HK1240236B - Cd3 binding domain - Google Patents

Description

CD3 binding domain

Technical Field

The present invention relates to an antigen-binding protein comprising a novel CD3 binding site. In particular, the present invention relates to a multispecific antigen-binding protein. The novel CD3 binding site comprises a humanized VH domain and a VL domain.

Background Art

The CD3 antigen is associated with the T cell receptor complex on T cells. Multispecific antigen binding proteins that are specific for CD3 and target cell antigens can trigger the cytotoxic activity of T cells against target cells. That is, through multispecific binding of the antigen binding protein to CD3 and to target cells (e.g., tumor cells), cell lysis of target cells can be induced. Antigen binding proteins with CD3 binding sites and their production are known in the art (and described in, for example, Kipriyanov et al., 1999, Journal of Molecular Biology 293: 41-56; Le Gall et al., 2004, Protein Engineering, Design & Selection, 17/4: 357-366).

In addition to antibodies derived from quadromas, various forms of multispecific recombinant antibody fragments have been designed. Specific forms of multivalent antibody fragments and optionally multispecific antibody fragments are called "tandem diabodies" because their design is based on the intermolecular pairing of the _VH variable domain and the _VL variable domain of two different polypeptides, as described for diabodies (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 6444-6448). The antibodies described are bispecific for tumor antigens and CD3. In contrast to bivalent scFv-scFv (scFv) ₂ tandems, tandem diabodies are tetravalent because they have four antigen binding sites. Tandem diabodies lack immunoglobulin constant domains. Tandem diabodies have been reported to have advantages such as high affinity, higher avidity, lower clearance rate, and show favorable in vitro and in vivo efficiencies (Kipriyanov et al., J. Mol. Biol. (1999) 293, 41-56 and Kipriyanov Meth. Mol. Biol. (2009) 562, 177-193).

This bispecific tandem diabody can form a bridge between tumor cells (e.g., B-CLL cells) and CD3 ⁺ T cells of the human immune system, thereby allowing the killing of tumor cells. The tight binding of tumor cells and T cells induces the destruction of tumor cells. Although this tandem diabody has been shown to be beneficial for therapeutic applications (e.g., therapeutic concepts for treating tumors), there is still a need for improved antigen-binding molecules.

To facilitate the toxicological evaluation of multispecific antibody fragments that recruit T cells in non-human primates (NHPs) during preclinical development, cross-reactive CD3-binding domains are required.

Murine IgG clone SP34 (EMBO J. 1985. 4(2): 337-344; J. Immunol. 1986, 137(4): 1097-100; J. Exp. Med. 1991, 174: 319-326; J. Immunol. 1991, 147(9): 3047-52) binds to humans, and cynomolgus monkey CD3ε has been selected for humanization.

While humanization by grafting the murine VH CDRs onto the most homologous human VH framework (human VH3_72) resulted in molecules that remained functional, grafting the murine VL CDRs onto the human VL framework with the highest homology (human vλ7_7a) surprisingly resulted in poorly expressed tandem diabodies or tandem diabodies that failed to recognize CD3.

Summary of the Invention

It is therefore an object of the present invention to provide CD3 binding domains with functional VL/VH pairing, good stability, good expression and other biophysical properties.

This problem has been solved by the subject matter of the claims.

It has been found that functional VL/VH pairing can be achieved by another people's VL framework. Surprisingly, the change from the mouse VL framework of lambda chain to the people's VL framework of kappa chain (people Vκ1_39) results in the binding protein showing superior CD3 binding affinity, expression ability and cytotoxicity efficacy in the target cell lysis induced by bispecific series double antibody. However, such molecules are weaker in terms of stability and other biophysical properties. Further, it has been found that the amino acid in position VH111 and position VL49 (they directly contact each other according to the model) is critical for the combination and stability properties of the CD3 binding site of, for example, bispecific antibody molecules, particularly multimeric antigen-binding proteins (such as double antibody or series double antibody). The mutation of VH111 to Y or H and the mutation of VL49 from G to A in addition surprisingly produce a binding domain with improved stability properties, while retaining original binding affinity and/or cytotoxicity. Improved 7-day stability at 40°C was observed for such proteins, particularly when expressed as dimeric tandem diabodies. Furthermore, clones demonstrated increased levels of correctly dimerized tandem diabodies and improved recovery.

Thus, in one aspect, the present invention provides an antigen binding protein comprising at least one CD3 binding site, wherein the CD3 binding site comprises:

(a) a heavy chain variable domain (VH) selected from the VH depicted in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 and a light chain variable domain (VL) selected from the VL depicted in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or,

(b) a heavy chain variable domain (VH) having at least 95% sequence identity compared to the VH as depicted in SEQ ID NO: 8, wherein the amino acid residue at position 111 is Y or H; and a light chain variable domain (VL) having at least 95% sequence identity compared to the VL as depicted in SEQ ID NO: 3, wherein the amino acid residue at position 49 is G or A; or,

(c) a heavy chain variable domain (VH) incorporating 1 to 5 conservative amino acid substitutions compared to the VH as depicted in SEQ ID NO: 8, wherein the amino acid residue at position 111 is Y or H; and a light chain variable domain (VL) incorporating 1 to 5 conservative amino acid substitutions compared to the VL as depicted in SEQ ID NO: 3, wherein the amino acid residue at position 49 is G or A.

In certain embodiments, the antigen binding protein comprising at least one CD3 binding site as defined above is not a bispecific binding protein that specifically binds to human CD33 and human CD3, wherein the CD3 binding site comprises at least one antibody heavy chain variable domain and at least one light chain variable domain that form a human CD3 antigen binding site, and the anti-CD3 light chain variable domain is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, and the anti-CD3 heavy chain variable domain is selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8. In a specific embodiment, the antigen binding protein is not a bispecific tandem diabody that binds to CD33 and CD3, wherein the anti-CD3 light chain variable domain is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, and the anti-CD3 heavy chain variable domain is selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8. Such bispecific CD33 and CD3 binding proteins are disclosed in PCT/US2015/038666, filed June 30, 2015, and U.S. Application No. 14/642,497, filed March 9, 2015, which claim priority from U.S. Application No. 62/019,795, filed July 1, 2014, and U.S. Application No. 62/111,470, filed February 3, 2015. In other embodiments of the present invention, the antigen binding protein comprising at least one CD3 binding site as defined above is not a bispecific binding protein that specifically binds to human CD33 and human CD3.

The term "antigen binding protein" refers to an immunoglobulin derivative with antigen binding properties, i.e., an immunoglobulin polypeptide or fragment thereof containing an antigen binding site. The binding protein comprises the variable domains of an antibody or fragment thereof. Each antigen binding site is formed by an antibody (i.e., immunoglobulin) heavy chain variable domain (VH) and an antibody light chain variable domain (VL) that bind to the same epitope, and the heavy chain variable domain (VH) comprises three heavy chain complementary determining regions (CDRs): CDR1, CDR2, and CDR3; and the light chain variable domain (VL) comprises three light chain complementary determining regions (CDRs): CDR1, CDR2, and CDR3. In some cases, the binding protein according to some embodiments herein lacks immunoglobulin constant domains. In some cases, the light chain variable domain and the heavy chain variable domain that form the antigen binding site are covalently linked to each other, for example, by a peptide linker, or in other cases, the light chain variable domain and the heavy chain variable domain are non-covalently associated with each other to form an antigen binding site. The term "antigen binding protein" also refers to monoclonal antibodies and antibody fragments or antibody derivatives of the IgA, IgD, IgE, IgG or IgM classes, including, for example, Fab, Fab', F(ab') ₂ , Fv fragments, single-chain Fv, tandem single-chain Fv ((scFv) ₂ ), diabodies, flexibodies (WO 03/025018) and tandem diabodies (Kipriyanov et al., 1999, J. Mol. Biol., 293:41-56; Cochlovius et al., 2000, Cancer Res., 60:4336-4341; Reusch et al., 2004, Int. J. Cancer, 112:509-518, Kipriyanov, 2009, Methods Mol Biol., 293:41-56; Cochlovius et al., 2000, Cancer Res., 60:4336-4341; Reusch et al., 2004, Int. J. Cancer, 112:509-518; ... et al., 2009, Methods Mol Biol., 293:41-56; Cochlovius et al., 2000, Cancer Res., 60:4336-4341; Reusch et al., 2004, Int. J. Cancer, 112:509- Biol, 562: 177-93; McAleese and Eser, 2012, Future Oncol. 8: 687-95). "TandAb" is a trademark of Affimed Therapeutics used to designate tandem diabodies. In the context of the present invention, "TandAb" and "tandem diabodies" are used as synonyms.

In certain embodiments, the CD3 binding site comprises a VH and a VL, wherein the VH framework is derived from the human VH3_72 framework and the VL framework is derived from human Vκ1_39. In a specific embodiment, the CD3 binding site comprises a VH selected from the VH depicted in SEQ ID NO: 8 or SEQ ID NO: 9 and a VL selected from the VL depicted in SEQ ID NO: 3 or SEQ ID NO: 4. In some cases, the CD3 binding site comprises (i) a VH depicted in SEQ ID NO: 8 and a VL depicted in SEQ ID NO: 3 or (ii) a VH depicted in SEQ ID NO: 9 and a VL depicted in SEQ ID NO: 4. In alternative embodiments, the heavy chain domain and the light chain domain incorporate homologs or variants of the sequences described herein that specifically bind to CD3. Thus, in some embodiments, the VL or VH sequence of CD3 is similar, but not identical, to the amino acid sequence depicted in SEQ ID NO: 3 or SEQ ID NO: 8, wherein (i) the amino acid residue at position VH111 is F, Y, or H, or (ii) the amino acid residue at position VH111 is F, Y, or H, and the amino acid residue at position VL49 is G or A. In certain embodiments, the VH variant sequence or VL variant sequence has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 81% or 80% sequence identity compared to the sequence depicted in SEQ ID NO:3 or SEQ ID NO:8 and that specifically binds to CD3, wherein (i) the amino acid residue at position VH111 is F, Y or H, or (ii) the amino acid residue at position VH111 is F, Y or H and the amino acid residue at position VL49 is G or A.

In another embodiment, the VH variant and/or VL variant incorporates 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitutions. Conservative substitutions include: the interchange of alanine, valine, leucine and isoleucine in aliphatic amino acids, the interchange of hydroxyl residues serine and threonine, the exchange of acidic residues aspartic acid and glutamic acid, the substitution between amide residues asparagine and glutamine, the exchange of basic residues lysine and arginine, and the replacement between aromatic residues phenylalanine and tyrosine. In some cases, the VH variant and/or VL variant incorporates such 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitutions, provided that (i) the amino acid residue at position VH111 is F, Y or H, or (ii) the amino acid residue at position VH111 is F, Y or H, and the amino acid residue at position VL49 is G or A. In some cases, such substitutions are not within a CDR.

In additional embodiments, the VH variant or VL variant comprises substitutions that enhance the properties of the CDR (e.g., increased stability, expression, recovery, binding affinity for CD3, and/or cytotoxic potency).

Furthermore, in certain embodiments, the antigen binding protein is multivalent, ie, has two, three, or more CD3 binding sites.

In some cases, the antigen-binding proteins according to the present invention are multifunctional. As used herein, the term multifunctional refers to a binding protein of the present invention having two, three or more different biological functions, one of which is binding to CD3. For example, the different biological functions are different specificities for different antigens. In some cases, the multifunctional antigen-binding protein is multispecific, i.e., has binding specificity for CD3 and one, two or more other antigens. Such multispecific antigen-binding proteins include, for example, multispecific F(ab') ₂ , Fv fragments, single-chain Fv, tandem single-chain Fv ((scFv) ₂ ), diabodies, flexosomes (WO 03/025018) and tandem diabodies.

In certain embodiments, the antigen binding protein comprises at least one CD3 binding site and at least one additional antigen binding site that is specific for an antigen present on a bacterial substance, a viral protein, an autoimmune marker, or a specific cell (e.g., a cell surface protein of a B cell, a T cell, a natural killer (NK) cell, a bone marrow cell, a phagocyte, or a tumor cell). Such an antigen binding molecule is capable of cross-linking two cells and can be used to direct T cells to a specific target.

Examples of such targets may be tumor cells or infectious agents, such as viral or bacterial pathogens, e.g. dengue virus, herpes simplex, influenza virus, HIV, or cells carrying autoimmune targets, e.g. IL-2, autoimmune markers or autoimmune antigens.

In certain embodiments, at least one additional antigen binding site is specific for an antigen on a tumor cell. The antigen on a tumor cell can be a tumor antigen and a cell surface antigen on the corresponding tumor cell, such as a specific tumor marker. This multispecific antigen binding protein binds to both CD3 on tumor cells and T cells, thereby triggering a cytotoxic response induced by T cells. The term "tumor antigen" as used herein includes tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). "Tumor-associated antigens" (TAAs) as used herein refer to proteins present on tumor cells, as well as proteins present on normal cells during fetal life (once-fetal antigens) and in selected organs after birth, but are present at concentrations far lower than those present on tumor cells. TAAs can also be present in the matrix near tumor cells, but are expressed in lower amounts in the matrix elsewhere in the body. In contrast, the term "tumor-specific antigen" (TSA) refers to proteins expressed by tumor cells. The term "cell surface antigen" refers to any antigen or fragment thereof that can be recognized by antibodies on the cell surface.

Examples of antigens for tumor cells include, but are not limited to, CD19, CD20, CD30, laminin receptor precursor protein, EGFR1, EGFR2, EGFR3, EGFRvIII, Ep-CAM, PLAP, Thomsen-Friedenreich (TF) antigen, MUC-1 (mucin), IGFR, CD5, IL4-Rα, IL13-R, FcεRI, and IgE, as described in the art.

In another embodiment, the antigen binding protein may comprise at least one CD3 binding site and at least one additional antigen binding site specific for a molecule selected from the group consisting of a drug, a toxin, a radionucleotide, an enzyme, an albumin (e.g., serum albumin) and a lipoprotein, a naturally occurring ligand (e.g., a cytokine or chemokine). If the target molecule is albumin, the albumin or serum albumin may be selected from the group consisting of human, bovine, rabbit, canine, and mouse origin.

In certain embodiments, the binding protein is multispecific, having a first specificity for CD3 and a second specificity for CD19, CD30, EGFRvIII, or HSA. In a specific embodiment, the multispecific binding protein having a first specificity for CD3 does not have a second specificity for CD33.

In another aspect, the antigen-binding proteins according to the present invention are multimeric, i.e., comprise two, three, or more polypeptides that form at least one CD3 antigen-binding site. As used herein, "multimer" refers to a complex of two or more polypeptides. In certain embodiments, the polypeptides of a multimer are non-covalently associated with one another, particularly provided that there is no covalent bonding between the polypeptides. In certain embodiments, the multimer is homomeric, i.e., comprises identical polypeptides. The term "polypeptide" refers to a polymer of amino acid residues linked by amide bonds. In certain embodiments, the polypeptide is a single-chain fusion protein that is not branched. In the polypeptide, the antibody variable domains are linked sequentially. In other embodiments, the polypeptide may have contiguous amino acid residues in addition to the N-terminal and/or C-terminal residues of the variable domains. For example, in certain embodiments, such contiguous amino acid residues may comprise a tag sequence at the C-terminus, which is expected to be useful for purification and detection of the polypeptide. In certain embodiments, the multimer is dimeric, i.e., comprises two polypeptides. Examples of multimers encompassed by the present invention are diabodies, tandem diabodies, and flexobodies.

In certain embodiments, the multimer is an antigen-binding protein in the form of a tandem diabody. Such tandem diabodies are constructed by linking four antibody variable binding domains (e.g., two VH and two VL) in a single gene construct capable of non-covalent dimerization. In such tandem diabodies, the linker length is such that it prevents intramolecular pairing of the variable domains so that the molecule cannot fold back on itself to form a single-chain diabody, but is instead forced to pair with the complementary domain of the other chain. The domains are also configured so that the corresponding VH domain and VL domain pair during this dimerization. After expression from the gene construct, the two polypeptide chains fold head-to-tail to form a functional non-covalent dimer of approximately 105 kDa (Kipriyanov Meth. Mol. Biol. (2009) 562, 177-193, McAleese and Eser, 2012, Future Oncol. 8: 687-95). Despite the absence of intermolecular covalent bonds, the dimer, once formed, is highly stable, remains intact and does not revert back to monomeric form.

Tandem diabodies have many characteristics that provide advantages over traditional monoclonal antibodies and other smaller Fv molecules. Tandem diabodies contain only antibody variable domains and therefore lack any side effects that may be associated with the Fc portion. Because tandem diabodies are multivalent and allow for bivalent binding to CD3, the avidity is the same as that of IgG. The size of the tandem diabodies is approximately 105 kDa in some embodiments, which is smaller than the size of IgG, allowing for enhanced tumor penetration. However, such a size is much higher than the renal threshold for first-pass clearance, providing a pharmacokinetic advantage compared to smaller antibody forms based on antibody binding sites or non-antibody scaffolds. Tandem diabodies are well expressed in host cells, such as mammalian CHO cells. It is anticipated that robust upstream and downstream manufacturing methods can be used for tandem diabodies (e.g., Kipriyanov, Meth. Mol. Biol. (2009) 562, 177-193).

In some cases, the multispecific antigen-binding proteins (e.g., tandem diabodies) described herein are designed to allow specific targeting of tumor cells by recruiting cytotoxic T cells. Compared to conventional antibodies, this improves ADCC (antibody-dependent cell-mediated cytotoxicity). Antibodies cannot directly recruit cytotoxic T cells. On the contrary, by binding to CD3 molecules specifically expressed on these cells, multispecific antigen-binding proteins (e.g., tandem diabodies) can crosslink cytotoxic T cells with tumor cells in a highly specific manner, thereby significantly increasing the cytotoxic efficacy of such molecules.

In one aspect, the multimer is a bispecific tandem diabody, wherein each polypeptide of the bispecific tandem diabody comprises four variable domains, the VL and VH of CD3 having a second specificity different from CD3. In certain embodiments, the four variable domains are connected by peptide linkers L1, L2, and L3, and in some cases are arranged from N-terminus to C-terminus as follows:

(i) VL(CD3)-L1-VH(second antigen-binding site)-L2-VL(second antigen-binding site)-L3-VH(CD3); or

(ii) VH(CD3)-L1-VL(second antigen-binding site)-L2-VH(second antigen-binding site)-L3-VL(CD3); or

(iii) VL(second antigen-binding site)-L1-VH(CD3)-L2-VL(CD3)-L3-VH(second antigen-binding site); or

(iv) VH (second antigen-binding site)-L1-VL (CD3)-L2-VH (CD3)-L3-VL (second antigen-binding site).

In certain embodiments, the "another antigen binding site" is specific for a tumor antigen, such as CD19, CD30, or EGFRvIII. In a specific embodiment, the tumor antigen is not CD33.

The length of the joint affects the flexibility of the tandem diabody. Therefore, in some embodiments, the length of the peptide joint L1, L2 and L3 is such that the domain of a polypeptide can associate with the domain molecule of another polypeptide to form a dimerized antigen-binding tandem diabody. In certain embodiments, such a joint is "short", i.e., composed of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Therefore, in some cases, the joint is composed of about 12 or less amino acid residues. In the case of 0 amino acid residues, the joint is a peptide bond. Such a short joint is conducive to the intermolecular dimerization of two polypeptides by combining and forming a correct antigen binding site between the antibody light chain variable domain and the antibody heavy chain variable domain of different polypeptides. Shortening the joint to about 12 or less amino acid residues generally prevents the adjacent domains of the same polypeptide chain from interacting intramolecularly with each other. In some embodiments, these linkers consist of about 3 to about 12, in particular about 3 to about 10, such as 4, 5, 6, 7, 8 or 9 consecutive amino acid residues.

Regarding the amino acid composition of the linker, a peptide that does not interfere with the dimerization of the two polypeptides is selected. For example, a linker comprising a glycine residue and a serine residue generally provides protease resistance. The amino acid sequence of the linker can be optimized, for example, by phage display methods to improve antigen binding and the yield of antigen-binding polypeptide dimers. Examples of peptide linkers suitable for tandem diabodies according to the present invention are GGSGGS (SEQ ID NO: 16), GGSG (SEQ ID NO: 17), or GGSGG (SEQ ID NO: 18).

In some embodiments, the multimeric antigen-binding proteins described herein are produced by expressing a polynucleotide encoding a polypeptide of a tandem diabody (which associates with another identical polypeptide to form an antigen-binding tandem diabody). Thus, another aspect is a polynucleotide, such as DNA or RNA, encoding a polypeptide of a multimeric antigen-binding protein (e.g., a tandem diabody) described herein.

By known methods, for example, by combining genes encoding antibody variable domains (these antibody variable domains are separated by peptide linkers or directly connected by peptide bonds in other embodiments) into a single genetic construct operably connected to a suitable promotor and optionally a suitable transcription terminator, and expressing in bacteria or other suitable expression systems (e.g., Chinese hamster ovary celI), polynucleotides are constructed. Depending on the vector system and host employed, any number of suitable transcription and translation elements can be used, including constitutive and inducible promoters. Such a promoter is selected: it drives the expression of the polynucleotides in the corresponding host cell.

In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents another embodiment of the present invention. The recombinant vector can be constructed according to known methods.

A variety of expression vector/host systems can be used to contain and express polynucleotides encoding polypeptides such as the tandem diabodies described herein. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

Thus, in some embodiments, the antigen-binding tandem diabodies described herein are produced by introducing a vector encoding the above-described polypeptide into a host cell and culturing the host cell under conditions where the polypeptide chains are expressed, which can be isolated and optionally further purified. Taq' is not required for isolation and purification of the polypeptide, which is an advantage for in vivo administration.

In other aspects, provided herein are pharmaceutical compositions comprising an antigen-binding protein according to the present invention (e.g., a tandem diabody), a vector comprising a polynucleotide encoding a polypeptide of the antigen-binding protein or a host cell transformed by the vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is non-toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions (e.g., oil-in-water emulsions), various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and can be administered to a subject at a suitable dose. Preferably, the composition is sterile. These compositions may also contain adjuvants, such as preservatives, emulsifiers, and dispersants. By including various antibacterial and antifungal agents, it is possible to ensure that the behavior of microorganisms is prevented. The administration of suitable compositions can be achieved in different ways, such as by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. Of course, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound being administered, the time and route of administration, the type of treatment, general health, and other drugs being administered concurrently.

The present invention also provides medical uses or methods, wherein the antigen binding proteins as described above are administered to a subject (e.g., a patient) at an effective dose for immunosuppressive therapy (e.g., immunosuppressive therapy in transplantation); treatment of autoimmune diseases, inflammatory diseases, infectious diseases, allergies, or cancer (e.g., non-Hodgkin's lymphoma; chronic lymphocytic leukemia; Hodgkin's lymphoma; solid tumors, such as those arising in breast cancer, ovarian cancer, colon cancer, kidney cancer, or bile duct cancer; minimal residual disease; metastatic tumors, such as those metastasized in the lung, bone, liver, or brain). The antigen binding proteins can be used alone or in combination with current treatments for the prevention or treatment of these conditions.

Cancers that can be treated using the multispecific antigen binding proteins of the invention include, but are not limited to, primary and metastatic adrenocortical carcinoma, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, CNS tumors, peripheral CNS cancer, breast cancer, giant lymphadenopathy, cervical cancer, childhood non-Hodgkin lymphoma, colon and rectal cancer, endometrial cancer, esophageal cancer, Ewing family of tumors (e.g., Ewing sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, childhood leukemia, disease, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid, non-Hodgkin lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (soft tissue cancer in adults), melanoma skin cancer, non-melanoma skin cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer (e.g., uterine sarcoma), vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

"Effective dose" refers to the amount of active ingredient sufficient to affect the course and severity of the disease, resulting in alleviation or relief of such pathology. "Effective dose" for treating and/or preventing these diseases or conditions can be determined using methods known to those skilled in the art (see, e.g., Fingl et al., The Pharmacological Basis of Therapeutics, Goddman and Gilman, eds., Macmillan Publishing Co., New York, pp. 1-46 (1975)).

In another aspect of the invention, the antigen binding proteins described herein above are used in the manufacture of immunosuppressive agents or medicaments for treating autoimmune diseases, inflammatory diseases, infectious diseases, allergies, or cancer (e.g., non-Hodgkin's lymphoma; chronic lymphocytic leukemia; Hodgkin's lymphoma; solid tumors, such as those arising in breast cancer, ovarian cancer, colon cancer, kidney cancer, or bile duct cancer; minimal residual disease; metastatic tumors, such as those metastasizing to the lung, bone, liver, or brain). In certain cases, multispecific binding proteins have been described above as having particular utility in treating specific diseases, and these antigen binding proteins can also be used in the manufacture of medicaments for those specific diseases.

Methods for preparing pharmaceutical compositions (ie, medicaments) and the clinical use of antigen binding proteins in preventing and/or treating diseases (eg, cancer) are known to those skilled in the art.

In a specific aspect of the invention, the antigen binding protein is multispecific and is used for cancer treatment, because such multispecific antigen binding proteins can be used to retarget cytotoxic effector cells against tumor cells. This therapeutic concept is well known in the art. For example, clinical studies have shown tumor regression in patients treated with anti-CD3x anti-tumor bispecific antibodies (e.g., Canevari, S et al., J. Natl. Cancer Inst., 87: 1463-1469, 1996) or in patients treated with anti-CD16x anti-tumor bispecific antibodies (e.g., Hartmann et al.; Clin Cancer Res. 2001; 7(7): 1873-81). Proof of concept has been shown for a variety of recombinant bispecific antibody molecules comprising only the variable domain (Fv) (Cochlovius et al.; Cancer Research, 2000, 60: 4336-4341) or more recently in clinical studies using monomeric single-chain Fvs in a format (two single-chain antibodies of different specificities linked together; Amgen Germany; Bargou R. et al., Science, 2008, 321(5891): 974-977; Baeuerle PA and Reinhardt C., Cancer Res. 2009, 69(12): 4941-4944). The dimeric antigen binding proteins described herein can be used as pharmaceutical agents and applied in therapeutic approaches in a manner similar to bispecific antibodies in the art, as they can redirect therapeutic (e.g., cytotoxic) mechanisms using the same combined antibody specificities. In addition, immunosuppressive antibodies monospecific for CD3 (e.g., muromonab-CD3) are known for use in the treatment of transplant rejection, and acute rejection of kidney transplants (allografts), liver transplants, and heart transplants is known. Thus, antigen binding proteins specific for albumin and CD3 can be used in the same therapeutic approaches as known monospecific anti-CD3 antibodies.

Antigen binding proteins and compositions thereof can be in the form of oral, intravenous, intraperitoneal or other pharmaceutically acceptable dosage forms. In some embodiments, the composition is orally administered and the dosage form is a tablet, capsule, caplet or other oral form. In some embodiments, the composition is parenteral, for example, intravenous, intraperitoneal, intramuscular or subcutaneous, and is administered via a solution containing the antigen binding molecule.

Those skilled in the art will be able to easily and without undue burden construct and obtain the antigen binding proteins described herein by utilizing established techniques and standard methods known in the art, see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) New York; The Protein Protocols Handbook, edited by John M. Walker, Humana Press Inc. (2002); or Antibody engineering: methods and protocols/edited by Benny K.C. Lo; Benny K.C. Series II: Methods in molecular biology (Totowa, New Jersey).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Binding and cytotoxic activity of tandem diabodies containing different anti-CD3 domains. (A) Human CD19 ⁺ Raji cells, human CD3 ⁺ Jurkat cells, and cynomolgus monkey CD3 ⁺ HSC-F cells were stained on ice with 10 μg/mL of the tandem diabody TandAbB (containing a combination of the CD3 binding domain var_w (SEQ ID NO: 1 and SEQ ID NO: 6) and the CD19 binding domain). Cell surface-bound tandem diabodies were detected using an anti-His mAb followed by FITC-conjugated goat anti-mouse IgG. Mean fluorescence intensity determined by flow cytometry was modeled as a sigmoidal dose-response using nonlinear regression to calculate _KD . (B) EC50 values were determined in a cytotoxicity assay using calcein-labeled EGFRvIII ⁺ F98 target cells and PBMCs as effector cells at an E:T ratio of 50:1 and a 4- _hour incubation time. _EC50 values were calculated using nonlinear regression of the data modeled as a sigmoidal curve.

Figure 2: Biophysical stability of TandAb G (SEQ ID NO: 14), TandAb D (SEQ ID NO: 11), and TandAb E (SEQ ID NO: 12) incubated at 40°C for up to seven (7) days. (A) Homodimer tandem diabody content, (B) % recovery.

Figure 3: Binding affinity and cross-reactivity of EGFRvIII/CD3 tandem diabodies with humanized anti-CD3 domains. Human CD3+ Jurkat cells (A) and cynomolgus CD3+ HSC-F cells (B) were stained with increasing concentrations of TandAb _D (SEQ ID NO: 11) ( _KD = 1.822 nM on Jurkat, _KD = 3.4 nM on HSC-F) and TandAb E (SEQ ID NO: 12) (KD ⁼ 27.74 nM on Jurkat, _KD ⁼ 24.3 nM on HSC-F). Cell surface-bound tandem diabodies were detected by anti-His mAb followed by FITC-conjugated goat anti-mouse IgG. To calculate _KD , mean fluorescence intensity determined by flow cytometry was modeled as a sigmoidal dose-response by nonlinear regression.

Figure 4: Cytotoxic activity of an EGFRvIII/CD3 tandem diabody with humanized anti-CD3 domains. EC50 values were determined at an E:T ratio of 50:1 in a 4-hour cytotoxicity assay using calcein-labeled EGFRvIII ⁺ F98 cells as target cells and PBMCs as effector cells. _EC50 values were calculated by nonlinear regression _of the data modeled as a sigmoidal curve.

DETAILED DESCRIPTION

Examples of specific VL and VH comprising CD3 binding sites for use in generating antigen binding proteins according to the present invention are described below:

In the first step, the murine VH CDRs of SP34 (BD Bioscience; J. Immunol. Methods., 1994, 178:195) were grafted onto the most homologous human framework of IgG1 (human VH3-72), generating a chimeric binding domain composed of the newly humanized VH and the parental murine VLλ chain. Binding data showed that the chimeric molecule retained function. Therefore, the humanized VH chain was not altered during the subsequent humanization step of the VL chain.

For humanization of the murine SP34 VL sequence, the murine CDRs were grafted onto a human VL framework (human Vλ7-7a) with the highest homology to the parental murine framework. Constructs containing this closely related lambda chain were generated by introducing back mutations and assayed as tandem diabodies in combination with the CD19 binding domain. Back mutations were selected based on a comparison of the model of the original murine SP34 and a model of a humanized variant containing the Vλ7-7a framework; back mutations were selected to reduce steric hindrance and maintain the donor murine antibody CDR configuration when grafted onto the recipient human framework. However, all of these tandem diabodies containing lambda chains either expressed poorly or failed to recognize the antigen.

In the present invention, the humanization VL chain of the present invention is carried out to replace the humanized VL chain of the present invention.Therefore, the alternative method for humanization VL chain is selected.In this second strategy, the data about the best pairing of heavy chain and light chain are analyzed.Mouse CDR is transplanted on fixed people's framework (people Vκ1_39).Therefore, lambda chain is replaced by kappa chain.It is found that the Vκ1_39 framework of light chain can be compatible with the humanized VH3_72 framework (as described above) for heavy chain.After this, the combination of test Vκ1_39 framework and VH3_72 framework causes the Fv with improved VL/VH pairing, and whether can produce the humanized domain with good stability, expression and other biophysical properties.

Some tandem double antibodies were produced, which contained a small group of single mutations or their combinations. When transplanted onto the acceptor human framework, back mutations were selected to reduce steric hindrance and maintain the donor mouse antibody CDR configuration. Most surprisingly, the antigen-binding proteins containing κ chains showed acceptable expression and excellent properties for binding to human and cynomolgus monkey CD3. Based on their biophysical and functional properties and their respective expression yields, Vκ binding domain var_w was identified as the most promising candidate and was therefore selected for further development. The initial stability of var_w can be significantly improved by the following mutations in VL: D72→T72, K73→D73 and A74→F74, which leads to candidate var_x.

In the step of carrying out successively of humanized CD3 binding domain, var_w and var_x are combined with other antigen binding sites (i.e., anti-EGFRvIII, anti-EpCAM and anti-CD30) except anti-CD19, to measure the ability of these two variants and various anti-tumor antigen site pairing. The combination of var_w and var_x with these tumor target binding sites produces well-expressed and stable tandem diabodies, which show target binding and T cell-mediated cytotoxicity. Figure 1A shows the binding activity of TandAb B (antibody containing the combination of CD3 binding domain var_w and CD19 binding domain) measured by flow cytometry. The binding affinity ( _KD ) of TandAb B to human cell line Raji, CD3 ⁺ human T cell line (Jurkat) and cynomolgus monkey CD3 expressing cell line (HSC-F) expressing CD19 is shown. Cross-reactivity is observed, while there is no substantial difference in the combination ( _KD ) of cynomolgus monkey and human CD3. In Fig. 1 B, present cytotoxicity test, wherein series connection diabody contains CD3 binding domain var_w or var_x and EGFRvIII binding domain combination.In using human PBMC as 4 hour cytotoxicity test (calcein labeling) of effector cell, the cell line F98 expressing EGFRvIII is effectively dissolved.The target cell lysis of the series connection diabody mediation of TandAb G (SEQ ID NO:14) containing var_w is suitable with the target cell lysis of the series connection diabody mediation of control TandAb F (SEQ ID NO:13) containing parent mouse CD3 binding domain of SP34.

The sequence of clone var_w and var_x and modeling analysis have found the key role of the combination and stability properties of these variants in the series connection diabody to (according to model they directly contact each other) amino acid in position VH111 and VL49.Introduce some different sudden changes at these positions, and measure individually or in the series connection diabody with the combination of EGFRvIII binding domains.On position VH111, sudden change is from W to T, Q, N, S, F, Y, R or H; On position VL49, sudden change is from G to A, V, S, T or N.Measure the binding properties, cytotoxicity and the stability of all series connection diabodies obtained.

Mutation of VH111 from W to Y and VL49 from G to A generated the CD3 binding domain var_y, while a single mutation of VH111 from W to H generated the domain var_z; these binding domains resulted in the tandem diabodies TandAb D (var_y) (SEQ ID NO: 11) and TandAb E (var_z) (SEQ ID NO: 12), which exhibit improved stability properties relative to the parent var_w. According to Shirai's rule (Kuroda et al., 2008, Proteins, 73: 608), VH111 occupies a specific position in CDRH3 that determines whether the conformation adopted by CDRH3 is extended or kinked. Experimental results indicate that permissible substitutions for W at this position must contain large aromatic rings, such as F, Y, or H; otherwise, loss of binding is expected. In addition, simulated direct contacts of the backbone near VL49 and VH111 also impose significant constraints on the nature of permissible residues. Using size exclusion chromatography (SEC) to monitor homodimer tandem diabody content, improved 7-day stability of these proteins at 40°C was observed. Both clones exhibited increased desired tandem diabody homodimer content relative to TandAb G (SEQ ID NO: 14) containing the parental var_w, as well as improved recovery. Figures 2A and 2B depict the stability of TandAb D (SEQ ID NO: 11), TandAb E (SEQ ID NO: 12), and TandAb G (SEQ ID NO: 14) at 40°C for 7 days.

Surprisingly, functional assays further demonstrated that the binding activity and cytotoxicity of these tandem diabodies were retained. Figures 3A and 3B show the binding of titrated tandem diabodies TandAb D containing var_y and TandAb E containing var_z (SEQ ID NO: 12) to CD3 ⁺ human T cell lines (Jurkat) and CD3 ⁺ cynomolgus monkey cell lines (HSC-F), as determined by flow cytometry analysis. _KD values were calculated by nonlinear regression and are summarized in the table below. Comparable binding affinities were observed for human and cynomolgus monkey CD3, indicating cross-reactivity of the CD3 binding domains. The affinity of TandAb D (SEQ ID NO: 11) for human CD3 ( _KD = 1.8 nM) was >10-fold that of TandAb E ( _KD = 27.7 nM) (SEQ ID NO: 12).

In a 4-hour calcein release cytotoxicity assay, TandAb D (SEQ ID NO: 11), containing var_y, and its precursor, TandAb G (SEQ ID NO: 14), containing var_w, were directly compared using the EGFRvIII-expressing cell line F98 as target cells and human PBMCs as effector cells. Target cell lysis induced by both antibodies was observed in a concentration-dependent manner, demonstrating comparable potency (TandAb G EC ₅₀ = 129 pM, TandAb D EC ₅₀ = 122 pM). Titration curves are shown in Figure 4.

In summary, starting from mouse clone SP34, VH chain and VL chain are progressively humanized by CDR-transplantation. The stability of the humanized molecule is further increased in the tandem diabody by point mutation of framework residues and the selection of those substitutions that retain binding activity and cytotoxicity. Two optimized CD3 binding domains var_y and var_z show very good cross-reactivity to cynomolgus monkey CD3. The target affinity difference of the optimized domain is about 10 times, which makes it possible to produce tandem diabodies with different affinities for T cell recruitment. The VH amino acid sequence and the VL amino acid sequence of the comparison of the CD3 binding domains (var_y and var_z), two intermediates (var_w and var_x) and the parent mouse clone (SP34) are shown in Table 1.

Sequence Listing

<110> Evermede Co., Ltd.

<120> CD3 binding domain

<130> A 3268

<150> PCT/EP2014/002177

<151> 2014-08-07

<160> 18

<170> BiSSAP 1.0

<210> 1

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..110

<223> /mol_type="Protein"

/note="var_w的VL"

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<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 2

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..110

<223> /mol_type="Protein"

/note="var_x's VL"

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<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 3

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..110

<223> /mol_type="Protein"

/note="var_y的VL"

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<400> 3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Ala Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 4

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..110

<223> /mol_type="Protein"

/note="var_z的VL"

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<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 5

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..109

<223> /mol_type="Protein"

/note="Rat SP34 VL"

/organism="Artificial sequence"

<400> 5

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 6

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..125

<223> /mol_type="Protein"

/note="var_w的VH"

/organism="Artificial sequence"

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 7

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..125

<223> /mol_type="Protein"

/note="var_x的VH"

/organism="Artificial sequence"

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 8

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..125

<223> /mol_type="Protein"

/note="var_y的VH"

/organism="Artificial sequence"

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Tyr Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 9

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..125

<223> /mol_type="Protein"

/note="var_z's VH"

/organism="Artificial sequence"

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser His Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 10

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..125

<223> /mol_type="Protein"

/note="Rat SP34 VH"

/organism="Artificial sequence"

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 11

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..494

<223> /mol_type="Protein"

/note="TandAb D"

/organism="Artificial sequence"

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Ala Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

115 120 125

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

130 135 140

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

145 150 155 160

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

165 170 175

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

180 185 190

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

195 200 205

Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe

210 215 220

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser

225 230 235 240

Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser

245 250 255

Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys

260 265 270

Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

275 280 285

Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe

290 295 300

Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val

305 310 315 320

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser

325 330 335

Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

340 345 350

Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

355 360 365

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

370 375 380

Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly

385 390 395 400

Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr

405 410 415

Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

420 425 430

Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

435 440 445

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly Asn

450 455 460

Ser Tyr Val Ser Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

465 470 475 480

Val Ser Ser Ala Ala Ala Gly Ser His His His His His

485 490

<210> 12

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..494

<223> /mol_type="Protein"

/note="TandAb E"

/organism="Artificial sequence"

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

115 120 125

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

130 135 140

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

145 150 155 160

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

165 170 175

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

180 185 190

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

195 200 205

Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe

210 215 220

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser

225 230 235 240

Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser

245 250 255

Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys

260 265 270

Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

275 280 285

Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe

290 295 300

Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val

305 310 315 320

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser

325 330 335

Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

340 345 350

Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

355 360 365

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

370 375 380

Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly

385 390 395 400

Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr

405 410 415

Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

420 425 430

Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

435 440 445

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly Asn

450 455 460

Ser Tyr Val Ser His Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

465 470 475 480

Val Ser Ser Ala Ala Ala Gly Ser His His His His His

485 490

<210> 13

<211> 493

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..493

<223> /mol_type="Protein"

/note="TandAb F"

/organism="Artificial sequence"

<400> 13

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Ser

100 105 110

Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

115 120 125

Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

130 135 140

Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

145 150 155 160

Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser

165 170 175

Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

180 185 190

Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met

195 200 205

Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe Asp

210 215 220

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser Gly

225 230 235 240

Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro

245 250 255

Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln

260 265 270

Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val

275 280 285

Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser

290 295 300

Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln

305 310 315 320

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Gly

325 330 335

Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly

340 345 350

Gly Ser Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

355 360 365

Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe

370 375 380

Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys

385 390 395 400

Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala

405 410 415

Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp

420 425 430

Asp Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu

435 440 445

Asp Thr Ala Met Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser

450 455 460

Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

465 470 475 480

Ser Ser Ala Ala Ala Gly Ser His His His His His

485 490

<210> 14

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..494

<223> /mol_type="Protein"

/note="TandAb G"

/organism="Artificial sequence"

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ser Arg

50 55 60

Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

115 120 125

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

130 135 140

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

145 150 155 160

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

165 170 175

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

180 185 190

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

195 200 205

Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe

210 215 220

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser

225 230 235 240

Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser

245 250 255

Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys

260 265 270

Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

275 280 285

Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe

290 295 300

Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val

305 310 315 320

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser

325 330 335

Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

340 345 350

Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

355 360 365

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

370 375 380

Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly

385 390 395 400

Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr

405 410 415

Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

420 425 430

Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

435 440 445

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly Asn

450 455 460

Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

465 470 475 480

Val Ser Ser Ala Ala Ala Gly Ser His His His His His

485 490

<210> 15

<211> 494

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..494

<223> /mol_type="Protein"

/note="TandAb H"

/organism="Artificial sequence"

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr

20 25 30

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

65 70 75 80

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Leu Trp Tyr Ser

85 90 95

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Ser Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

115 120 125

Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser

130 135 140

Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly

145 150 155 160

Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr

165 170 175

Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile

180 185 190

Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala

195 200 205

Met Tyr Tyr Cys Ala Arg Leu Gly Ser Ser Trp Thr Asn Asp Ala Phe

210 215 220

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Ser

225 230 235 240

Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser

245 250 255

Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys

260 265 270

Gln Tyr Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

275 280 285

Val Ile Tyr Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe

290 295 300

Ser Gly Ser Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val

305 310 315 320

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser

325 330 335

Gly Thr Pro Leu Ile Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

340 345 350

Gly Gly Ser Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

355 360 365

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

370 375 380

Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly

385 390 395 400

Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr

405 410 415

Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg

420 425 430

Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr

435 440 445

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Asn Phe Gly Asn

450 455 460

Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

465 470 475 480

Val Ser Ser Ala Ala Ala Gly Ser His His His His His

485 490

<210> 16

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..6

<223> /mol_type="Protein"

/note="Peptide linker"

/organism="Artificial sequence"

<400> 16

Gly Gly Ser Gly Gly Ser

1 5

<210> 17

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..4

<223> /mol_type="Protein"

/note="Peptide linker"

/organism="Artificial sequence"

<400> 17

Gly Gly Ser Gly

1

<210> 18

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> SOURCE

<222> 1..5

<223> /mol_type="Protein"

/note="Peptide linker"

/organism="Artificial sequence"

<400> 18

Gly Gly Ser Gly Gly

1 5

Claims (16)

1. An antigen-binding protein comprising at least one CD3 binding site, wherein the CD3 binding site comprises: a) the heavy chain variable domain VH as described in SEQ ID NO: 8 and the light chain variable domain VL as described in SEQ ID NO: 3; or b) The heavy chain variable domain VH as described in SEQ ID NO: 9 and the light chain variable domain VL as described in SEQ ID NO: 4; and, The antigen-binding protein having a) is not a bispecific binding protein that specifically binds to human CD33 and human CD3. 2. The antigen-binding protein according to claim 1, wherein the antigen-binding protein is not a bispecific tandem double antibody binding CD33 and CD3. 3. The antigen-binding protein according to claim 1 or 2, wherein the protein comprises at least one additional functional domain. 4. The antigen-binding protein according to claim 3, wherein the at least one additional functional domain is an additional antigen-binding site. 5. The antigen-binding protein according to claim 4, wherein the additional antigen-binding site is specific to antigens of tumor cells, viral pathogens, bacterial pathogens, or autoimmune antigens. 6. The antigen-binding protein of claim 5, wherein the additional antigen-binding site is not specific to CD33. 7. The antigen-binding protein according to any one of claims 1, 4, 5 and 6, wherein the antigen-binding protein is multivalent. 8. The antigen-binding protein according to claim 7, wherein the antigen-binding protein is multispecific. 9. The antigen-binding protein according to claim 8, wherein the antigen-binding protein is polymeric. 10. The antigen-binding protein of claim 9, wherein the antigen-binding protein is dimer, comprising a first polypeptide and a second polypeptide, each polypeptide having at least four sequentially linked chain variable domains, wherein the antigen-binding protein comprises at least one CD3 binding site as described in claim 1 and at least one additional antigen-binding site specific to the second antigen. 11. The antigen-binding protein of claim 10, wherein each polypeptide has at least four variable domains fused together with each other via peptide linkers L1, L2 and L3 in the following order: i) VL(CD3)-L1-VH(second antigen)-L2-VL(second antigen)-L3-VH(CD3); ii) VH(CD3)-L1-VL(second antigen)-L2-VH(second antigen)-L3-VL(CD3); iii) VL (second antigen)-L1-VH(CD3)-L2-VL(CD3)-L3-VH (second antigen); or iv) VH (second antigen)-L1-VL(CD3)-L2-VH(CD3)-L3-VL(second antigen). 12. The antigen-binding protein according to claim 11, wherein linkers L1, L2 and L3 consist of 12 or fewer amino acid residues. 13. A polynucleotide encoding an antigen-binding protein according to any one of claims 1 to 12. 14. A pharmaceutical composition comprising i) an antigen-binding protein according to any one of claims 1 to 12, a polynucleotide according to claim 13, or a mediator comprising the polynucleotide according to claim 13, and ii) a pharmaceutically acceptable carrier. 15. A method for producing an antigen-binding protein according to any one of claims 1 to 12, comprising: i) Introducing the polynucleotide according to claim 13 into the host cell. ii) Culture the host cells under conditions expressing the antigen-binding protein, and iii) Purify the expressed antigen-binding protein. 16. Use of the antigen-binding protein according to any one of claims 1 to 12 in the preparation of a pharmaceutical agent.