures, optionally in an automated fashion. Clones can e.g. be cultured in 96-well or other multi-well plates, e.g. in arrayed format, and screened for production of a desired mixture. Robotics may be suitably employed for this purpose. Methods to implement high-throughput culturing and assays are generally available and known to the person skilled in the art.

In specific embodiments of the present invention, said mixture of single chain antibodies according to the method of identifying at least one host cell according to the invention, comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more single antibodies having different specificities and/or affinities. A potential advantage of the method will be that it will allow exploring many possible combinations simultaneously, the combinations inherently including the presence of bispecific single chain antibodies in the produced mixture. Therefore more combinations can be tested than by just mixing purified known monoclonal single chain antibodies, both in number of combinations and in ratios of presence of different antibodies in these combinations.

Method of producing a mixture of single chain antibodies

The clone that has been identified by the method according to the invention, can be used for producing a desired mixture of antibodies. It is therefore another aspect of the present invention to provide a method of producing a mixture of single chain antibodies, the method comprising the step of: culturing a host cell clone identified by the method of identifying at least one host cell clone that produces a mixture of single chain antibodies according to the invention, said culturing being under conditions conducive to expression of the nucleic acid molecules encoding the at least three different heavy chains. The produced single chain antibodies may be recovered from the host cells and/or from the host cell culture, e.g. from the culture medium. The mixture of single chain antibodies can be recovered according to a variety of techniques known to the person skilled in the art.

Mixtures of different isotypes

Human antibodies are capable of eliciting effector function via binding to immunoglobulin receptors on immune effector cells. Human IgG, and in particular IgGl and IgG3 fix complement to induce CDC and interact with Fey receptors to induce antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis, endocytosis, induction of respiratory burst and release of inflammatory mediators and cytokines. Human IgA interacts with FcaR, also to result in efficient activation of ADCC and phagocytosis of target cells. Hence, due to the differential distribution of FcyR and FcaR on peripheral blood cells (Huls et al., 1999), using a mixture of single chain antibodies directed against the target and consisting of both IgG and IgA would potentially maximize the recruitment and activation of different immune effector cells. Such a mixture of both IgG and IgA could be obtained by producing the IgG and IgA single chain antibody in a separate production process using two distinct production cell lines, but could also be obtained from a single cell line producing both the IgG and the IgA single chain antibody. This would have the advantage that only a single production process has to be developed. Thus when different heavy chains are mentioned, also heavy chains differing in their constant regions are encompassed in the invention. The principle of using single chain antibodies can also be used for the production of a mixture of single chain antibodies of different isotypes from a host cell. It is therefore yet another aspect of the present invention to provide a method for producing a mixture of single chain antibodies comprising different isotypes from a host cell, the method comprising the step of: culturing a host cell comprising nucleic acid sequences encoding at least three different single chain antibodies of at least two different isotypes under conditions conducive to expression of said nucleic acid sequences. According to this aspect of the invention, different single chain antibodies may have identical variable regions, and only differ in their constant regions (i.e. be of different isotype and have the same specificity). In a particular embodiment, said isotypes comprise at least an IgG and an IgA and/or IgM, preferably IgGl or IgG3 and IgA. Other combinations of IgGl, IgG2, IgG3 and IgG4 can also be used. In these embodiments, bispecific single chain antibodies will not be produced because the variable regions are the same.

Homodimers

In other embodiments according to this aspect of the invention, not only the constant regions of the single chain antibodies may differ, but also the variable regions, thereby giving rise to different specificities. When bispecific single chain antibodies are not desired for a given purpose, e.g. because the mixtures of single chain antibodies are less efficacious because of the presence of the bispecific antibodies, it is possible to use at least three single chain antibodies according to the invention wherein said single chain antibodies differ sufficiently in their constant regions to reduce or prevent pairing between the different single chain antibodies, e.g. by using single chain antibodies of different isotypes, e.g. an IgGl and an IgG3 (see Fig for a schematic representation). It is anticipated that the single chain antibodies of different isotype will pair much less efficient, if at all, compared to single chain antibodies of one isotype. Alternatively, it is also possible to engineer the different single chain antibodies in their constant region such that homodimerization is favored over heterodimerization, e.g. by introducing self-complementary interactions (see e.g. WO 98/50431 for possibilities, such as "protuberance- into-cavity" strategies (see WO 96/27011)). It is therefore another aspect of the present invention to provide a method for producing a mixture of single chain antibodies in a recombinant host, the method including the step of: expressing in a recombinant host cell a nucleic acid sequences encoding at least three different single chain antibodies that differ in the variable region, wherein said single chain antibodies further differ in their constant regions sufficiently to reduce or prevent pairing between the different single chain antibodies. In one embodiment, said single chain antibodies are of different isotype. In specific embodiments, 3, 4, 5, 6, 7, 8, 9,10, or more different single chain antibodies are expressed. Mixtures of single chain antibodies obtainable by this method are also embodied in the present invention. Such mixtures will comprise mainly monospecific single chain antibodies.

Method to identify mixture of single chain antibodies It is yet another aspect of the present invention to provide a method for identifying a mixture of single chain antibodies having a desired effect in a functional assay, the method comprising the steps of i) adding a mixture of single chain antibodies in a functional assay, and ii) determining the effect of said mixture in said assay. In a preferred embodiment said mixture is comprised in a composition according to the present invention.

Composition comprising a mixture of recombinantly produced single chain antibodies

It is another aspect of the present invention to provide a composition comprising a mixture of recombinantly produced single chain antibodies, wherein at least three different single chain antibody sequences are represented in the mixture of recombinant single chain antibodies. The present invention discloses mixtures of single chain antibodies useful for diagnosis or treatment in various fields.

The compositions according to the invention comprise mixtures of at least three different single chain antibodies. The mixtures may comprise bispecific single chain antibodies. The mixtures may be produced from a clone that was derived from a single host cell, i.e. from a population of cells containing the same recombinant nucleic acid sequences. The mixtures can be obtained by methods according to the invention, or be produced by host cells according to the invention. In other embodiments, the number of heavy chain antibodies represented in said mixture is 4, 5, 6, 7, 8, 9, 10, or more. The optimal mixture for a certain purpose may be determined empirically by methods known to the person skilled in the art, or by methods provided by the present invention. Such compositions according to the invention may have several of the advantages of a polyclonal antibody mixture, without the disadvantages usually inherently associated with polyclonal antibody mixtures, because of the manner in which they are produced. It is furthermore expected that the mixture of single chain antibodies is more efficacious than separate monoclonal antibodies. Therefore the dosage, and hence the production capacity required, may be less for the mixtures of antibodies according to the invention than for monoclonal antibodies. It has for instance been described that although no single monoclonal antibody to botulinum neurotoxin (BoNT/A) significantly neutralized toxin, a combination of three such monoclonal antibodies (oligoclonal antibody) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyper immune globulin (Nowakowski et al, 2002). This result demonstrates that oligoclonal mixtures of antibodies comprising only 2 to 3 different specificities may have very high potency. Furthermore, the chances of a mixture of the invention losing its activity due to target or epitope loss is reduced, when compared to a single monoclonal antibody. In particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the single chain antibodies present in the mixture according to the invention have different specificities. Said different specificities may be directed to different epitopes on the same antigen and/or may be directed to different antigens present in one antigen comprising mixture. A composition according to the invention further may also comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more single chain antibodies having different affinities for the same epitope. Antibodies with differing affinities for the same epitope may for instance be generated by methods of affinity maturation, known to the person skilled in the art.

In a particularly preferred embodiment, the composition according to the invention has an effect that is greater than the effect of each individual monospecific single chain antibody present in said composition. Said effect can be measured in a functional assay. A "functional assay" according to the present invention is an assay that can be used to determine one or more desired parameters of the antibody or the mixture of antibodies subject to the assay conditions. Suitable functional assays may be binding assays, apoptosis assays, antibody dependent cellular cytotoxicity (ADCC) assays, complement dependent cytotoxicity (CDC) assays, inhibition of cell growth or proliferation (cytostatic effect) assays, cell killing (cytotoxic effect) assays, cell signaling assays, assays for measuring inhibition of binding of pathogen to target cell, assays to measure the secretion of vascular endothelial growth factor (VEGF) or other secreted molecules, assays for bacteriostasis, bactericidal activity, neutralization of viruses, assays to measure the attraction of components of the immune system to the site where single chain antibodies are bound, including in situ hybridization methods, labeling methods, and the like. Clearly, also in vivo assays such as animal models, including mouse tumor models, models of autoimmune disease, virus-infected or bacteria-infected rodent or primate models, and the like, can be used for this purpose. The efficacy of a mixture of single chain antibodies according to the invention can be compared to individual antibodies in such models by methods generally known to the person skilled in the art.

Transgenic animals or plants for production It is yet another aspect of the present invention to provide a transgenic non-human mammal or a transgenic plant comprising a nucleic acid sequence or nucleic acid sequences encoding at least three different single chain antibodies.

Besides cell culture as a production system for recombinant proteins, the art also discloses the use of transgenic animals, transgenic plants, and for instance transgenic chickens to produce proteins in the eggs, and the like to produce recombinant proteins of interest (Pollock et al, Journal of Immunological Methods. 231: 147-157, 1999; Larrick and Thomas, Curr. Opin. Biotechnol. 12: 411-4182001, 2001; WO 91/08216). These usually comprise the recombinant gene or genes encoding one or more proteins of interest in operable association with a tissue specific promoter. It has for instance been shown that recombinant antibodies can be produced at high levels in the milk of transgenic animals, that contain the nucleic acids encoding a heavy and a light chain behind a mammary gland specific promoter (e.g. Pollock et al, 1999, supra; WO95/17085). Particularly useful in this respect are cows, sheep, goats, pigs, rabbits, mice, and the like, which can be milked to obtain antibodies. Useful promoters are the casein promoters, such as the p-casein promoter, the a Sl-casein promoter, the whey acidic protein (WAP) promoter, the p-lactoglobulin promoter, the a-lactalbumin promoter, and the like. Production of biopharmaceutical proteins in the milk of transgenic mammals has been extensively described (e.g. Pollock et al, 1999, supra). Besides mammary gland specific promoters, also other tissue specific promoters may be used, directing the expression to the blood, urine, saliva, and the like. The generation of transgenic animals comprising recombinant nucleic acid molecules has been extensively documented, and may include micro-injection of oocytes (see e.g. Wilmut and Clark, Expenentia, 15;47(9):905-12, 1991), nuclear transfer after transfection (e.g. Schnieke et al, Science, 19;278(5346):2130-3, 1997), infection by recombinant viruses (e.g. US Patent No. 6,291,740), and the like. Nuclear transfer and cloning methods for mammalian cells are known to the person skilled in the art, and are e.g. described in (WO 95/17500 and WO 98/39416). It is nowadays possible to clone most of these animals, to generate lines of animals that are genetically identical, which renders it possible for a person skilled in the art to create such a line once an individual animal producing the desired mixture of single chain antibodies has been identified. Alternatively, classical breeding methods can be used to generate transgenic offspring. Strategies for the generation of transgenic animals for production of recombinant proteins in milk are described in Brink et al, Theriogenology, 2000, 53(1): 139-48. Other transgenic systems for producing recombinant proteins have also been described, including the use of transgenic birds to produce recombinant proteins in eggs (e.g WO 97/47739), and the use of transgenic fish (e.g. WO 98/15627), and can be used in combination with the teachings of the present invention to obtain mixtures of antibodies. It is also possible to use an in vitro transciption/translation or in vitro translation system for the expression of mixtures of single chain antibodies according to the present invention. It will be clear to the skilled person that the teachings of the current invention will allow producing mixtures of single chain antibodies in systems where recombinant nucleic acid encoding single chain antibodies can be introduced and expressed. Preferably such systems are able to produce single chain antibodies encoded by said nucleic acid sequences, without the use of amplification of said nucleic acid sequences in said systems.

A repertoire of antigen- specific single chain antibodies can also be produced in transgenic plants. Transgenic plants or plant cells producing antibodies have been described (Hiatt et al., 1989, supra; Peeters et al., Vaccine, 2001, 19(17):2756-61), the entirety of which are incorporated herein by reference) and useful plants for this purpose include corn, maize, tobacco, soybean, alfalfa, rice, and the like. Constitutive promoters that can, for instance, be used in plant cells are the CaMV 35S and 19S promoters and Agrobacterium promoters nos and ocs. Other useful promoters are light-inducible promoters such as rbcS. Tissue-specific promoters can, for instance, be seed- specific, such as promoters from zein, napin, beta-phaseolin, ubiquitin, or tuber-specific, leaf- specific (e.g., useful in tobacco), root- specific, and the like. It is also possible to transform the plastid organelle by homologous recombination to express proteins in plants.

In another aspect of the invention, a cell from a transgenic non-human animal or a transgenic plant according to the invention is provided. Such cells can be used to generate the animals or plants according to the invention, using techniques known to the person skilled in the art, such as nuclear transfer or other known methods of cloning whole organisms from single cells. The cells according to the invention may also be obtained by introducing at least three different single chain antibody sequences into isolated cells of non-human animals or plants, which cells are capable of becoming part of a transgenic animal or plant. Particularly useful for such purposes are embryonic stem cells. These can contribute to the germ line, and therefore the genetic information introduced into such cells can be passed to future generations. In addition, plant cell cultures of cotton, corn, tomato, soybean, potato, petunia, and tobacco can be utilized as hosts, when transformed with the nucleic acid molecules encoding single chain antibodies, e.g. by use of the plant transforming bacterium Agrobacterium tumefaciens or by particle bombardment, or by infecting with r-ecombinant plant viruses.

Pharmaceutical composition

It is another aspect of the present invention to provide a pharmaceutical composition comprising a mixture of recombinantly produced single chain antibodies and a suitable carrier, wherein at least two different heavy chains are represented in said mixture of recombinantly produced single chain antibodies. Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof. In particular embodiments, 3, 4, 5, 6, 7, 8, 9,10, or more different single chain antibodies are represented in said mixture. Said mixture can be obtained by mixing recombinantly produced monoclonal single chain antibodies, but may also be obtained by methods according to the present invention. Said mixture may comprise bispecific antibodies. Said mixture may be produced from a clone that was derived from a single host cell, i.e. from a population of cells containing the same recombinant nucleic acid molecules. The term "recombinantly produced" as used herein refers to production by host cells that produce single chain antibodies encoded by recombinant nucleic acids introduced in such host cells or ancestors thereof. It does therefore not include the classical method of producing polyclonal antibodies, whereby a subject is immunized with an antigen or antigen comprising mixture, after which the antibodies produced by this subject are recovered from the subject, e.g. from the blood.

Treatment and diagnosis It is another aspect of the present invention to provide a mixture of antibodies wherein at least three different single chain antibodies are represented, for use in the treatment or diagnosis of a human or animal subject. In another aspect, the invention provides the use of a mixture of single chain antibodies wherein at least three different single chain antibodies are represented, for the preparation of a medicament for use in the treatment or diagnosis of a disease or disorder in a human or animal subject. In particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9, la, or more single chain antibodies are represented in said mixture. Said mixtures of single chain antibodies may be mixtures of antibodies according to the invention, or obtained by methods according to the invention. The mixtures may comprise bispecific single chain antibodies, and may be recombinantly produced from a clone that was derived from a single host cell, i.e. from a population of cells containing the same recombinant nucleic acid molecules.

Targets and use

The targets may be used to immunize genetically engineered animals, as described supra, to obtain 2, 3, 4, 5, 6, 7,8, 9, 10, or more single chain antibodies that bind to the target, and produce a mixture of these according to the teachings of the present invention. Virtually any area of medicine where monoclonal antibodies can be used is amenable for the use of the mixtures according to the invention. This can e.g. include treatment of auto-immune diseases and cancer, including solid tumors of the brain, head- and neck, breast, prostate, colon, lung, and the like, as well as hematologic tumors such as B-cell tumors. Neoplastic disorders which can be treated with the mixtures according to the present invention include leukemias, lymphomas, sarcomas, carcinomas, neural cell tumors, squamous cell carcinomas, germ cell tumors, metastases, undifferentiated tumors, seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors, neoplasias caused by infectious agents, and other malignancies. Targets for single chain antibody mixtures may include, but are not limited to, the HER-2/Neu receptor, other growth factor receptors such as VEGFR1 and VEGFR2 receptor, B-cell markers such as CD19, CD20, CD22, CD37, CD72, etc, T-cell markers such as CD3, CD25, etc, other leukocyte cell surface markers such as CD33 or HLA-DR, etc, cytokines such as TNF, interleukins, receptors for these cytokines such as members of the TNF receptor family, and the like. It is anticipated that the use of such mixtures of single chain antibodies in the treatment of cancerous tissues or other complex multi-antigen comprising cells such as microorganisms or viruses will give rise to less occurrence of epitope-loss escape variants than the use of single monoclonalantibodies. Several treatments nowadays use polyclonal mixtures of antibodies, which are derived from immunized humans or animals. These treatments may be replaced by use of the mixtures according to the present invention.

Use of these mixtures can also include use in graft- versus-host rejections, known in the art of transplantation, e.g. by use of anti-thymocyte antibodies. It is anticipated that the mixtures of antibodies are superior to monoclonal antibodies in the treatment of complex antigens or antigen comprising mixtures such as bacteria or viruses. Therefore, use according to the invention can also include use against strains of bacteria and fungi, e.g. in the treatment of infectious diseases due to pathogenic bacteria such as multidrug resistant S.aureus and the like, fungi such as Candida albicans and Aspergillus species, yeast and the like. The mixtures according to the invention may also be used for post exposure profylaxis against viruses, such as members of the genus Lyssavirus e.g. rabies virus, or for therapeutic or prophylactic use against viruses such as Varicella-Zoster Virus, Adenoviruses, Respiratory Syncitium Virus, Human Immunodeficiency Virus, Human Metapneumovirus, Influenzavirus, West Nile Virus, the virus causing Severe Acute Respiratory Syndrome (SARS), and the like. Mixtures according to the inventions can also be used to protect against agents, both bacteria and viruses, and against toxic substances that are potential threats of biological warfare. Therefore, use according to the invention can also include use against strains of bacteria such as Bacillus anthracis, Clostridium botulinum toxin, Clostridium perfringens epsilon toxin Yersinia Pestis, Francisella tulariensis, Coxiell burnetii, Brucella species, Staphylococcus enterotoxin B, or against viruses such as Variola major, alpha viruses causing meningoencephalitis syndromes (EEEV, VEEV, and WEEV) , viruses known to cause hemorrhagic fevers such as Ebola, Marburg and Junin virus or against viruses such as Nipah virus, Hantaviruses, Tickborne encephalitis virus and Yellow fever virus or against toxins e.g. Ricin toxin from Ricinus communis and the like. Use of the mixtures according to the invention can also include use against unicellular or multicellular parasites. Recombinant mixtures of antibodies according to the invention may become a safe alternative to polyclonal antibodies obtained from pools of human sera for passive immunization, or from sera of hyper- immunized animals. The mixtures may be more efficacious than recombinant monoclonal antibodies in various therapeutic applications, including cancer, allergy, viral diseases, chronic inflammation, and the like.

It has been described that homodimerization of tumorreactive monoclonal antibodies markedly increases their ability to induce growth arrest or apoptosis of tumor cells (Ghetie et al, Proc Natl Acad Sci U S A. 1997 94(14):7509-14). Possibly, when single chain antibodies against receptors or other surface antigens on target cells, such as tumor cells or infectious microorganisms, are produced according to the present invention, the bispecific single chain antibodies present in mixtures according to the invention may also crosslink different receptors or other antigens on the surface of target cells, and therefore such mixtures may be very suitable for killing such cells. Alternatively, when bispecific antibodies are less desirable, the present invention also provides methods to recombinantly produce mixtures of single chain antibodies comprising mainly monospecific single chain antibodies. It has been described that the efficacy of treatment with Rituximab™ (anti-CD20 monoclonal antibody) was increased when anti-CD59 antibodies were added (Herjunpaa et al, 2000). Therefore, it is expected that inclusion of antibodies against CD59 in a mixture according to the invention comprising anti-tumor antibodies in the form of B-cell receptor recognizing antibodies increases the sensitivity of tumor cells to complement attack. It has also been shown that a triple combination cocktail of anti-CD19, anti-CD22, and anti-CD38-saporin immunotoxins is much more effective than the individual components in the treatment of human B-cell lymphoma in an immunodeficient mouse model (Flavell et al, Cancer Re.1997, 57(21):4824-9). Many other combinations may also be feasible and can be designed by the person skilled in the art. In general, the use of single chain antibody mixtures that are capable of recognizing multiple B-cell epitopes will likely decrease the occurrence of escape variants.

Another possible target is a transmembrane tyrosine kinase receptor, encoded by the Her-2/Neu (ErbB2) protooncogene (see e.g. US Patent Nos. 5,772,997 and 5,783,186 for anti-Her2 antibodies). Her-2 is overexpressed on 30% of highly malignant breast cancers, and successful antibodies against this target, marketed under the name Herceptin™ (Trastuzumab), have been developed. It has been shown that targeting multiple Her-2 epitopes with a mixture of monoclonal antibodies results in improved anti-growth activity of a human breast cancer cell line in vitro and in vivo (Spiridon et al, 2002). Her-2 may therefore be a good target for single chain antibody mixtures according to the present invention. Single chain antibodies useful for this purpose can be obtained by methods described in the present invention, including antibody display methods and genetically engineered animals capable of expressing a diversified repertoire of single chain antibdodies. All publications (including patents and patent applications) cited herein are hereby expressly incorporated by reference in their entirety.

Further details of the invention are provided in the following non-limiting examples. Example 1: Generation of genetically engineered rats expressing heavy chain-only antibodies

Construction of modified human Is loci on YACs and BACs.

The human IgH locus was constructed and assembled in several parts, which involved the modification and joining of rat C region genes, which were then joined downstream of human V_H6 -D - J_H region. Two BACs with separate clusters of human V_H genes [BAC3 and BAC6] were then co-injected with a BAC encoding the assembled (human V_H6 -D - J_H-rat C) fragment.

For the rat constant region three BACs were identified [N12, M5 and 18]. These were individually shaved, while a 170 bp homology arm matching the 5' end of shaved M5 was added to the 3' end of shaved N12 and a 100 bp homology arm matching the 5' end of shaved 18 was added to the 3' end of shaved M5. These modified BACs when put together contain a large part of the rat constant (C) region including E(enhancer^, s(switch^, Cμ, C5, s^b, Cy2b, se, CE, sot, Coc and 3Έ. The CHI regions of rat Cμ and rat CY2b located in shaved N12 and 18, respectively, were removed. The modified N12 and M5 were then joined to yield the BAC N12M5. These BAC modifications were carried out using the Red^® ET Recombineering technology.

As multiple BAC modifications in E. coli frequently deleted repetitive regions such as switch sequences and enhancers, technologies were developed to assemble sequences with overlapping ends in S. cerevisiae as circular YAC (cYAC) and, subsequently, to convert such a cYAC into a BAC. Advantages of YACs include their large size, the ease of homologous alterations in the yeast host and the sequence stability, whilst BACs propagated in E. coli offer the advantages of easy preparation and large yield. Additionally, detailed restriction mapping and sequencing analysis can be better achieved in BACs than in YACs. Two self-replicating S. cerevisiaelE. Coli shuttle vectors, pBelo-CEN-URA, and pBelo-CEN-HYG were constructed. Briefly, S.

cerevisiae CEN4 was cut out as an Avrll fragment from pYAC-RC (Marchuk and Collins, 1988) and ligated to Spel-linearised pAP599. The resulting plasmid contains CEN4 cloned between URA3 and Hyg^R. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or, a Pmll-Sphl fragment containing Hyg^R followed by CEN4, was cut out, and ligated to ApaLI and BamHI or Hpal and Sphl digested pBACBelol 1 (New England Biolabs) to yield pBelo-CEN-URA and pBelo- CEN-HYG. Restriction analysis of the modified 18 revealed that the S 2b region in this BAC was 2.5 to 3 kb shorter than expected. To assemble the ~125 kb rat C region lacking CHI in C as well as C 2b (N12M5I8) and maintain its authentic configuration, equal moles of the following purified fragments were mixed: 51 kb Swal-Notl from modified

N12M5, 6.5 kb Xbal fragment encompassing the authentic S72b region to replace the shortened S 2b in the modified 18 (previously cloned into pBeloBACl 1), 81 kb Nrul fragment from the modified 18, and the PCR-amplified pBelo-CEN-URA containing homology arms (65 bp) at either end corresponding to the sequence immediately downstream of rat JH in N12 and the 3' end of the rat C region in 18 (primers 321 and 322). The DNA mix, in which each fragment overlaps from 65 bp to 15 kb with its neighbouring fragment at both the 5' and 3' end, was transformed into S. cerevisiae

AB1380 cells using the standard spheroplast transformation procedure to select for URA⁺ clones (Nelson and Brownstein, 1994). Through homologous recombination in yeast associated with the transformation, a cYAC containing N12M5I8 in expected

configuration was assembled. The overlapping junctions of the neighbouring fragments in the resulting cYAC were confirmed by PCR analysis using yeast genomic DNA as template. After purification the cYAC was transformed into E. coli DH10 competent cells (Invitrogen) via electroporation. The correct BAC N12M5I8 was identified by extensive restriction mappings and sequencing.

BAC1 was modified in 3 steps to yield BACl-Shaved containing the human V_H6 -D - J_H region. Firstly, BAC1 was partially digested by Pvul and re-ligated to remove the sequence upstream of human V_H6-1. Secondly, the resulting shortened BAC1 was digested at a Pad site immediately downstream of the human J_Hs as well as an Ascl site in the vector backbone to remove a 41 kb fragment. Subsequently, a 2.5 kb fragment located immediately downstream of the rat J_Hs was amplified from rat genomic DNA and flanked by Pad and Ascl sites (primers 140 and 141). This Pacl-Ascl fragment which provides the overlap to the 5' end of modified N12 was ligated with Pad and Ascl double digested shortened BAC1 to yield BACl-Shaved.

Subsequently, BAC 3-1N12M5I8 was constructed via the cYAC/BAC strategy. This BAC contains the following regions from 5' to 3': the 11.3 kb sequence from the 3' end of BAC3 (providing the overlap to BAC3 when co-injected into the rat genome), the entire BACl-Shaved followed by the entire N12M5I8. Conveniently, the 3' end of BAC3 overlaps 5.5 kb with the 5' end of BACl-Shaved. To assemble 3-1N12M5I8, the 11.3 kb BAC3 fragment was amplified by PCR , and then mixed with the Pvul-Ascl fragment from BACl-Shaved, the Mlul fragment encompassing the entire N12M5I8, and the amplified pBelo-CEN-URA with homology arms at both ends corresponding to the 5' end of the 11.3 kb BAC3 fragment and the 3' end of 18 . This DNA mix was used to transform AB 1380 cells. Correct joining of each fragment in the transformation was confirmed by PCR analysis. After converting the assembled 3-1N12M5I8 region into a BAC, it was thoroughly checked by restriction mapping. The heavy chain gene region in BAC 3-1N12M5I8 can be cut out entirely together with the S. cerevisiae URA3 gene at its 3' end as a Notl fragment. When integrated into the rat genome, the existence of the URA3 in this large DNA fragment facilitates the identification of the transgene.

Finally, a 10.6 kb fragment located at the 5' end of human V_H loci in BAC3 was amplified using primers 411, 412 and integrated into BAC6 to provide overlap to BAC3. This modified BAC was named BAC6(+3). The 3' of the human V_H loci in BAC6 contains highly repetitive sequences which renders the manipulation via recombination very difficult in this region. Hence, we chose to integrate the BAC3 fragment into the vector backbone of BAC6. To achieve this, the pBelo-CEN+URA vector with 65 bp homology arms added to either end which overlaps with the 3' end of the 10.6 kb BAC3 fragment as well as the 5 'end of human V_H loci in BAC6, respectively, was amplified using primers 427 and 414. The DNA mix including equal moles of the 10.6 kb BAC3 fragment, the amplified pBelo-CEN+URA, and uncut B AC6 was transformed into AB1380 S. cerevisiae spheroplasts, and URA⁺ transformants were selected. PCR analysis were used to identify the correct integrants that contain the BAC3 fragment followed by pBelo-BAC+URA located between the vector backbone of BAC6 and the 5' end of B AC6 human V_H loci . After converting this cYAC into a BAC, it was thoroughly checked by restriction mapping. Digesting B AC6(+3) with Ascl releases a fragment approximately 220 kb containing the entire BAC6 human V_H loci and at its 3' end, the 10.6 kb overlapping BAC3 fragment. DNA purification

Linear YACs, circular YACs and BAC fragments after digests, were purified by electro-elution using Elutrap™ (Schleicher and Schuell)(Gu et al., 1992) from strips cut from 0.8% agarose gels run conventionally or from pulsed-field-gel electrophoresis (PFGE). The DNA concentration was usually several ng/μΐ in a volume of ~100μ1. For fragments up to -200 kb the DNA was precipitated and re-dissolved in micro-injection buffer (10 mM Tris-HCl pH 7.5, 100 mM EDTA pH 8 and 100 mM NaCl but without Spenriine/Sperrnidine) to the desired concentration.

The purification of circular YACs from yeast was carried out using Nucleobond AX silica-based anion-exchange resin (Macherey-Nagel, Germany). Briefly, spheroplasts were made using zymolyase or lyticase and pelleted (Davies et al., 1996). The cells then underwent alkaline lysis, binding to AX 100 column and elution as described in the Nucleobond method for a low-copy plasmid. Contaminating yeast chromosomal DNA was hydolyzed using Plamid -Safe™ ATP-Dependent DNase (Epicentre

Biotechnologies) followed by a final cleanup step using SureClean (Bioline). An aliquot of DH10 electrocompetent cells (Invitrogen) was then transformed with the circular YAC to obtain BAC colonies. For the separation of the insert DNA for microinjection, 150- 200 kb, from BAC vector DNA, ~10 kb, a filtration step with sepharose 4B-CL was used (Yang et al., 1997). Gel analyses

Purified YAC and BAC DNA was analysed by restriction digest and separation on conventional 0.7% agarose gels (Sambrook and Russell, 2001). Larger fragments, 50-200 kb, were separated by PFGE (Biorad Chef Mapper™) at 8°C, using 0.8% PFC Agaraose in 0.5% TBE, at 2-20 sec switch time for 16 h, 6V/cm, 10mA. Purification allowed a direct comparison of the resulting fragments with the predicted size obtained from the sequence analysis. Alterations were analysed by PCR and sequencing.

Microinjection

Outbred SD/Hsd strain animals were housed in standard microisolator cages under approved animal care protocols in animal facility that is accredited by the Association for the Assessment and Accreditation for Laboratory Animal Care (AAALAC). The rats were maintained on a 14-10 h light/dark cycle with ad libitum access to food and water. Four to five week old SD/Hsd female rats were injected with 20-25 IU PMSG (Sigma- Aldrich) followed 48 hours later with 20-25 IU hCG (Sigma- Aldrich) before breeding to outbred SD/Hsd males. Fertilized 1-cell stage embryos were collected for subsequent microinjection. Manipulated embryos were transferred to pseudopregnant SD/Hsd female rats to be carried to parturition.

Purified DNA encoding recombinant immunoglobulin loci was resuspended in microinjection buffer with 10 mM Spermine and 10 mM Spemidine. The DNA was injected into fertilized oocytes at various concentrations from 0.5 to 3 ng/μΐ.

Plasmid DNA or mRNA encoding ZFNs specific for rat immunoglobulin genes were injected into fertilized oocytes at various concentrations from 0.5 to 10 ng/ul.

Zinc-finger Nucleases (ZFNs)

ZFNs specific for rat immunoglobulin genes were generated. The ZFN specific for rat Ckappa had the following binding site:

ATGAGCAGCACCCTCtcgttgACCAAGGCTGACTATGAA (SEQ ID NO: 1)

ZFNs specific for rat J- locus sequences had the following binding sites:

CAGGTGTGCCCATCCagctgaGTTAAGGTGGAG (SEQ ID NO: 2) and

CAGGACCAGGACACCTGCAgcagcTGGCAGGAAGCAGGT (SEQ ID NO: 3) Rats with transloci.

Transgenic rats carrying artificial heavy chain immunoglobulin loci in unrearranged configuration were generated. The included constant region genes encode IgM, IgD, IgG2b, IgE, IgA and 3'enhancer. RT-PCR and serum analysis (ELISA) of transgenic rats revealed productive rearrangement of transgenic immunoglobulin loci and expression of heavy chain only antibodies of various isotypes in serum. Immunization of transgenic rats resulted in production of high affitnity antigen-specific heavy chain only antibodies.

Novel Zinc-finger-nuclease knock-out technology.

For further optimization of heavy chain-only antibody generation in transgenic rats, knockout rats with inactivated endogenous rat immunoglobulin loci were generated.

For the inactivation of rat heavy immunoglobulin heavy chain expression and rat K light chain expression, ZFNs were microinjected into single cell rat embryos. Subsequently, embryos were transferred to pseudopregnant female rats and carried to parturition. Animals with mutated heavy chain and light chain loci were identified by PCR. Analysis of such animals demonstrated inactivation of rat immunoglobulin heavy and light chain expression in mutant animals.

Example 2: Generation of antigen-specific heavy chain-only antibodies in rats

For the generation of antigen-specific heavy chain-only antibodies in rats, genetically engineered rats expressing heavy chain only antibodies are immunized in various ways.

Immuniziation with inactivated virus

Influenza viruses with various different hemagglutinin and neuraminidase genes is provided by the Immunology and Pathogenesis Branch, Influenza Division, CDC, Atlanta, GA. Virus stock is propagated in the allantoic cavities of 10-day-old

embryonated chicken eggs and purified through a 10%-50% sucrose gradient by means of ultracentrifugation. Viruses are resuspended in phosphate-buffered saline and inactivated by treatment with 0.05% formalin at 4oC for 2 weeks. Inactivated virus and alumn solution (Pierce) are mixed in a 3:1 ratio and incubated at room temperature for 1 h before immunization. Genetically engineered rats expressing heavy chain-only antibodies are immunized with whole inactive. Immunization with proteins or peptides

Typically immunogens (proteins or peptides) are diluted to 0.05-0.15 ml with sterile saline and combined with adjuvant to a final volume of 0.1 -0.3ml. Many appropriate adjuvants are available (i.e. heat inactivated Bordetella pertussis, aluminium hydroxide gel, Quil A or saponin, bacterial lipopolysaccharide or anti-CD40 ) but none have the activity of Complete Freund' s Adjuvant (CFA) and Incomplete Freund' s

Adjuvant (IFA). The concentration of soluble immunigens such as proteins and peptides may vary between 5 μg and 5mg in the final preparation. The first immunization

(priming) with immunogen in CFA is administered intraperitoneally and/or

subcutaneously and/or intramuscularly. If intact cells are used as immunogens they are best injected intraperitoneally and/or intraveneously. Cells are diluted in saline and 1 to 20 million cells are administered per injection. Cells that survive in the rat will yield best immunization results. After the first immunization with immunogens in CFA a second immunization in IFA (booster) is usually delivered 4 weeks later. This sequence leads to the development of B cells producing high affinity antibodies. If the immunogen is weak booster immunizations are administered every 2 weeks until a strong humoral response is achieved. The immunogen concentrations can be lower in booster

immunizations and intraveneous routes can be used. Serum is collected from rats every 2 weeks to determine the humoral response.

DNA-based immunization protocols Gene vaccines, or the use of antigen-encoding DNAs to immunize, represent an alternative approach to the development of strong antibody responses in rats.

The route of DNA inoculation is in general the skin, muscle and any other route that supports transfection and expression of the antigen. Purified plasmid DNAs that have been designed to express antigens such an influenza virus hemagglutinin glycoprotein or other human or viral antigens are used. Routes of DNA inoculation include the following: intravenous (tail vein), intraperitoneal; intramuscular (both quadriceps), intranasal, intradermal (such as foot pad), and subcutaneous (such as scruff of the neck). In general, 10-100 g of DNA is administered in 100 μΐ of saline per inoculation site or DNA is administered with appropriate vehicles such as gold particles or certain formulations (ht^://www.incellart.com/index.php?page=genetic- immunization&menu=3.3) that facilitate uptake and transfection of cells. The immunization scheme is similar to the protocol described above; primary immunization followed by booster immunizations.

Purification of heavy chain only antibodies

For the purification of antibodies, blood is collected from immunized rats and serum or plasma is obtained by centrifugation which separates the coagulated cell pellet from the liquid top phase containing serum antibodies. Antibodies from serum of plasma are purified by standard procedures. Such procedure include precipitation, ion exchange chromatography, and/or affinity chromatography. For the purification of IgG protein A or potein G can be used (Briiggemann et al., JI, 142, 3145, 1989).

Example 3: Isolation of antibody expressing B cells from rats.

Isolation ofB cells from spleen, lymph nodes or peripheral blood

A single-cell suspension is prepared from the spleen or lymph nodes of an immunized rat. Cells can be used without further enrichment, after removal of erythrocytes or after the isolation of B cells, memory B cells, antigen- specific B cells or plasma cells. Enrichment can lead to better results and as a minimum removal of erythrocytes is recommended. Memory B cells are isolated by depletion of unwanted cells and subsequent positive selection. Unwanted cells, for example, T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells are depleted using a cocktail of antibodies against CD2, CD 14, CD 16, CD23, CD36, CD43, and CD235a (Glycophorin A). Positive selection with antibodies specific for IgG or CD 19 results in highly enriched B cells (between 50%-95%). Antigen- specific B cells are obtained by exposing cells to antigen(s) tagged with fluorescent markers and/or magnetic beads. Subsequently, cells tagged with fluorochrome and/or magnetic beads are separated using (flow cytometry or a fluorescence activated cell sorter [FACS]) a FACS sorter and/or magnets. As plasma cells may express little surface Ig, intracellular staining may be applied. IgM positive B cell memory cells are isolated using antibodies specific for IgM and CD27.

Isolation ofB cells by fluorescence activated cell sorting

FACS-based methods are used to separate cells by their individual properties. It is important that cells are in a single-cell suspension. Single cell suspensions prepared from peripheral blood, spleen or other immune organs of immunized rats are mixed with fluorochrome-tagged antibodies specific for B cell markers such as CD19, CD138, and CD27. Alternatively, cells are incubated with fluorochrome-tagged antigens. The cell concentration is between 1-20 million cells/ml in an appropriate buffer such as PBS. For example, memory B cells cells can be isolated by selecting cells positive for CD27 and negative for CD45R. Plasma cells can be isolated by selecting for cells positive for CD 138 and negative for CD45R. Cells are loaded onto the FACS machine and gated cells are deposited into 96 well plates or tubes containing media. If necessary positive controls for each fluorochrome are used in the experiment which allows background subtraction to calculate the compensation. Isolation ofB cells from bone marrow

Bone marrow plasma cells (BMPCs) are isolated from immunized animals as described (Reddy et al., 2010). Muscle and fat tissue are removed from the harvested tibias and femurs. The ends of both tibias and femurs are clipped with surgical scissors and bone marrow is flushed out with a 26-gauge insulin syringe (Becton Dickinson, BD). Bone marrow is collected in sterile-filtered buffer no. 1 (PBS, 0.1% BSA, 2 mM EDTA). Bone marrow cells are collected by filtration through a cell strainer (BD) with

mechanical disruption and washed with 20 ml PBS and collected in a 50ml tube (Falcon, BD). Bone marrow cells are centrifuged at 335g for 10 min at 4oC. Supernatant is decanted and the cell pellet is resuspended in 3 ml of red cell lysis buffer (eBioscience) and shaken gently at 25oC for 5 min. Cell suspension is diluted with 20 ml of PBS and centrifuged at 335g for 10 min at 4oC. Supernatant is decanted and cell pellet

resuspended in 1 ml of buffer no.l

Bone marrow cell suspensions are incubated with biotinylated anti-CD45R and anti-CD49b antibodies. The cell suspension is then rotated at 4oC for 20 min. This is followed by centrifugation at 930g for 6 min at 4oC, removal of supernatant and re- suspension of the cell pellet in 1.5 ml of buffer no. 1. Streptavidin conjugated M28 magnetic beads (Invitrogen) are washed and resuspended according to the manufacturer's protocol. Magnetic beads (50 ul) are added to each cell suspension and the mixture is rotated at 4oC for 20 min. The cell suspensions are then placed on Dynabead magnets (Invitrogen) and supernatant (negative fraction, cells unconjugated to beads) are collected and cells bound to beads are discarded.

Prewashed streptavidin M280 magnetic beads are incubated for 30 min at 4oC with biotinylated anti-CD 138 with 0.75 ug antibody per 25ul of magnetic beads mixture. Beads are then washed according to the manufacturer's protocol and resuspended in buffer no. 1. The negative cell fraction (depleted of CD45R+ and CD49b+ cells) collected as above is incubated with 50 ul of CD138-conjugated magnetic beads and the suspension is rotated at 4oC for 30 min. Beads with CD 138+ bound cells are isolated by the magnet, washed 3 times with buffer no.l, and the negative (CD138-) cells unbound to beads are discarded . The positive CD 138+ bead-bound cells are collected and stored at 4oC until further processed.

Example 4: Generation of hvbridomas

Isolated B cells are immortalized by fusion with myeloma cells such as X63 or YB2/0 cells as described (Kohler and Milstein, Nature, 256, 495, 1975 ) . Hybridoma cells are cultured in selective media and antibody producing hybridoma cells are generated by limiting dilution or single cell sorting. Example 5: Isolation of cDNAs encoding heavy chain only antibodies Generation ofcDNA sequences from isolated cells

Isolated cells are centrifuged at 930g at 4oC for 5 min. Cells are lysed with TRI reagent and total RNA is isolated according to the manufacturer's protocol in the

Ribopure RNA isolation kit (Ambion). mRNA is isolated from total RNA with oligo dT resin and the Poly(A) purist kit (Ambion) according to the manufacturer's protocol. mRNA concentration is measured with an ND-1000 spectrophotometer (Nanodrop).

The isolated mRNA is used for first-strand cDNA synthesis by reverse

transcription with the Maloney murine leukemia virus reverse transcriptase (MMLV-RT, Ambion). cDNA synthesis is performed by RT-PCR priming using 50ng of mRNA template and oligo dT) primers according to the manufacturer's protocol of Retroscript (Ambion). After cDNA construction, PCR amplification is performed to amplify heavy chain only antibodies. A list of primers is shown in Table 1:

Table 1:

A 50ul PCR reaction consists of 0.2 mM forward and reverse primer mixes, 5 ul of Thermopol buffer (NEB), 2 ul of unpurified cDNA, 1 ul of Taq DNA polymerase (NEB) and 39 ul of double-distilled H₂0. The PCR thermocycle program is 92oC for 3 min; 4 cycles (92oC for 1 min, 50oC for 1 min, 72oC for 1 min); 4 cycles (92oC for 1 min, 55oC for 1 min, 72oC for 1 min), 20 cycles (92oC for 1 min, 63oC for 1 min, 72oC for 1 min); 72oC for 7 min, 4oC storage. PCR gene products are gel purified and DNA sequenced.

Example 6: Cloning and expression of recombinant heavy chain-only antibodies PCR products are subcloned into a plasmid vector. For expression in eukaryotic cells cDNA encoding heavy chain only antibody are cloned into an expression vector as described (Tiller et al., 2008).

Alternatively, the genes encoding heavy chain only antibodies are cloned into a minicircle producing plasmid as described (Kay et al., 2010). Alternatively, genes encoding heavy chain only antibodies are synthesized from overlapping oligonucleotides using a modified thermodynamically balanced inside-out nucleation PCR (Gao at al., 2003) and cloned into an eukaryotic expression vector.

Alternatively, genes encoding heavy chain-only antibodies are synthesized and cloned into a plasmid. For the assembly of multiple expression cassettes encoding various heavy chain only antibodies in an artificial chromosome, multiple expression cassettes are ligated with each other and subsequently cloned into a BAC vector, which is propagated in bacteria. For transfection ElectroMAX DH10B cells from Invitrogen are used

(http;//tools.invitrogen.com/content/sfs/manuals/18290015.pdf) . Alternatively, ligated expression cassettes are further ligated with yeast artificial chromosome arms, which are propagated in yeast cells (Davies et al., 1996) .

Plasmid purification

GenElute™ plasmid miniprep kits from Sigma- Aldrich are used for plasmid isolation from ~5ml (or larger) overnight bacterial culture

(http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna- purification/plasrrdd-miniprep-kit.html). This involves harvesting bacterial cells by centrifugation followed by alkaline lysis. DNA is then column-bound, washed and eluted and ready for digests or sequencing.

BAC purification

NucleoBond^R BAC 100 from Clontech is a kit designed for BAC purification (http://www.clontech.com/products/detail.asp?tabno=2&product id=186802). For this bacteria are harvested from 200 ml culture and lysed by using a modified alkaline/SDS procedure. The bacterial lysate is cleared by filtration and loaded onto the equilibrated column, where plasmid DNA binds to the anion exchange resin. After subsequent washing steps, the purified plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. The plasmid DNA is reconstituted in TE buffer for further use.

YAC purification

Linear YACs, circular YACs and BAC fragments after digests, are purified by electro-elution using Elutrap™ (Schleicher and Schuell)(Gu et al., 1992) from strips cut from 0.8% agarose gels run conventionally or from pulsed-field-gel electrophoresis (PFGE). The purified DNA is precipitated and re-dissolved in buffer to the desired concentration.

The purification of circular YACs from yeast is carried out using Nucleobond AX silica-based anion-exchange resin (Macherey-Nagel, Germany). Briefly, spheroplasts are made using zymolyase or lyticase and pelleted (Davies et al., 1996). The cells then undergo alkaline lysis, binding to AX 100 column and elution as described in the

Nucleobond method for a low-copy plasmid. Contaminating yeast chromosomal DNA is hydolyzed using Plamid -Safe™ ATP-Dependent DNase (Epicentre Biotechnologies) followed by a final cleanup step using SureClean (Bioline). An aliquot of DH10 electrocompetent cells (Invitrogen) is then transformed with the circular YAC to obtain BAC colonies (see above). For the separation of the insert DNA, 150-200 kb, from BAC vector DNA, ~10 kb, a filtration step with sepharose 4B-CL is used (Yang et al., 1997).

Transfection of cells with plasmid or BAC DNA

For the expression of recombinant heavy chain only antibodies, eukaryotic cells are transfected as described (Andreason and Evans, 1989; Baker and Cotton, 1997; http://www.millipore.com/cellbiology/cb3/mam^ . Cells expressing heavy chain only antibodies are isolated using various selection methods. Limiting dilution or cell sorting are used for the isolation of single cells. Clones are analyzed for heavy chain only antibody expression. Example 7: Characterization of antibody expression in cell clones expressing recombinant polyclonal heavy chain only antibodies

The composition of purified recombinant heavy chain only antibodies is analyzed by analytical cation exchange chromatography as well as mass spectrometry based techniques as described (Persson et al 2010). Heavy chain only antibodies are desalted and equilibrated in water. Subsequently, the sample is dried in a vaccum centrifuge and reconstituted in 6 M Gnd-HCl, 0.2 M Tris, pH 8.4 at a final concentration of about 10 mg/ml. Reduction is performed by addition of DTT and incubation at 65oC . Reduced heavy chain only antibodies are alkylated by addition of iodoacetic acid and incubation in the dark at room temperature. Heavy chain only antibodies are digested proteolytically using various enzymes including trypsin, papain, pepsin of endoproteinase Asp-N.

Marker peptides are analyzed by reverse phase-HPLC/MS analysis using a Agilent 1100 HPLC system coupled to an Agilent 1100 LC/MSD mass spectrometer. The enzymatic digest is applied onto a Zorbax 300 SB-C18 column in 80% mobile phase A (water, 0.1% TFA) and 20% mobile phase B (acetonitrile, 0.08% TFA) at 40oC using a flow rate of O.lmL/min. The peptides are separated using a gradient of mobile phase. Peptides are detected with electrospray ionization-quadropose (ESI-q) MS. The mass spectrometer is set up to run in a positive mode with an m/z range of 400-2000. A mixture of propionic acit and isopropanol is added to the mobile phase prior to mass detection at a flow rate of 10 uL/min for fixation of TFA. Collected peptides are prepared with Prosorb sample cartridges (Applied Biosystems, Carlsbad, CA) according to the manufaturer's recommendations and sequenced on a Procise 494 sequenator (Applied Biosystems).

References for the Examples:

Popov, A.V., Butzler, C, Frippiat, J.-P., Lefranc, M.P., and Briiggemann, M. (1996) Assembly and extension of yeast artificial chromosomes to build up a large locus. Gene, 177, 195-205

Popov, A.V., Zou, X., Xian, J., Nicholson, I.C. and Briiggemann, M. (1999) A human immunoglobulin λ locus is similarly well expressed in mice and humans. J. Exp. Med., 189, 1611-1619

Marchuk. D. and Collins, F.S. (1988) pYAC-RC, a yeast artificial chromosome vector for cloning DNA cut with infrequently cutting restriction endonucleases. Nucl. Acids Res., 16, 7743.

Ma, B., Pan, S.J., Zupancic, M.L. and Cormack, B.P. (2007) Assimilation of NAD⁺ precursors in Candida glabrata. Mol. Microbiol., 66, 14-25.

Gietz D and Woods RA (1998) Transformation of yeast by the lithium acetate single- stranded carrier DNA/PEG method. Methods in Microbiology 26 53-66.

Peterson KR (2007) Preparation of intact yeast artificial chromosome DNA for transgenesis of mice. Nature Protocols 2 (11) 3009-3015.

Beverly SM (1988) Characterization of the 'unusual' mobility of large circular DNAs in pulsed field-gradient electrophoresis. Nucleic Acids Research 16(3) 925-939.

Kawasaki K, Minoshima S, Nakato E, Shibuya K, Shintani A, Asakawa S, Sasaki T, Klobeck HG, Combriato G, Zachau HG, Shimizu N. Evolutionary dynamics of the human immunoglobulin κ locus and the germline repertoire of the VK genes. (2001) Eur J Immunol. 31, 1017-1028.

Wagner, S.D., Gross, G., Cook, G.P., Davies, S.L. and Neuberger, M.S. (1996) Antibody expression from the core region of the human IgH locus reconstructed in transgenic mice using bacteriophage PI clones. Genomics, 35, 405-414.

Nelson, D.J. and Brownstein, B.H. (1994) YAC libararies, a user's guide. Freeman and Compny NY

Gu H, Wilson D and Inselburg J (1992) Recovery of DNA from agarose gels using a modified Elutrap™. Journal of Biochemical and Biophysical Methods 24, 45-50.

Davies, N.P., Popov, A.V., Zou, X. and Briiggemann, M. (1996) Human antibody repertoires in transgenic mice: Manipulation and transfer of YACs. Antibody

Engineering: A Practical Approach, eds. J. McCafferty, H.R. Hoogenboom and D.J. Chiswell, pp. 59-76, IRL, Oxford.

Yang XW, Model P and Heintz N (1997) Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nature Biotechnology 15, 859-865.

Sambrook, J. andf Russell, D.W. Molecular Cloning: a laboratory manual. Cold Spring Harbor, NY, 2001. Reddy, S.T., Ge, X., Miklos, A.E., Hughes, R.A., Kang, S.H. Hoi, K.H., Chrysostomou, C, Hunicke-Smith, S.P., Iverson, B.L. and Tucker, P.W., 2010. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nature biotechnology 28(9):965-969

Gao, X., Yo, P., Keith, A., Ragan, T.J., and Harris, T.K., 2003. Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acid Research 31, el43. Tiller, T., Meffre, E., Yurasov, S., Tsuiji, M., Nussenzweig, M.C., and Wardemann, H., 2008. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol. Methods 329: 112-124

Kay, M.A., He, C-Y., Chen Z-Y., 2010. A robust system for production of minicircle DNA vectors. Nature biotechnology 28(12): 1287-1289

Persson, P., Engstrom, A., Rassmussen, L.K., Holmberg, E., and Frandsen, T.P., 2010. Development of mass spectrometry based techniques for the identification and determination of compositional variability in recombinant polyclonal antibody products. Anal. Chem 82: 7274-7282

Peterson KR (2007) Preparation of intact yeast artificial chromosome DNA for

transgenesis of mice. Nature Protocols 2 (11) 3009-3015.

Sambrook, J. andf Russell, D.W. Molecular Cloning: a laboratory manual. Cold Spring Harbor, NY, 2001.

Andreason, G. L. and Glen Evans, G. A. Analytical Biochemistry, 180, 269, 1989 Baker, A. and Cotton, M. NAR 25, 1950, 1997

Claims

CLAIMS:

1. A method for production of a mixture of single chain antibodies, the method comprising

(b) isolating nucleic acid molecules encoding at least three non-identical antigen- specific single chain antibodies from said transgenic animal;

(c) introducing said nucleic acid molecules in a single recombinant host cell and

(d) producing a mixture of single chain antibodies.

2. The method of claim 1 wherein said animal is naturally capable of expressing said diversified repertoire of single chain antibodies.

3. The method of claim 2 wherein said animal is a camelid.

4. The method of claim 3 wherein said animal is selected from the group consisting of camel, lama, alpaca.

5. The method of claim 2 wherein said animal is a shark.

6. The method of claim 1 wherein said animal is genetically engineered to express a diversified repertoire of single chain antibodies.

7. The method of claim 6 wherein said animal is selected from the group consisting of rodents, rabbits, birds, goats, cattle and sheep.

8. The method of claim 7 wherein the rodent is a transgenic mouse or a rat.

9. The method of claim 8 wherein the transgenic animal is a transgenic mouse or rat carrying artificial heavy chain immunoglobulin loci in unrearranged configuration.

10. The method of claim 9 wherein said transgenic animal has been engineered to produce heavy chain only antibodies of various isotypes.

11. The method of claim 1 wherein the single chain antibodies are heavy chain only antibodies.

12. The method of claim 1 wherein the single chain antibodies are selected from the group consisting of scFv, VHH antibodies and fragments thereof, and domain antibodies (dAbs) and fragments thereof.

13. The method of claim 1 wherein in step (b) nucleic acid encoding three non- identical antigen-specifics single chain antibodies is isolated.

14. The method of claim 1 wherein in step (c) the recombinant host cell is an eukaryotic or prokaryotic cell.

15. The method of claim 14 wherein the recombinant host cell is a mammalian cell.

16. The method of claim 1 wherein the recombinant host cell expresses a first, a second and a third non-identical antigen specific single chain antibody.

17. The method of claim 16 wherein said first, second and third non- identical antibodies have differing specificities for the same target antigen.

18. The method of claim 16 wherein said first, second and third non- identical antibodies have differing affinities for the same target epitope.

19. The method of claim 16 wherein said first, second and third single chain antibodies bind to at least two different antigens.

20. The method of claim 19 wherein said first, second and third single chain antibodies bind to three different antigens.

21. The method of claim 16 wherein the mixture of single chain antibodies produced in step (d) comprises homodimers and heterodimers of said single chain antibodies.

22. The method of claim 16 wherein the mixture of single chain antibodies produced in step (c) is composed predominantly of homodimers of said single shain antibodies.

23. The method of claim 16 wherein the mixture of single chain antibodies produced in step (c) is composed predominantly of heterodimers of said single chain antibodies.

24. A recombinant host cell comprising nucleic acid encoding at least three non-identical antigen- specific single chain antibodies.

25. The recombinant host cell of claim 24, comprising three non- identical antigen- specific single-chain antibodies.

26. The recombinant host cell of claim 24, wherein said single chain antibodies are heavy chain only antibodies.

27. The recombinant host cell of claim 24, wherein said single chain antibodies are selected from the group consisting of scFv, VHH antibodies and fragments thereof, and domain antibodies (dAbs) and fragments thereof.

28. The recombinant host cell of claim 24, wherein at least two of said single chain antibodies differ in their iso types.

28. The method of claim 24, wherein at least two of said single chain antibodies differ in their binding specificities.

29. The method of claim 24, wherein at least two of said single chain antibodies differ in their binding affinities.

30. The method of any one of claims 24 to 29, wherein said recombinant host cell is an eukaryotic or a prokaryotic host cell.

31. The method of any one of claims 24 to 29, wherein said recombinant host cell is a mammalian cell.

32. The method according to any one of claims 24 to 29, wherein said recombinant host cell is a plant cell.

33. A composition comprising a mixture of at least three non-identical single chain antibodies.

34. The composition of claim 33, which is a pharmaceutical composition.

35. The composition of claim 33, which is a diagnostic composition.

36. The composition of claim 34, wherein the composition has a biological activity exceeding the biological activity of each single chain antibody present in the composition.

37. A transgenic non-human animal comprising a nucleic acid sequence or nucleic acid sequnces encoding at least three different single chain antibodies.

38. The method of claim 37, wherein said single chain antibodies are heavy chain only antibodies.

39. The method of claim 37, wherein said single chain antibodies are selected from the group consisting of scFv, VHH antibodies and fragments thereof, and domain antibodies (dAbs) and fragments thereof.