CD3-specific binding molecules

Abstract

The present invention relates to specific binding molecules, in particular antibodies and fragments thereof, which bind to CD3 with improved properties.

Description

Technical Field

[0001] The present invention relates to specific binding molecules, in particular antibodies and fragments thereof, which bind to CD3 with improved properties. Background Art

[0002] CD3 (cluster of differentiation 3) is a T cell co-receptor that helps activate cytotoxic T cells (CD8+T cells) and T helper cells (CD4+T cells). CD3 associates with the T-cell receptor (TCR) and the zeta chain (zeta chain; CD247) to generate activation signals in T lymphocytes. TCR, zeta chain, and CD3 molecules together constitute the TCR complex.

[0003] Antibodies that bind to CD3 are known and can be used as immunosuppressive drugs. Examples include muromonab-CD3 (Janssen-Cilag), otelixizumab (also known as TRX4), teplizumab (also known as PRV-031), and visilizumab.

[0004] Anti-CD3 antibodies can also be used as T cell recruiting agents in a class of protein-based therapies widely known as T cell engagement bispecific therapies (Baeuerle et al., Cancer Res. 2009 Jun 15; 69 (12): 4941-4). This type of therapy combines a target cell recognition domain with an anti-CD3 domain. Independent of T cell receptor specificity, simultaneous engagement of target cells (e.g., cancer cells) and CD3+ cytotoxic T cells leads to activation of the CD3 signaling pathway and ultimately to target cell death. The bispecific antibody blinatumomab (Amgen) is an example of a commercially available T cell engagement therapeutic for the treatment of acute lymphoblastic leukemia (ALL). Many other T cell-engaging bispecific antibodies are being studied in clinical trials for the treatment of various cancers and infectious diseases (e.g., see Yuraszeck et al., Clin Pharmacol Ther. 2017 May; 101(5): 634-645 and Husain et al., BioDrugs. 2018 Oct; 32(5): 441-464). Most T cell-engaging bispecific antibodies recognize cell surface antigens on target cells. In addition, T cell-engaging bispecific antibodies are also known to recognize short peptides derived from intracellular antigens and presented on the cell surface in complex with MHC (pMHC) (Liddy et al., Nat Med. 2012 Jun; 18(6): 980-7).

[0005] Antibody UCHT1 is a clinically relevant anti-CD3 antibody known in the art (Shalaby et al., J Exp Med. 1992 Jan 1; 175(1): 217-25; US5821337). The scFv fragment of the humanized UCHT1 antibody has been fused to a soluble T cell receptor to construct a T cell engaging bispecific antibody that binds to pMHC on target cells (see, for example, WO2011001152). Summary of the invention

[0006] The present inventors have surprisingly found that the introduction of certain mutations in the amino acid sequence of UCHT1 leads to the generation of T cell engaging bispecific molecules with unexpected properties that are particularly advantageous for clinical use.

[0007] In a first aspect, a specific binding molecule is provided, which binds to CD3 and comprises a polypeptide having an immunoglobulin VL domain and an immunoglobulin VH domain, wherein the VL domain comprises complementarity determining regions (CDRs) VLCDR1, VLCDR2 and VLCDR3, and wherein the VH domain comprises complementarity determining regions (CDRs) VHCDR1, VHCDR2, VHCDR3, each complementarity determining region having:

[0008] The following amino acid sequences, wherein

[0009] VLCDR1 is QDIRNY

[0010] VLCDR2 for YTS

[0011] VLCDR3 is QQGNTLPWT

[0012] VHCDR1 is GYSFTGYA

[0013] VHCDR2 is INPYKGVS

[0014] VHCDR3 is ARSGYYGDSDWYFDV;

[0015] or an amino acid sequence that is at least 70% identical thereto.

[0016] CDR is based on the International ImMunoGeneTics information system. ) definition (LeFranc et al., Nucleic Acids Res. 2009Jan; 37 (Database issue): D1006-12).

[0017] In particular, the specific binding molecules of the present invention comprise an alanine at the C-terminus of VHCDR1, which corresponds to No. 38 in the numbering (which is designated as No. 165 in this article and Figure 1 (exemplified and shown in bold in the heavy chain sequence v1 of ). It has been found that molecules comprising this mutation have improved properties, including an improved specificity window, compared to molecules without this mutation.

[0018] CDR can be provided in antibody variable domain framework sequence.Framework sequence can be mouse framework sequence or people framework sequence or humanized framework sequence or any other suitable framework.Preferred framework is people framework sequence or humanized framework sequence.People framework or humanized framework basically has the amino acid sequence of human immunoglobulin.In some cases, mouse framework, people framework and humanized framework can be mixed in any combination.

[0019] Preferably, the immunoglobulin VL comprises the full sequence VLFW1-VLCDR1-VLFW2-VLCDR2-VLFW3-VLCDR3-VLFW4, wherein VLFW1, VLFW2, VLFW3 and VLFW4 are VL framework (VL framework, VLFW) sequences 1 to 4, respectively, and the immunoglobulin VH comprises the full sequence VHFW1-VHCDR1-VHFW2-VHCDR2-VHFW3-VHCDR3-VHFW4, wherein VHFW1, VHFW2, VHFW3 and VHFW4 are VH framework (VH framework, VHFW) sequences 1 to 4, respectively, optionally wherein the VLFW sequence and the VHFW sequence are mouse, human or humanized framework sequences.

[0020] Preferably, the immunoglobulin VL comprises the following full sequence: VLFW1-VLCDR1-VLFW2-VLCDR2-VLFW3-VLCDR3-VLFW4, wherein VLFW1, VLFW2, VLFW3 and VLFW4 are framework (FW) sequences 1 to 4, respectively, and VLFW1, VLFW2, VLFW3 and VLFW4 each have:

[0021] The following amino acid sequences, wherein:

[0022] VLFW1 is AIQMTQSPSSLSASVGDRVTITCRAS or DIQMTQSPSSLSASVGDRVTITCRAS

[0023] VLFW2 is LNWYQQKPGKAPKLLIY

[0024] VLFW3 is RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC

[0025] VLFW4 is FGQGTKVEIK;

[0026] or an amino acid sequence that is at least 70% identical thereto.

[0027] Preferably, the immunoglobulin VH comprises the following full sequence: VHFW1-VHCDR1-VHFW2-VHCDR2-VHFW3-VHCDR3-VHFW4, wherein VHFW1, VHFW2, VHFW3 and VHFW4 are framework (FW) sequences 1 to 4, respectively, and VHFW1, VHFW2, VHFW3 and VHFW4 each have:

[0028] The following amino acid sequences, wherein:

[0029] VHFW1 is EVQLVESGGGLVQPGGSLRLSCAAS

[0030] VHFW2 is MNWVRQAPGKGLEWVAL

[0031] VHFW3 is TYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYC or TYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYC

[0032] VHFW4 is WGQGTLVTVSS;

[0033] or an amino acid sequence that is at least 70% identical thereto.

[0034] In particular, the specific binding molecules of the present invention are in the region corresponding to The 78th position in the numbering contains phenylalanine (which is designated as position 202 herein and Figure 1 It has been found that molecules including this mutation in addition to the alanine at position 38 have increased T cell activation efficiency compared to molecules without these mutations.

[0035] Preferably, the immunoglobulin VL comprises the following sequence or an amino acid sequence that is at least 70% identical to the following sequence:

[0036] AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK; or

[0037] DIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK.

[0038] Preferably, the immunoglobulin VH comprises the following sequence or an amino acid sequence that is at least 70% identical to the following sequence:

[0039] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS;

[0040] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS.

[0041] Preferably, the specific binding molecule is in the form of a scFv fragment.

[0042] Preferably, the immunoglobulin VL and VH domains are connected by a linker. The linker can be any amino acid sequence, preferably about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Optionally, in addition to other amino acids, preferred linkers include sequences with the formula (GGGGS)n. Therefore, a single-chain specific binding molecule having the following sequence or an amino acid sequence at least 70% identical to the following sequence is provided:

[0043] AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGS OR

[0044] AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGS GGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS.

[0045] The specific binding molecule can also be a part of a fusion protein comprising other domains. The fusion protein can be constructed by fusion of the N-terminal or C-terminal with a VL or VH immunoglobulin domain. Other domains can be fused by a joint. Joint sequences are usually flexible because they are mainly composed of amino acids (such as glycine, alanine and serine without the bulky side chains that may limit flexibility). Alternatively, joints with greater rigidity may be desirable. Available or optimal length joint sequences can be easily determined. Usually the length of the joint sequence is less than about 12 amino acids, for example less than 10, or less than 2-10 amino acids. The joint can be any amino acid sequence, preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Examples of suitable linkers include, but are not limited to, GGGSGGGG, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP and GGEGGGSEGGGS (as described in WO2010 / 133828). Preferred linkers include sequences having the formula (GGGGS)n, optionally with other amino acids.

[0046] Preferably, when the specific binding molecule is used as a part of a bispecific molecule (preferably a T cell engaging bispecific antibody as described herein), the specific binding molecule exhibits one or more improved therapeutic properties relative to a non-mutated molecule. Preferably, the improved therapeutic properties are selected from an improved therapeutic window and / or an increased maximum T cell activation at a given concentration. The improved therapeutic window can achieve a higher dosage while minimizing the toxicity caused by off-target activation. At a given bispecific molecule concentration, the increase in maximum T cell activation can more effectively kill target cells at a given drug dose. Methods for determining T cell activation are known in the art, including the release of immune activation cytokines and T cell-mediated cell death. The therapeutic window of a T cell engaging bispecific antibody can be determined by measuring T cell activation in the presence of antigen-positive cells and antigen-negative cells and calculating the difference between the two measured values. More details of the preferred method are described in Example 2.

[0047] Preferably, the anti-CD3 antibodies of the present invention bind to the CD3ε subunit of CD3.

[0048] The specific binding molecules according to the present invention can be used for a method of treating or diagnosing a human or animal body, such as a method of treating a patient (preferably a human) condition, the method comprising administering an effective amount of the specific binding molecules of the present invention to the patient. The present invention also provides specific binding molecules of the present invention for use in medicine, and uses of the specific binding molecules of the present invention in the preparation of a drug for diagnosing or treating a tumor.

[0049] These and other aspects of the invention are described in further detail below.

[0050] As used herein, "treatment" includes any regimen that can benefit a human or non-human animal, preferably a mammal. Treatment may be for an existing condition, or may be preventative (prophylactic treatment).

[0051] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen binding site that specifically binds to an antigen, whether natural or partially or wholly synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is an antibody binding domain or homologous to an antibody binding domain. These antibodies may be from natural sources, or they may be partially or wholly synthetically produced. Examples of antibodies are immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and isotype subclasses thereof; fragments comprising antigen binding domains such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. Monoclonal antibodies may be referred to as "mabs" herein.

[0052] Monoclonal antibodies and other antibodies can be used, and recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such technology may involve introducing DNA encoding the immunoglobulin variable region or complementary determining region (CDR) of the antibody into the constant region or constant region plus framework region of different immunoglobulins. For example, see EP-A-184187, GB2188638A or EP-A-239400. Hybridomas or other cells that produce antibodies may undergo genetic mutations or other changes, which may or may not change the binding specificity of the antibody produced.

[0053] Since antibodies can be modified in a variety of ways, the term "antibody" should be interpreted as covering any specific binding molecule or substance having a binding domain with desired specificity. Therefore, the term covers antibody fragments, derivatives, functional equivalents and antibody homologues, humanized antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or fully or partially synthesized. Therefore, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. Humanized antibodies can be modified antibodies with non-human (e.g., murine) antibody variable regions and human antibody constant regions. For example, U.S. Patent No. 5,225,539 describes a method for preparing humanized antibodies.

[0054] It has been shown that fragments of intact antibodies can perform the function of binding to antigens. Examples of binding fragments are: (i) Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH domains (Ward, ES et al., Nature 341: 544-546 (1989)); (v) isolated CDR regions; (vi) F(ab')2 fragments: bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al., Science 242: 423-426 (1988); Huston et al., PNAS USA85:5879-5883 (1988)); (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965); and (ix) "diabodies": multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0055] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain containing an immunoglobulin light chain binding region and a second domain containing an immunoglobulin heavy chain binding region, the two domains being connected (e.g., via a peptide linker) but unable to associate with each other to form an antigen binding site: the antigen binding site is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO94 / 13804).

[0056] Where bispecific antibodies are used, these may be conventional bispecific antibodies (Hollinger & Winter, Current Opinion Biotechnol. 4:446-449 (1993)) that can be made in various ways, such as chemical preparation or hybridoma hybridization, or may be any of the bispecific antibody fragments described above. It may be preferred to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs can be constructed using only the variable domains without an Fc region, potentially reducing the effect of anti-idiotypic responses. Other forms of bispecific antibodies include single-chain "Janusins" described in Traunecker et al., EMBO Journal 10:3655-3659 (1991).

[0057] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be easily selected using phage display from libraries (WO 94 / 13804). If one arm of the diabody is to be kept constant, e.g., a constant specificity for antigen X, a library can be prepared in which the other arm is varied, and antibodies with the appropriate specificity selected.

[0058] An "antigen binding domain" is a portion of an antibody that contains a region that specifically binds to and is complementary to part or all of an antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided by one or more antibody variable domains. The antigen binding domain can include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0059] "Specificity" is generally used to refer to situations where one member of a specific binding pair does not exhibit any significant binding to molecules other than its specific binding partner, e.g., the member has less than about 30%, preferably 20%, 10% or 1% cross-reactivity with any other molecule. The term also applies, for example, to situations where an antigen binding domain is specific for a particular epitope carried by many antigens, in which case a specific binding molecule carrying the antigen binding domain will be able to bind to a variety of antigens carrying that epitope.

[0060] According to the present invention, "isolated" refers to the state in which the specific binding molecules of the present invention or nucleic acids encoding such binding molecules are preferably located. Molecules and nucleic acids will generally be free of or substantially free of materials with which they are naturally associated, for example, other polypeptides or nucleic acids in their natural environment, or other polypeptides or nucleic acids found in the environment in which they are prepared (e.g., cell culture medium) when the preparation is carried out by recombinant DNA techniques implemented in vitro or in vivo. Specific binding molecules and nucleic acids can be formulated with diluents or adjuvants, but are still isolated for practical purposes - for example, the molecules will usually be mixed with gelatin or other carriers if used to coat microtiter plates for immunoassays, or with pharmaceutically acceptable carriers or diluents when used for diagnosis or treatment. Specific binding molecules may be naturally glycosylated or glycosylated by heterologous eukaryotic cell systems, or they may be (e.g., if produced by expression in prokaryotic cells) unglycosylated.

[0061] "Essentially as listed" means that the CDR regions of the invention are Figure 1 a and Figure 1The designated regions of cb are identical or highly homologous. "Highly homologous" means that 1 to 5, 1 to 4, 1 to 3, 2 or 1 substitutions can be made in the CDR.

[0062] The present invention also includes within its scope Figure 1 and Figure 2 Polypeptides of the amino acid sequence shown and sequences substantially identical thereto, such as sequences having at least 70%, at least 80%, at least 85%, at least 90, at least 95% or 99% identity thereto.

[0063] The variable domain can be derived from any germline or rearranged human variable domain, or can be a synthetic variable domain based on the consensus sequence of a known human variable domain. Recombinant DNA technology can be used, for example, the disclosed reorganization or combination techniques of Stemmer (Nature 370: 389-391 (1994), which describes the technology associated with the beta-lactamase gene, and observes that the method can be used to produce antibodies), to introduce a sequence of the present invention into a variable domain library lacking a CDR3 region.

[0064] Another alternative is to use random mutagenesis of, for example, the SC104 VH or VL gene to generate mutations throughout the variable domain, thereby generating new VH or VL regions carrying the sequences of the invention. This technique is described by Gram et al. (Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992)), who used error-prone PCR.

[0065] Another approach that can be used is to directly mutagenesis the CDR regions of the VH or VL genes. This technique is disclosed by Barbas et al. (Proc. Natl. Acad. Sci. USA 91:3809-3813 (1994)) and Schier et al. (J. Mol. Biol. 263:551-567 (1996)).

[0066] The main part of the immunoglobulin variable domain will generally include at least three CDR regions and their intervening framework regions. The portion may also include at least about 50% of one or both of the first framework region and the fourth framework region, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The additional residues at the N-terminal or C-terminal end of the main part of the variable domain may be those residues that are not usually associated with the naturally occurring variable domain region. For example, the construction of the specific binding molecule of the present invention by recombinant DNA technology may result in the introduction of N-terminal or C-terminal residues encoded by a joint introduced to facilitate cloning or other operating steps, including the introduction of a joint to connect the variable domain of the present invention and other protein sequences, which include immunoglobulin heavy chains, other variable domains (e.g., in the production of double-lifting antibodies) or protein tags, as discussed in more detail below.

[0067] Preferably, the specific binding molecule comprises a Figure 1 A pair of binding domains based on the amino acid sequences of the VL and VH regions listed above. A single binding domain based on any of these sequences forms a further aspect of the invention. In a method based essentially on Figure 1 In the case of binding domains of the listed VH region amino acid sequences, such binding domains can be used as targeting agents, since it is known that immunoglobulin VH domains are able to bind to target antigens in a specific manner.

[0068] The specific binding molecules of the present invention may also comprise antibody constant regions or portions thereof. Figure 1 The specific binding molecules of the VL region shown can be linked at their C-terminal end to the antibody light chain constant domain, including human Cκ chain or Cλ chain. Figure 1 The specific binding molecules of the VH region shown can be linked at its C-terminus to all or part of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE and IgM) and any isotype subclass (particularly IgG1 and IgG4).

[0069] The specific binding molecules of the present invention may additionally be labeled with a functional marker. Such functional markers include toxins (e.g., ricin) and enzymes (e.g., bacterial carboxypeptidases or nitroreductases) that are capable of converting prodrugs into active drugs. In addition, the specific binding molecules may be combined with chemotherapeutic or cytotoxic agents (e.g., calicheamicin) or radioactive markers (e.g., 90 Y or 131 I) connected or otherwise associated.

[0070] In addition, the specific binding molecules of the present invention can be associated with additional therapeutic agents or targeting moieties. The therapeutic agents that can be associated with the specific binding molecules include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforins) or chemotherapeutic agents (e.g., cisplatin). In order to ensure that toxic effects are exerted at the desired location, the toxin can be in the liposome connected to the specific binding molecule so that the compound is slowly released. This will prevent the damage during the in vivo transport process and ensure that the toxin has the maximum effect after the specific binding molecule is combined with the relevant antigen presenting cells.

[0071] Examples of suitable therapeutic agents include, but are not limited to:

[0072] Small molecule cytotoxic agents, i.e. compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e. compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate arbourate, auristatin E, vincristine, and doxorubicin;

[0073] Peptide cytotoxins, i.e. proteins or fragments thereof that have the ability to kill mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, deoxyribonucleases and ribonucleases;

[0074] Radionuclides, i.e., unstable isotopes of elements that decay with the simultaneous emission of one or more of alpha or beta particles or gamma rays. For example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213; chelating agents may be used to promote the association of these radionuclides with high-affinity TCRs or multimers thereof;

[0075] Immunostimulants, i.e. immune effector molecules that stimulate immune responses. For example, cytokines such as IL-2 and IFN-γ;

[0076] Superantigens and their mutants;

[0077] TCR-HLA fusions, e.g., with peptide-HLA complexes, wherein the peptide is derived from a common human pathogen, e.g., Epstein Barr Virus (EBV);

[0078] Chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.

[0079] Antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28 or anti-CD16);

[0080] Alternative protein scaffolds with antibody-like binding characteristics;

[0081] Complement activators;

[0082] Heterologous protein domains, allologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0083] Targeting moieties that can be associated with specific binding molecules include:

[0084] TCR (including α / β and γ / δ TCR)

[0085] Antibodies or fragments thereof that recognize and bind to antigens presented on target cells, including peptides derived from intracellular antigens presented on the cell surface and in complex with MHC / HLA;

[0086] Alternative protein scaffolds with antibody-like binding characteristics.

[0087] In addition, depending on the disease to be treated, the specific binding molecules of the present invention can be administered alone or with other treatments, which can be administered simultaneously or sequentially. Therefore, the present invention further provides a product containing the specific binding molecules of the present invention and an activating agent, which is used as a combined preparation for simultaneous, separate or sequential use in tumor therapy. The activating agent can include a chemotherapeutic agent or a cytotoxic agent, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin (oxaliplatin) and tamoxifen (tamoxifen), which can synergize with the binding molecules of the present invention. Other activating agents can include an analgesic of suitable dose, such as nonsteroidal anti-inflammatory drugs (such as aspirin, paracetamol, ibuprofen or ketoprofen) or opiates (such as morphine) or antiemetics.

[0088] The specific binding molecules of the present invention will generally be administered in the form of a pharmaceutical composition, which may include at least one component in addition to the specific binding molecules. In addition to the active ingredient, the pharmaceutical composition may also include a pharmaceutically acceptable excipient, diluent, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration, which may be oral or injection, such as intravenous injection.

[0089] The primary route of therapeutic administration of the composition is expected to be injection, but it can also be delivered using a catheter or other surgical infusion tube. Some suitable routes of administration include intravenous, subcutaneous, and intramuscular administration. The powder formulation can be reconstituted into a liquid formulation for use.

[0090] For intravenous injection or injection at the site of affliction, the active substance will be in the form of a pyrogen-free, pH-appropriate, isotonic and stable enterally acceptable aqueous solution. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic excipients such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[0091] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. Tablets can include solid carriers, such as gelatin or adjuvants. Liquid pharmaceutical compositions generally include liquid carriers, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included. When the preparation is liquid, it can be, for example, a physiological saline solution containing a non-phosphate buffer of pH 6.8-7.6, or a lyophilized powder.

[0092] The composition can also be administered via microspheres, liposomes, other microparticle delivery systems, or sustained-release formulations placed in certain tissues (including blood). Suitable examples of sustained-release vehicles include semipermeable polymer matrices in the form of shared articles, such as suppositories or microcapsules. Implantable or microcapsule sustained-release matrices include polylactides (US Patent No. 3,773,919; EP-A-0058481) copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al, Biopolymers 22 (1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langeret al, J. Biomed. Mater. Res. 15: 167-277, 1981 and Langer, Chem. Tech. 12: 98-105, 1982). Liposomes containing polypeptides are prepared by the following well-known methods: DE 3,218,121A; Epstein et al, PNAS USA, 82:3688-3692, 1985; Hwang et al, PNAS USA, 77:4030-4034, 1980; EP-A-0052522; EA-0036676; EP-A-0088046; EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Pat. Nos. 4,485,045 and 4,544,545. Typically, the liposomes are small (about 200 angstroms to 800 angstroms) unilamellar types, wherein the lipid content is greater than about 30 mol% cholesterol, the selected ratio being adjusted according to the optimal rate of polypeptide leakage.

[0093] The composition may be administered in a localized manner to the desired site or may be delivered in such a way that it targets the relevant cells.

[0094] The composition is preferably administered to a subject in a "therapeutically effective amount", which is an amount sufficient to show benefit to the subject. The actual amount administered, the rate of administration and the time course will depend on the nature and severity of the treatment being treated. Treatment prescription, such as dosage decisions, etc., is the responsibility of general practitioners and other physicians, and generally requires consideration of the condition being treated, the individual patient's condition, the delivery site, the method of administration and other factors known to the physician.

[0095] The optimal dose can be determined by the physician based on a number of parameters, including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered, and the route of administration. Generally, serum concentrations of polypeptides and antibodies that allow receptor saturation are desirable. Concentrations in excess of about 0.1 nM are generally sufficient. For example, a 100 mg / m2 antibody dose provides a serum concentration of about 20 nM for about eight days.

[0096] As a rough guide, the antibody dose that can be given weekly may be 10 mg / m 2 -300mg / m 2 Equivalent doses of the antibody fragment should be used at more frequent intervals to maintain serum levels above the concentration that saturates Sialyltetraosyl carbohydrateceramide.

[0097] The dosage of the composition will depend on the properties of the binding molecule, for example, its binding activity and plasma half-life in vivo, the concentration of the polypeptide in the formulation, the route of administration, the site of administration and the dose, the clinical tolerance of the participating patients, the pathological conditions suffered by the patients, etc., which are completely within the skill of the physician. For example, a dosage of 300 μg of antibody per administration per patient is preferred, although the dosage range may be about 10 μg to 6 mg per dose. Different doses are used during a series of sequential vaccinations; the practitioner may perform an initial vaccination and then boost with a relatively smaller dose of the antibody.

[0098] The binding molecules of the present invention can be generated in whole or in part by chemical synthesis. The binding molecules can be easily prepared according to the standard liquid phase peptide synthesis method or the preferred solid phase peptide synthesis method of complete establishment, and the general description of the method is available everywhere (for example, see JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984); M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California); or, they can be prepared by liquid phase method in solution, or prepared by any combination of solid phase method, liquid phase method and solution chemistry, for example, by first completing each peptide part, then as needed and whether appropriate, after removing any protecting group present, by the reaction of each carbonic acid or sulfonic acid or its reactive derivative to introduce residue X.

[0099] Another convenient way to produce a binding molecule according to the invention is to express a nucleic acid encoding the binding molecule by using the nucleic acid in an expression system.

[0100] The present invention also provides nucleic acids encoding the separation of specific binding molecules of the present invention. Nucleic acids include DNA and RNA. In preferred aspects, the present invention provides nucleic acids encoding specific binding molecules of the present invention as described above. Those skilled in the art can determine that replacement, deletion and / or addition of such nucleic acids will still be provided for specific binding molecules of the present invention.

[0101] The present invention also provides a construct in the form of a plasmid, vector, transcription or expression assembly containing at least one of the above nucleic acids. The present invention also provides a recombinant host cell comprising one or more of the above constructs. As described above, the nucleic acid encoding the specific binding molecule of the present invention constitutes one aspect of the present invention; the method for producing the specific binding molecule also constitutes one aspect of the present invention, the method comprising expressing the encoding nucleic acid of the specific binding molecule. Expression can be conveniently achieved by culturing a recombinant host cell containing the nucleic acid under appropriate conditions. After production by expression, the specific binding molecule can be separated and / or purified by any suitable technique as appropriate and then used.

[0102] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many other cells. A common preferred bacterial host is E. coli. Expression antibodies and antibody fragments in prokaryotic cells (e.g., E. coli) are well established in the art. For review, see, e.g., Plückthun, Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture is also an option for producing specific binding molecules available to those skilled in the art, and recent reviews are seen, e.g., Reff, Curr. Opinion Biotech. 4:573-576 (1993); Trill et al., Curr. Opinion Biotech. 6:553-560 (1995).

[0103] Suitable vectors containing appropriate regulatory sequences can be selected or constructed, and the regulatory sequences may include promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. The vector may be a plasmid, a virus such as a phage or a phagemid as appropriate. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual: 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Ausubel et al. eds., Short Protocols in Molecular Biology, 2nd Edition, John Wiley & Sons (1992) describes in detail the known techniques and protocols of many nucleic acid operations, such as the preparation of nucleic acid constructs, mutagenesis, sequencing, DNA introduction into cells and gene expression and protein analysis.

[0104] Therefore, another aspect of the present invention provides a host cell containing nucleic acids disclosed herein. In yet another aspect, a method is provided, which includes introducing such nucleic acids into host cells. Any available technique can be used for introduction. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (e.g., vaccinia virus, or for insect cells, baculovirus). For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophage. Expression of the nucleic acid can be initiated or allowed after introduction, for example, by culturing host cells under expression conditions of the gene.

[0105] The nucleic acid of the invention can be integrated into the genome (eg chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome according to standard techniques.

[0106] The present invention also provides a method, which comprises using the above construct in an expression system to express the above specific binding molecule or polypeptide.

[0107] In another aspect, a bifunctional binding molecule is provided, comprising:

[0108] i) T cell receptor (TCR) or antibody; and

[0109] ii) A specific binding molecule that binds to CD3 according to the first aspect.

[0110] It will be appreciated that any and all features described above with respect to the first aspect are equally applicable to this other aspect.

[0111] The targeting moiety can be a T cell receptor (TCR), an antibody or an antibody fragment.

[0112] The arrangement of the anti-CD3 and targeting moieties may be in any known form (e.g., Brinkman et al., MAbs .2017Feb-Mar;9(2):182–212 Figure 2 described in ).

[0113] The T cell receptor (TCR) may be a heterodimeric α / β or γ / δ TCR polypeptide pair. The T cell receptor (TCR) may be a single chain TCR polypeptide.

[0114] The specific binding molecule bound to CD3 can be fused to the C-terminus or N-terminus of the α chain or β chain of the T cell receptor (TCR). Preferably, the CD3 effector is fused to the N-terminus of the β chain of the TCR. The bifunctional binding molecule can be a diabody in which TCR-Va is connected to anti-CD3-VL and TCR-Vb is connected to anti-CD3-VH, or vice versa.

[0115] In some cases, the specific binding molecule that binds to CD3 can be fused to the C-terminus or N-terminus of the targeting moiety. In other cases, the specific binding molecule that binds to CD3 is fused to the C-terminus or N-terminus of the targeting moiety via a linker.

[0116] The specific binding molecule bound to CD3 can be fused to a T cell receptor (TCR) or a TCR-like antibody via a linker, which can be a polypeptide linker. Polypeptide linker sequences are generally flexible because they are composed of amino acids (such as glycine, alanine, and serine, which do not have bulky side chains that may limit flexibility). Available or optimal length linker sequences are easily determined. Typically, the length of the linker sequence will be less than about 12 amino acids, for example, less than 10 or 5 to 10 amino acids. The length of the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. Preferably, in addition to other amino acids, a preferred linker includes a sequence having the formula (GGGGS)n, optionally with other amino acids.

[0117] The TCR may comprise a non-native disulfide bond between the α chain constant region and the β chain constant region.

[0118] The TCR may bind to an MHC that forms a complex with a peptide antigen. Preferably, the peptide antigen is any disease-associated antigen. Preferably, the peptide antigen is any tumor-associated antigen. Preferably, the peptide antigen is a peptide derived from GP100, NYESO, MAGEA4 or PRAME as described in WO2011001152, WO2017109496, WO2017175006 and WO2018234319.

[0119] Suitable TCRs may have amino acid sequences as defined in WO2011001152, WO2017109496, WO2017175006 and WO2018234319.

[0120] The TCR may have the following amino acid sequence, or an amino acid sequence that is at least 70% identical to the following amino acid sequence:

[0121] DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGSAPIRFGPGTRLTVTEDLKNVFP PEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

[0122] The bifunctional binding molecule may have the following amino acid sequence or an amino acid sequence that is at least 70% identical to the following amino acid sequence:

[0123] AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGG GSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGSAPIRFGPGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

[0124] Alternatively, the bifunctional binding molecule may have the following amino acid sequence, or an amino acid sequence that is at least 70% identical to the following amino acid sequence:

[0125] AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGG GSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SSGGGGSDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGSAPIRFGPGTRLTVTEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

[0126] Phenotypic silent variants of specific binding molecules or bifunctional binding molecules are also disclosed herein. As used herein, the term "phenotypical silent variant" is understood to refer to a specific binding molecule or bifunctional binding molecule variable domain that incorporates one or more other amino acid changes (including substitutions, insertions and deletions), which has a phenotype similar to a corresponding molecule or polypeptide that does not contain the changes.

[0127] Phenotypically silent variants may comprise one or more conservative substitutions and / or one or more harmless substitutions (toleratedsubstitutions). Harmless substitutions are substitutions that do not fall within the conservative definition provided below but are still phenotypically silent. The skilled artisan knows that various amino acids have similar properties and are therefore "conservative". One or more such amino acids of a protein, polypeptide, or peptide may generally be replaced by one or more other such amino acids without eliminating the desired activity of the protein, polypeptide, or peptide.

[0128] Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine (amino acids with aliphatic side chains) are generally interchangeable. Of these possible substitutions, glycine and alanine are preferably used to replace each other (because they have relatively short side chains), and valine, leucine, and isoleucine are preferably used to replace each other (because they have larger hydrophobic aliphatic side chains). Other amino acids that are generally interchangeable include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains). It should be understood that amino acid substitutions within the scope of the present invention may be performed using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on alanine may be substituted with an ethyl group, and / or that minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0129] Replacements of this nature are generally referred to as "conservative" or "semi-conservative" amino acid replacements. Therefore, the present invention extends to the use of specific binding molecules or bifunctional binding molecules as follows, which comprise any of the above amino acid sequences, but have one or more conservative replacements and / or one or more harmless replacements in the sequence, so that the amino acid sequence of the specific binding molecule or bifunctional binding molecule has at least 80% identity, or at least 90% identity, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the above specific binding molecule or bifunctional binding molecule.

[0130] "Identity" as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences determined by comparing sequences. In the art, identity also means the degree of sequence correlation between polypeptide or polynucleotide sequences (as the case may be), as determined by the matching between the strings of such sequences. Although there are many methods for measuring the identity between two polypeptide sequences or two polynucleotide sequences, the method generally used to determine identity is encoded in a computer program. Preferred computer programs for determining the identity between two sequences include but are not limited to GCG program packages (Devereux, et al., Nucleic Acids Research, 12, 387 (1984, BLASTP, BLASTN and FASTA (Atschul et al., J.Molec.Biol.215, 403 (1990)).

[0131] Amino acid sequences can be compared using a program such as the CLUSTAL program. The program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. Amino acid identity or similarity (identity plus conservation of amino acid type) can be calculated for the optimal alignment. Programs such as BLASTx compare the longest stretch of similar sequences and assign values ​​to the fit. Therefore, comparisons can be obtained when several similar regions are found, each with different scores. Two types of identity analysis are considered in the present invention.

[0132] The percent identity of two amino acid sequences or two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence to optimally align with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is the alignment of two sequences that achieves the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the compared sequences (i.e., % identity = number of identical positions / total number of positions x 100).

[0133] The determination of the percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, which was improved in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The BLASTn and BLASTp programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated this algorithm. The BLASTn program can be used to determine the percent identity between two nucleotide sequences. The BLASTp program can be used to determine the percent identity between two protein sequences. In order to obtain gap alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches to detect long-distance relationships (Id.) between molecules. When using BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of each program (e.g., BLASTp and BLASTp) can be used. See http: / / www.ncbi.nlm.nih.gov. Default conventional parameters can include, for example, font size = 3, expected threshold = 10. Parameters can be selected to automatically adjust to input short sequences. Another example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) as a part of the CGC sequence alignment software package has incorporated this algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10: 3-5; and FASTA as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-8. In FASTA, ktup is a control option for setting the sensitivity and speed of the search. For the purpose of evaluating the percentage identity in the present invention, BLASTp and default parameters are used as the comparison method. In addition, when the percentage identity provides a non-integer value of an amino acid (i.e., a value provided by a sequence of 25 amino acids with 90% sequence identity is "22.5"), the resulting value is rounded down to the next integer, i.e. "22".Thus, in the example provided, sequences having 22 matches out of 25 amino acids are within 90% sequence identity.

[0134] It will be apparent to those skilled in the art that it is possible to truncate or extend the sequence provided at its C-terminus and / or N-terminus by 1, 2, 3, 4, 5 or more residues without substantially affecting the functional properties of the specific binding molecule. The sequence provided at its C-terminus and / or N-terminus may be truncated or extended by 1, 2, 3, 4 or 5 residues. All of these variants are encompassed in the present invention.

[0135] Any suitable method can be used to introduce mutations (including conservative and harmless substitutions, insertions and deletions) into the provided sequence, including but not limited to methods based on polymerase chain reaction (PCR), restriction enzyme-based cloning or ligation independent cloning (LIC) steps. These methods are described in detail in many standard molecular biology texts. For more details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning-A Laboratory Manual (3rd Ed.) CSHL Press. More information on ligation-independent cloning (LIC) steps can be found in Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6. The sequences provided by the present invention can be obtained from solid-state synthesis or any other suitable method known in the art.

[0136] The targeting moiety may be an antibody or a fragment thereof. Preferably, the antibody, including fragments, derivatives and variants thereof, binds to an antigen presented on a diseased cell or cancerous cell.

[0137] The specific binding molecules or bifunctional binding molecules of the present invention can be used for methods of diagnosing and treating cancer or infectious diseases in human or animal subjects. Examples of cancer include, but are not limited to, liquid tumors such as leukemia, lymphoma, and myeloma; and solid tumors including bladder, breast, cervix, colorectum, esophagus, endometrium, stomach, glioblastoma, liver, melanoma, lung, ovary, pancreas, prostate, sarcoma, thyroid. Examples of infectious diseases include, but are not limited to, HIV, HBV, TB, HCV.

[0138] When used diagnostically, the specific binding molecules or bifunctional binding molecules of the invention can be labeled with a detectable marker, such as a radioactive marker, such as 131 I or 99Tc, these detectable labels can be attached to the specific binding molecules of the present invention using conventional chemical methods known in the field of antibody imaging. Labels also include enzyme labels, such as horseradish peroxidase. Labels also include chemical moieties, such as biotin, which can be detected by binding to a specific homologous detectable moiety (e.g., labeled avidin).

[0139] Specific binding molecules or bifunctional binding molecules of the present invention can be suitable for high yield purification, particularly specific binding molecules of soluble form.Productivity (that is, the amount of the correct folded material obtained at the end of the purification process relative to the amount of the water-soluble material obtained before refolding) can be determined based on the amount of substance retained during the purification process, and / or the yield can be based on the amount of the correct folded material obtained at the end of the purification process relative to the original culture volume.High yield means greater than 1%, or more preferably greater than 5%, or higher yield.High yield means greater than 1mg / ml, or more preferably greater than 3mg / ml, or greater than 5mg / ml, or higher yield.

[0140] Determine the binding affinity (and equilibrium constant K D Methods for determining binding affinity and binding half-life (expressed as T1 / 2) are known to those skilled in the art. Preferably, binding affinity and binding half-life are determined using surface plasmon resonance (SPR) or bio-layer interferometry (BLI), for example using a BIAcore instrument or an Octet instrument, respectively. It should be understood that a doubling of affinity results in a K D Halved. According to ln2 divided by the dissociation rate (k off ) to calculate T1 / 2. Therefore, doubling T1 / 2 results in k off Halve. D and k off Value. In order to take into account the changes between independent measurements, especially the changes between the measurements of interactions when the dissociation time exceeds 20 hours, the binding affinity and / or binding half-life of a given molecule can be measured several times (e.g., 3 times or more) using the same assay protocol, and the results are averaged. In order to compare the binding data between two samples (i.e., two different molecules and / or two preparations of the same molecule), it is preferred to use the same assay conditions (e.g., temperature) for measurement, such as those described in WO2018234319.

[0141] TCR described herein can be an αβ heterodimer.α-β heterodimer TCR generally comprises an α chain TRAC constant domain sequence and / or a β chain TRBC1 or TRBC2 constant domain sequence. Constant domains can be full length, which means that there are extracellular domains, transmembrane domains and cytoplasmic domains, or they can be soluble forms (i.e., without transmembrane domains or cytoplasmic domains). Relative to natural TRAC and / or TRBC1 / 2 sequences, one or both of the constant domains may comprise mutations, substitutions or deletions. The terms TRAC and TRBC1 / 2 also encompass natural polymorphic variants, such as N at position 4 of TRAC becomes K (Bragado et al International immunology. 1994 Feb; 6 (2): 223-30).

[0142] For soluble TCR, the α and β chain constant domain sequences can be modified by truncating or replacing the natural disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The α and / or β chain constant domain sequences can have an introduced disulfide bond between the residues of each constant domain, such as described in WO 03 / 020763. The α and β constant domains can be modified by replacing the Thr48 position of TRAC and the Ser57 position of TRBC1 or TRBC2 with a cysteine ​​residue, which forms a disulfide bond between the α and β constant domains of the TCR. TRBC1 or TRBC2 may additionally include a cysteine ​​to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant domains present in the αβ heterodimer may be truncated at the C-terminus or C-terminus, for example, truncated to 15, or 10, or 8 or less amino acids at most. One or both of the extracellular constant domains present in the αβ heterodimer may be truncated at the C-terminus or C-terminus, for example, to 15, or 10, or 8 amino acids at most. The C-terminus of the α chain extracellular constant domain may be truncated to 8 amino acids. The soluble TCR is preferably associated with a therapeutic agent and / or a detectable marker.

[0143] The constant domain of αβ heterodimer TCR can be full length, with transmembrane domain and cytoplasmic domain. Such TCR can include disulfide bonds corresponding to those found in nature between respective α and β constant domains. Additionally or alternatively, non-natural disulfide bonds can be present between extracellular constant domains. The non-natural disulfide bonds are further described in WO03020763 and WO06000830. Non-natural disulfide bonds can be between Thr48 position of TRAC and Ser57 position of TRBC1 or TRBC2. Relative to natural TRAC and / or TRBC1 / 2 sequences, one or both of the constant domains may include one or more mutations, substitutions or deletions. TCR with full-length constant domains is preferably used for adoptive therapy.

[0144] TCR described herein can be in single-chain form. Single-chain forms include but are not limited to αβTCR polypeptides of Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβ type, wherein Vα and Vβ are TCRα and TCRβ variable regions, respectively, Cα and Cβ are TCRα and TCRβ constant regions, respectively, and L is a joint sequence (Weidanz et al., (1998) J Immunol Methods. Dec 1; 221 (1-2): 59-76; Epel et al., (2002), Cancer Immunol Immunother. Nov; 51 (10): 565-73; WO 2004 / 033685; WO9918129). One or two of the constant domains (when present) can be full-length, or they can be truncated and / or contain mutations as described above. Preferably, the single-chain TCR is soluble. The single-chain TCR may have introduced disulfide bonds between the residues of each constant domain, as described in WO 2004 / 033685. WO2004 / 033685; WO98 / 39482; WO01 / 62908; Weidanz et al. (1998) J Immunol Methods 221 (1-2): 59-76; Hoo et al. (1992) Proc Natl Acad Sci USA 89 (10): 4759-4763; Schodin (1996) Mol Immunol 33 (9): 819-829 further describes single-chain TCR.

[0145] The specific binding molecule or bifunctional binding molecule may be associated (covalently or otherwise) with a PK modifying moiety. Examples of PK modifying moieties include, but are not limited to, PEG (Dozier et al., (2015) Int J Mol Sci. Oct 28; 16(10): 25831-64 and Jevsevar et al., (2010) Biotechnol J. Jan; 5(1): 113-28), PASylation (Schlapschy et al., (2013) Protein Eng Des Sel. Aug; 26(8): 489-501), albumin, and albumin binding domains, (Dennis et al., (2002) J Biol Chem. Sep 20; 277(38): 35035-43), and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12): 1186-90). Other PK modifying moieties include antibody Fc fragments.

[0146] As used herein, the term "antibody" encompasses such fragments and variants. Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, diabodies, Nanobodies, TM (These constructs are marketed by Ablynx (Belgium) and comprise: synthetic single immunoglobulin variable heavy domains derived from camelid (e.g. camel or llama) antibodies and domain antibodies (Domantis (Belgium) including affinity matured single immunoglobulin variable heavy domains or immunoglobulin variable light domains) or alternative protein scaffolds exhibiting antibody-like binding properties, such as Affibodies (Affibody (Sweden) comprising an engineered protein A scaffold) or Anticalins (Pieris (Germany)) comprising engineered anticalins), to name a few.

[0147] Antibodies also include TCR-like antibodies (Chang et al., Expert Opin Biol Ther. 2016 Aug; 16(8):979-87 and Dahan et al., Expert Rev Mol Med. 2012 Feb 24; 14:e6).

[0148] The connection of the targeting moiety of the first aspect and the specific binding molecule can be by covalent or non-covalent connection. Covalent connection can be direct, or indirectly by a linker sequence. Linker sequences are usually flexible, because they are mainly composed of amino acids (such as glycine, alanine and serine without the bulky side chain that may limit flexibility). Alternatively, it may be desirable to have a linker with greater rigidity. Available or optimal length linker sequences can be easily determined. Usually, the length of the linker sequence will be less than about 12 amino acids, for example, less than 10 or 2 to 10 amino acids, preferably 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 amino acids. Examples of suitable linkers that can be used in the TCRs of the invention include, but are not limited to, GGGSGGGG, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP, and GGEGGGSEGGGS (as described in WO2010 / 133828).

[0149] As is well known in the art, specific binding molecules or bifunctional binding molecules can be post-translationally modified. Glycosylation is such a modification, which includes the covalent attachment of oligosaccharide moieties to amino acids defined in an amino acid chain. For example, asparagine residues or serine / threonine residues are well-known oligosaccharide attachment positions. The glycosylation state of a particular protein depends on many factors, including protein sequence, protein conformation, and the availability of certain enzymes. In addition, the glycosylation state (i.e., oligosaccharide type, covalent bonds, and total number of connections) can affect protein function. Therefore, when producing recombinant proteins, it is generally desirable to control glycosylation. Controlled glycosylation has been used to improve antibody-based therapeutics (Jefferis et al., (2009) Nat Rev Drug Discov Mar; 8 (3): 226-34.). For soluble TCRs, glycosylation can be controlled, for example, by using specific cell lines (including but not limited to mammalian cell lines, such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells) or by chemical modification. Such modification may be desirable because glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic the native human protein (Sinclair and Elliott, (2005) Pharm Sci. Aug; 94(8): 1626-35).

[0150] For administration to a patient, the specific binding molecules, bifunctional binding molecules, nucleic acids, expression vectors or cells of the invention can be provided as part of a sterile pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition can be in any suitable form (depending on the desired method of administering it to the patient). It can be provided in unit dosage form, typically in a sealed container, and can be provided as part of a kit. Such a kit will typically (but not necessarily) include instructions for use. It can include a plurality of such unit dosage forms.

[0151] The pharmaceutical composition can be suitable for administration by any appropriate route, such as parenteral (including subcutaneous, intramuscular, intracapsular or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions can be prepared by any method known in the pharmaceutical field, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0152] The dosage of the substance of the invention may vary within wide limits, depending on the disease or condition to be treated, the age and condition of the individual to be treated, etc., and a suitable dosage range of the molecule of the invention may be in the range of 25 ng / kg to 50 μg / kg or 1 μg to 1 g. The physician will ultimately determine the appropriate dosage to be used.

[0153] The specific binding molecules, bifunctional binding molecules, pharmaceutical compositions, vectors, nucleic acids and cells of the invention can be provided in a substantially pure form, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

[0154] The method of treatment may further comprise administering, separately, in combination or sequentially, an additional anti-tumor agent. Examples of such agents are known in the art and may include immune activators and / or T cell modulators.

[0155] It is contemplated that the nucleic acids, expression vectors, host cells and production methods described in relation to the first aspect are also relevant to the other aspects described herein.

[0156] The preferred features of each aspect of the invention apply mutatis mutandis to each other aspect.The prior art documents mentioned herein are incorporated herein to the maximum extent permitted by law. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1 - The VH and VL amino acid sequences of the improved UCHT1 variants are provided. The CDRs are underlined. Mutations are shown in bold;

[0158] Figure 2- provides the amino acid sequences of exemplary TCR-anti-CD3 fusion proteins into which improved anti-CD3 scFv variants are incorporated;

[0159] Figure 3 - shows that TCR-CD3 fusions incorporating improved anti-CD3 scFv variant 1 have a better therapeutic window relative to non-mutated anti-CD3 (A), and TCR-CD3 fusions incorporating improved anti-CD3 scFv variant 2 have a higher Emax relative to non-mutated anti-CD3 (B);

[0160] Figure 4 - Improved T cell killing properties mediated by TCR-CD3 fusions combining UCHT1 variant 1 and variant 2 are shown.

[0161] The invention is further described in the following non-limiting examples. DETAILED DESCRIPTION

[0162] Example

[0163] The following examples describe the bifunctional binding molecules of the present invention, which can be referred to as TCR-anti-CD3 bispecific proteins.

[0164] 1) Preparation of TCR-anti-CD3 bispecific fusion protein with improved anti-CD3

[0165] Fusion proteins comprising a TCR and an anti-CD3 scFv are known in the art (see, for example, WO2011001152, WO2017109496, WO2017175006 and WO2018234319). These molecules comprise a humanized UCHT1 scFv fragment.

[0166] In this example, variants of the TCR-anti-CD3 fusion protein described in WO2018234319 were generated, which incorporated anti-CD3 variant 1 (T165A) or anti-CD3 variant 2 (T165A+I202F). Standard mutagenesis and cloning methods were used to introduce mutations (e.g., Sambrook, Joseph. (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Figure 1 The amino acid sequences of VL and VH of T165A and T165A+I202F are provided in. Figure 2 The merged Figure 1 The amino acid sequence of TCR-anti-CD3 is provided in the sequence.

[0167] TCR-anti-CD3 fusion proteins containing T165A, T165A+I202F or non-mutated (WT) UCHT1 scFv were expressed as inclusion bodies in E. coli and subsequently folded and purified using the method described in Example 2 of WO2018234319.

[0168] 2) Increased therapeutic window and maximal T cell activation mediated by TCR-anti-CD3 with variants T165A and T165A+I202F

[0169] A) IFN-γ release

[0170] The ability of the above TCR-anti-CD3 fusion proteins to mediate CD3+ T cell activation in the presence of antigen-positive and antigen-negative cells was assessed. Interferon-γ (IFN-γ) release was used as a readout of T cell activation.

[0171] The human IFN-γ ELISPOT kit (BD Biosciences) was used for detection according to the manufacturer's instructions. Briefly, Mel624 melanoma cells were used as antigen-positive target cells. Granta-519 B cell lymphoma cells were used as antigen-negative target cells. 1×10 6 Target cells were prepared at a density of 100 μl / ml and plated at 50,000 cells per well in a volume of 50 μl. Peripheral blood mononuclear cells (PBMCs) isolated from fresh donor blood were used as CD3+ effector cells and plated at 35,000 cells per well in a volume of 50 μl. TCR-anti-CD3 protein was titrated to a final concentration of 10 nM to 0.0001 nM and added to the wells in a volume of 50 μl.

[0172] Prepare the plate according to the manufacturer's instructions and make the plate color. The wells containing target cells, effector cells and fusion proteins are supplemented to a final volume of 200 μl with assay culture medium. All reactions are performed in triplicate. Control wells are also prepared, in which fusion proteins, effector cells or target cells are omitted. The plate is incubated overnight (37°C / 5% CO2). The next day, the plate is washed three times with wash buffer (1×PBS pouch, containing 0.05% Tween-20, prepared in deionized water). Then the detection primary antibody is added to each well with a volume of 50 μl. The plate is incubated at room temperature for 2 hours and then washed three times again. Secondary antibody detection is performed by adding 50 μl of diluted streptavidin-HRP to each well, incubating at room temperature for 1 hour, and repeating the washing steps. No more than 15 minutes before use, add one drop (20 μl) of AEC chromogen per 1 ml AEC substrate and mix, and add 50 μl to each well. The spot color development was monitored regularly and the plate was washed in tap water to stop the color development reaction. The plate was then dried at room temperature for at least 2 hours and then the spots were counted using a CTL analyzer (Cellular Technology Limited) by immunoblotting software. Data were prepared and analyzed using PRISM software.

[0173] The therapeutic window was calculated by determining the relative potency of T cell activation mediated by the TCR-anti-CD3 variant and WT on antigen-positive cells and comparing the Ec50 values ​​after curve fitting in Prism. Reliable Ec50 values ​​could not be obtained for antigen-negative cells, so a "minimum cross-reactive concentration" was determined by setting a threshold number of spots (e.g., 25 spots per well) and determining by interpolation the concentration that would first exceed this number.

[0174] result

[0175] T165A

[0176] To obtain a reliable determination of the therapeutic window, data from 4 Ag+ plates and 4 Ag- plates, each with non-mutated and T165A side by side, were averaged. Figure 3 A shows the curve obtained from one plate. The data for each individual plate are shown in the table below.

[0177]

[0178]

[0179] The relative potency for antigen-positive cells (i.e., Ec50 of WT divided by Ec50 of T165A) averaged 0.41. The relative cross-reactivity for antigen-negative cells (i.e., WT concentration that provides 25 spots / T165A concentration that provides 25 spots) averaged 0.19. These data indicate that the therapeutic window of T165A is approximately 2 times that of WT.

[0180] T165A+I202F

[0181] Figure 3 B shows that relative to WT, the T165A+I202F variant leads to higher maximum T cell activation (Emax). In this case, its therapeutic window is similar to WT. These data indicate that T165A+I202F is more effective in activating T cells.

[0182] B) T cell-mediated killing

[0183] The ability of TCR-anti-CD3 fusion proteins to mediate the killing of antigen-positive and antigen-negative tumor cells by redirected T cells was investigated using the IncuCyte platform (Essen BioScience). The assay allows for real-time detection of the release of the apoptosis marker Caspase-3 / 7 by microscopy.

[0184] method

[0185] The assay was performed using the CellPlayer 96-well Caspase-3 / 7 apoptosis assay kit (Essen BioScience, catalog number 4440) and implemented according to the manufacturer's protocol. In short, target cells (Mel624 (antigen positive) or MDA MB 231 (antigen negative)) were plated with 10,000 cells per well and incubated overnight to allow cells to attach. TCR-anti-CD3 fusion proteins were added at a concentration of 0.05nM to 0.0125nM (for antigen positive cells) and 20nM to 0.125nM (for antigen negative cells). CD3+ effector cells (PBMC) were used with an effector-target cell ratio of 10:1 (100,000 cells per well). 30 μM NucView assay reagents were prepared, and 25 μl was added to each well, and the final volume was 150 μl (obtaining a final concentration of 5 μM). The plate was placed into the IncuCyte instrument and images were taken periodically over 3 to 5 days. The number of apoptotic cells in each image was determined and recorded as counts per square millimeter of target. The assay was performed in triplicate. Images were prepared using PRISM software.

[0186] result

[0187] The killing curves of WT, T165A and T165A+I202F against antigen-positive and antigen-negative cells are shown in Figure 4 Note that for clarity, only the curves for the specified concentrations are shown.

[0188] T165A

[0189] At a concentration of 0.005 nM, T165A showed slightly reduced T cell killing of antigen-positive cells relative to WT. No killing of antigen-negative cells was observed at up to 20 nM, while WT killing was observed at a concentration of 1 nM. These data confirm the above findings that T165A has an improved therapeutic window.

[0190] T165A+I202F

[0191] T165A+I202F showed increased T cell-mediated killing of antigen-positive cells at a given concentration (e.g., 0.005 nM). Killing of antigen-negative cells was comparable to WT. These data confirm the above findings and demonstrate that T165A+I202F is more effective in activating T cells.

[0192] The data presented in the Examples demonstrate that TCR-anti-CD3 fusion proteins incorporating the T165A or T165A+I202F UCHT1 variants have improved therapeutic properties. Sequence Listing <110> Anglo-American Roco Ltd. <120> CD3-specific binding molecules <130> P121631WO <150> GB1901305.1 <151> 2019-01-30 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> homosapien <400> 1 Gln Asp Ile Arg Asn Tyr 1 5 <210> 2 <211> 9 <212> PRT <213> homosapien <400> 2 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 3 <211> 8 <212> PRT <213> homosapien <400> 3 Gly Tyr Ser Phe Thr Gly Tyr Ala 1 5 <210> 4 <211> 8 <212> PRT <213> homosapien <400> 4 Ile Asn Pro Tyr Lys Gly Val Ser 1 5 <210> 5 <211> 15 <212> PRT <213> homosapien <400> 5 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 1 5 10 15 <210> 6 <211> 26 <212> PRT <213> homosapien <400> 6 Ala 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 Ala Ser 20 25 <210> 7 <211> 26 <212> PRT <213> homosapien <400> 7 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 Ala Ser 20 25 <210> 8 <211> 17 <212> PRT <213> homosapien <400> 8 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 9 <211> 36 <212> PRT <213> homosapien <400> 9 Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 10 <211> 10 <212> PRT <213> homosapien <400> 10 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 11 <211> 25 <212> PRT <213> homosapien <400> 11 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 20 25 <210> 12 <211> 17 <212> PRT <213> homosapien <400> 12 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 15 Leu <210> 13 <211> 38 <212> PRT <213> homosapien <400> 13 Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys 1 5 10 15 Lys Asn Thr To Tyr Leu Gln Met Asn To Arg To Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 14 <211> 38 <212> PRT <213> homosapien <400> 14 Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Phe Ser Val Asp Lys 1 5 10 15 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 15 <211> 11 <212> PRT <213> homosapien <400> 15 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 16 <211> 107 <212> PRT <213> homosapien <400> 16 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> homosapien <400> 17 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 122 <212> PRT <213> homosapien <400> 18 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 Tyr Ser Phe Thr Gly Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 122 <212> PRT <213> homosapien <400> 19 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 Tyr Ser Phe Thr Gly Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Phe Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 253 <212> PRT <213> homosapien <400> 20 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn 180 185 190 Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 225 230 235 240 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 <210> 21 <211> 253 <212> PRT <213> homosapien <400> 21 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn 180 185 190 Gln Lys Phe Lys Asp Arg Phe Thr Phe Ser Val Asp Lys Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 225 230 235 240 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 <210> 22 <211> 8 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 22 Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 23 <211> 5 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 23 Gly Gly Gly Gly Ser 1 5 <210> 24 <211> 5 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 24 Gly Gly Gly Ser Gly 1 5 <210> 25 <211> 5 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 25 Gly Gly Ser Gly Gly 1 5 <210> 26 <211> 5 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 26 Gly Ser Gly Gly Gly 1 5 <210> 27 <211> 6 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 27 Gly Ser Gly Gly Gly Pro 1 5 <210> 28 <211> 5 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 28 Gly Gly Glu Pro Ser 1 5 <210> 29 <211> 7 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 29 Gly Gly Glu Gly Gly Gly Pro 1 5 <210> 30 <211> 12 <212> PRT <213> artificial sequence <220> <223> Linker sequence <400> 30 Gly Gly Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 1 5 10 <210> 31 <211> 244 <212> PRT <213> homosapien <400> 31 Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys Glu Gly 1 5 10 15 Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp Ala Met 20 25 30 Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr 35 40 45 Ser Gln Ile Met Gly Asp Glu Gln Lys Gly Asp Ile Ala Glu Gly Tyr 50 55 60 Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val Thr Ser 65 70 75 80 Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser Trp Trp 85 90 95 Thr Gly Gly Ser Ala Pro Ile Arg Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe 115 120 125 Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val 130 135 140 Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp 145 150 155 160 Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro 165 170 175 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser 180 185 190 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe 195 200 205 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 210 215 220 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 225 230 235 240 Gly Arg Ala Asp <210> 32 <211> 502 <212> PRT <213> homosapien <400> 32 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn 180 185 190 Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 225 230 235 240 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 245 250 255 Gly Ser Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 260 265 270 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 275 280 285 Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile 290 295 300 Tyr Tyr Ser Gln Ile Met Gly Asp Glu Gln Lys Gly Asp Ile Ala Glu 305 310 315 320 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 325 330 335 Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser 340 345 350 Trp Trp Thr Gly Gly Ser Ala Pro Ile Arg Phe Gly Pro Gly Thr Arg 355 360 365 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 370 375 380 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 385 390 395 400 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 405 410 415 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 420 425 430 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu 435 440 445 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn 450 455 460 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 465 470 475 480 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 485 490 495 Ala Trp Gly Arg Ala Asp 500 <210> 33 <211> 502 <212> PRT <213> homosapien <400> 33 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn 180 185 190 Gln Lys Phe Lys Asp Arg Phe Thr Phe Ser Val Asp Lys Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 225 230 235 240 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 245 250 255 Gly Ser Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 260 265 270 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 275 280 285 Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile 290 295 300 Tyr Tyr Ser Gln Ile Met Gly Asp Glu Gln Lys Gly Asp Ile Ala Glu 305 310 315 320 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 325 330 335 Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser 340 345 350 Trp Trp Thr Gly Gly Ser Ala Pro Ile Arg Phe Gly Pro Gly Thr Arg 355 360 365 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 370 375 380 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 385 390 395 400 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 405 410 415 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 420 425 430 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu 435 440 445 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn 450 455 460 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 465 470 475 480 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 485 490 495 Ala Trp Gly Arg Ala Asp 500 <210> 34 <211> 202 <212> PRT <213> homosapien <400> 34 Met Gly Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu 1 5 10 15 Glu Glu Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr 20 25 30 Asp Tyr Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr 35 40 45 Val Ile His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu 50 55 60 Ala Ile Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala 65 70 75 80 Thr Leu Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Ile Leu Gly His 85 90 95 Ser Arg Leu Gly Asn Tyr Ile Ala Thr Phe Gly Lys Gly Thr Lys Leu 100 105 110 Ser Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 115 120 125 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 130 135 140 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 145 150 155 160 Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 165 170 175 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 180 185 190 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr 195 200 <210> 35 <211> 502 <212> PRT <213> homosapien <400> 35 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn 180 185 190 Gln Lys Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 225 230 235 240 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 245 250 255 Gly Ser Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 260 265 270 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 275 280 285 Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile 290 295 300 Tyr Tyr Ser Gln Ile Met Gly Asp Glu Gln Lys Gly Asp Ile Ala Glu 305 310 315 320 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 325 330 335 Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser 340 345 350 Trp Trp Thr Gly Gly Ser Ala Pro Ile Arg Phe Gly Pro Gly Thr Arg 355 360 365 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 370 375 380 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 385 390 395 400 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 405 410 415 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 420 425 430 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu 435 440 445 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn 450 455 460 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 465 470 475 480 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 485 490 495 Ala Trp Gly Arg Ala Asp 500 <210> 36 <211> 502 <212> PRT <213> homosapien <400> 36 Ala 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 Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe 145 150 155 160 Thr Gly Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn 180 185 190 Gln Lys Phe Lys Asp Arg Phe Thr Phe Ser Val Asp Lys Ser Lys Asn 195 200 205 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe 225 230 235 240 Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 245 250 255 Gly Ser Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys 260 265 270 Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp 275 280 285 Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile 290 295 300 Tyr Tyr Ser Gln Ile Met Gly Asp Glu Gln Lys Gly Asp Ile Ala Glu 305 310 315 320 Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val 325 330 335 Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser Ser 340 345 350 Trp Trp Thr Gly Gly Ser Ala Pro Ile Arg Phe Gly Pro Gly Thr Arg 355 360 365 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 370 375 380 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 385 390 395 400 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 405 410 415 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 420 425 430 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu 435 440 445 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn 450 455 460 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 465 470 475 480 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 485 490 495 Ala Trp Gly Arg Ala Asp 500

Claims

1. A specific binding molecule that binds to CD3 and comprises a polypeptide having an immunoglobulin VL domain and an immunoglobulin VH domain, wherein the VL domain comprises complementarity determining regions (CDRs) VLCDR1, VLCDR2, and VLCDR3, and wherein the VH domain comprises complementarity determining regions (CDRs) VHCDR1, VHCDR2, and VHCDR3, wherein VLCDR1 is QDIRNY (SEQ ID NO: 1) VLCDR2 for YTS VLCDR3 is QQGNTLPWT (SEQ ID NO: 2) VHCDR1 is GYSFTGYA (SEQ ID NO: 3) VHCDR2 is INPYKGVS (SEQ ID NO: 4) VHCDR3 is ARSGYYGDSDWYFDV (SEQ ID NO: 5).

2. The specific binding molecule according to claim 1, wherein The immunoglobulin VL comprises the full sequence VLFW1-VLCDR1-VLFW2-VLCDR2-VLFW3-VLCDR3-VLFW4, wherein VLFW1, VLFW2, VLFW3 and VLFW4 are VL framework (VLFW) sequences 1 to 4, respectively; the immunoglobulin VH comprises the full sequence VHFW1-VHCDR1-VHFW2-VHCDR2-VHFW3-VHCDR3-VHFW4, wherein VHFW1, VHFW2, VHFW3 and VHFW4 are VH framework (VHFW) sequences 1 to 4, respectively.

3. The specific binding molecule according to claim 2, wherein the VLFW sequence and VHFW sequence are mouse, human or humanized framework sequences.

4. The specific binding molecule according to claim 1, wherein The immunoglobulin VL comprises the full sequence VLFW1-VLCDR1-VLFW2-VLCDR2-VLFW3-VLCDR3-VLFW4, wherein VLFW1, VLFW2, VLFW3 and VLFW4 are framework (FW) sequences 1 to 4, VLFW1, VLFW2, VLFW3 and VLFW4, respectively, wherein: VLFW1 is AIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 6) or DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 7) VLFW2 is LNWYQQKPGKAPKLLIY(SEQ ID NO:8) VLFW3 is RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYC(SEQ ID NO:9) VLFW4 is FGQGTKVEIK (SEQ ID NO: 10).

5. The specific binding molecule according to any one of claims 1 to 4, wherein the immunoglobulin VH comprises the full sequence VHFW1-VHCDR1-VHFW2-VHCDR2-VHFW3-VHCDR3-VHFW4, wherein VHFW1, VHFW2, VHFW3 and VHFW4 are framework (FW) sequences 1 to 4, VHFW1, VHFW2, VHFW3 and VHFW4, respectively, wherein: VHFW1 is EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:11) VHFW2 is MNWVRQAPGKGLEWVAL (SEQ ID NO:12) VHFW3 is TYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO:13) or TYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO:14) VHFW4 is WGQGTLVTVSS (SEQ ID NO: 15).

6. The specific binding molecule according to any one of claims 1 to 4, wherein The immunoglobulin VL comprises the following sequence: OR DIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVP SRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID NO: 17).

7. The specific binding molecule according to any one of claims 1 to 4, wherein The immunoglobulin VH comprises the following sequence: OR EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKG VSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQ GTLVTVSS (SEQ ID NO: 19).

8. The specific binding molecule according to any one of claims 1 to 4, wherein the immunoglobulin VL comprises the following sequence: AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSG TDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK (SEQ ID No: 16); and The immunoglobulin VH comprises the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFK DRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS (SEQ ID No: 19).

9. The specific binding molecule according to any one of claims 1 to 4, wherein The molecule is in the form of a scFv fragment.

10. The specific binding molecule according to any one of claims 1 to 4, wherein The immunoglobulin VL domain and the immunoglobulin VH domain are connected via a linker.

11. The specific binding molecule according to any one of claims 1 to 4, wherein the molecule is a single-chain specific binding molecule having the following sequence: AIQMTQSPSSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSG GGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYAMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTFSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS(SEQ ID No:21).

12. A bifunctional binding molecule comprising: i) targeting moiety; and ii) A specific binding molecule that binds to CD3 according to any one of claims 1 to 11.

13. The bifunctional binding molecule according to claim 12, wherein The targeting moiety is a T cell receptor (TCR), an antibody or an antibody fragment.

14. The bifunctional binding molecule according to claim 13, wherein The T cell receptor (TCR) is a heterodimeric α / β TCR polypeptide pair.

15. The bifunctional binding molecule according to claim 13, wherein The T cell receptor (TCR) is a single chain α / β TCR polypeptide.

16. The bifunctional binding molecule according to claim 13, 14 or 15, wherein The TCR comprises a non-native disulfide bond between the α chain constant region and the β chain constant region.

17. The bifunctional binding molecule according to any one of claims 12 to 15, wherein The specific binding molecule that binds to CD3 according to any one of claims 1 to 11 is fused to the C-terminus or N-terminus of the targeting moiety.

18. The bifunctional binding molecule according to any one of claims 12 to 15, wherein The specific binding molecule that binds to CD3 according to any one of claims 1 to 11 is fused to the C-terminus or N-terminus of the targeting moiety via a linker.

19. A pharmaceutical composition comprising a specific binding molecule that binds to CD3 according to any one of claims 1 to 11 or a bifunctional binding molecule according to any one of claims 12 to 18.

20. A nucleic acid encoding a specific binding molecule that binds to CD3 according to any one of claims 1 to 11 or a bifunctional binding molecule according to any one of claims 12 to 18. An expression vector comprising the nucleic acid of claim 20 .

22. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21, wherein: The nucleic acid encoding the specific binding molecule binding to CD3 according to any one of claims 1 to 11 or the bifunctional binding molecule according to any one of claims 12 to 18 is present as a single open reading frame or as two different open reading frames encoding the α chain and the β chain, respectively.

23. A method for preparing a specific binding molecule that binds to CD3 according to any one of claims 1 to 11 or a bifunctional binding molecule according to any one of claims 12 to 18, the method comprising maintaining the host cell according to claim 22 under optimal conditions for nucleic acid expression and isolating the specific binding molecule or bifunctional binding molecule that binds to CD3.

24. The specific binding molecule that binds to CD3 according to any one of claims 1 to 11 or the bifunctional binding molecule according to any one of claims 12 to 18 for use as a medicament.

25. Use of a specific binding molecule that binds to CD3 according to any one of claims 1 to 11 or a bifunctional binding molecule according to any one of claims 12 to 18 in the preparation of a medicament for treating cancer or an infectious disease, wherein the cancer is selected from leukemia, lymphoma, myeloma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, endometrial cancer, glioblastoma, liver cancer, melanoma, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and thyroid cancer, and the infectious disease is selected from HIV, HBV, TB and HCV.

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