CD3 binding molecules

Abstract

The present invention relates to heavy chain variable regions, binding domains, and antibodies specific for human CD3, as well as CD3 binding proteins. The present invention further relates to the use of the CD3 binding proteins, preferably antibodies, of the present invention in the treatment of cancer or autoimmune diseases.

Description

[Technical Field]

[0001] This invention relates to the field of antibodies, particularly to the field of therapeutic antibodies. Antibodies can be used to treat humans. More specifically, this invention relates to antibodies for the treatment of tumors, preferably bispecific antibodies or multispecific antibodies. [Background technology]

[0002] Monoclonal antibodies that bind to human CD3 were among the first antibodies developed for therapeutic use in humans. Monoclonal CD3-binding antibodies are typically used, for example, in transplant rejection reactions, due to their immunosuppressive properties. Antibodies that are bispecific to CD3 on T cells and to surface target antigens on cancer cells can link any type of T cell to cancer cells, regardless of T cell receptor specificity, co-stimulation, or peptide antigen presentation. Such bispecific T cell-engaging antibodies hold great promise in the treatment of various cancers and tumor growth.

[0003] The object of the present invention is to provide a novel antibody having improved properties, such as CD3 binding characteristics that are not necessarily perfect but are essentially CD3 binding, with higher cytotoxicity and relatively low affinity, suitable for immuno-oncological applications for the engagement of T cells and effector cells; and conversely, a novel antibody having lower cytotoxicity and relatively high affinity CD3 binding, suitable for autoimmune applications for the downregulation of T cells and effector cells. A further object of the present invention is to provide a T cell engagement CD3 binding protein and antibody having the above properties, which bind to at least one further membrane-bound molecule. [Overview of the project]

[0004] The present invention provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1:SFGIS CDR2:GFIPVLGTANYAQKFQG CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the array, X1 = K or R, Includes CDR1, CDR2, and CDR3 (where X2 = L or I). In a preferred embodiment, X1 = K and X2 = L. In a preferred embodiment, X1 = R and X2 = I.

[0005] In a preferred embodiment, the present invention provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region has the following amino acid sequence: CDR1:SKTLTIS, CDR2:GIIPIFGSITYAQKFQD, CDR3:RGNWNPFDP, or Amino acid sequence: CDR1: GSGIS, CDR2:GFIPFFGSANYAQKFRD, CDR3:RGNWNPX 13 DP (In the array, X 13 Includes CDR1, CDR2, and CDR3 (which are either L or F).

[0006] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Including EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0007] Further provided is an antigen-binding protein, preferably an antibody, that binds to human CD3 and includes an antibody-variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: RX3WIG, CDR2:IIYPGDSDTRYSPSFQG, CDR3:X4IRYFX5WSEDYHYYX6DV (In the array, X3 = F or Y, X4 = H or N, X5 = D or V Includes CDR1, CDR2, and CDR3 (where X6 = L or M).

[0008] In one embodiment, 、 In another embodiment, X3=F, X4=H, X5=D, and X6=L.

[0009] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Including EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0010] Further provided is an antigen-binding protein, preferably an antibody, that binds to human CD3 and includes an antibody-variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: SYALS, CDR2:GISGSGRTTWYADSVKG, Includes CDR1, CDR2, and CDR3, which contain CDR3:DGGYSYGPYWYFDL.

[0011] Further provided is an antigen-binding protein, preferably an antibody, that binds to human CD3 and includes an antibody-variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: SYALS, CDR2:AISGSGRTTWYADSVKG, Includes CDR1, CDR2, and CDR3, which include CDR3:DGGYTYGPYWYFDL.

[0012] Further provided are an antigen-binding protein, preferably an antibody, that binds to human CD3 and includes an antibody-variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS includes, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0013] An antigen-binding protein that binds to human CD3, preferably an antibody, is also provided, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1:DYTMH, CDR2:DISWSSGSIGYADSVKG, It comprises CDR1, CDR2 and CDR3 including CDR3: DHRGYGDYEGGGFDY.

[0014] There is also provided an antigen-binding protein, preferably an antibody, that binds to human CD3 and comprises an antibody variable domain including a heavy chain variable region and a light chain variable region. The heavy chain variable region has the following amino acid sequence: CDR1: DYTMH CDR2: DISWSX7GX8X9X 10 YADSVKG CDR3: DHX 11 GYGDYEGGGFDX 12 (In the sequence, X7 = S or G, X8 = S or T, X9 = I or T, X 10 = G or Y, X 11 = R or M, X 12 = H or Y). It comprises CDR1, CDR2 and CDR3.

[0015] In one embodiment, X7, X8, X9 and X 10 are S, S, I and G, and X 11 and X <​​​​​​​​​​​​​​​​​​EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, or EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, or EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, or Including EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0017] The antigen-binding protein of the present invention, preferably the light chain variable region in the antibody, preferably includes a common light chain variable region. The common light chain variable region preferably includes the IgVκ1-39 light chain variable region. The light chain variable region preferably includes germline IgVκ1-39. * 01 is the variable region. The light chain variable region is the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * It is preferable to include 01. In one embodiment, the light chain variable region is human germline kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39* 01 / IGJκ5 * Includes 01. The light chain variable region is preferably an amino acid sequence. Including DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK, It has 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0018] The antigen-binding protein is preferably an antibody, preferably a bispecific or multispecific antibody.

[0019] The antibody preferably comprises a combination of H / L chains that bind to human CD3 as shown herein and a combination of H / L chains that bind to a tumor antigen. The combination of H / L chains that binds to the tumor antigen preferably binds to human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA protein or variants thereof, and in preferred embodiments, binds to EGFR, PD-L1, or CLEC12A.

[0020] The antibody, bispecific antibody, or multispecific antibody of the present invention is preferably a human antibody or a humanized antibody.

[0021] A bispecific or multispecific antibody preferably comprises two different immunoglobulin heavy chains having compatible heterodimerization domains. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain.

[0022] A bispecific or multispecific antibody is an IgG antibody having a mutated CH2 and / or lower hinge domain such that the interaction between the bispecific or multispecific IgG antibody and the Fc gamma receptor is reduced. The mutated CH2 and / or lower hinge domain preferably includes an amino acid substitution at position 235 and / or 236 (according to EU numbering), preferably an L235G substitution and / or a G236R substitution.

[0023] Bispecific antibodies or multispecific antibodies preferably contain a common light chain.

[0024] The present invention further provides antigen-binding proteins or antibodies as shown herein for use in the treatment of subjects who require them, preferably those who have cancer or are being treated for cancer. Antigen-binding proteins or antibodies having the CDR and / or VH sequences of MF8057, MF8058, MF8078, or MF8508, or variants thereof having 0 to 10 amino acid substitutions, mutations, insertions, additions, or deletions, are preferred for treatment, particularly for treatments involving topical administration and / or topical release of antigen-binding proteins or antibodies. Antigen-binding proteins or antibodies having the CDR and / or VH sequences of MF9249, MF9267, or MF8397, or variants thereof having 0 to 10 amino acid substitutions, mutations, insertions, additions, or deletions, are preferred for the treatment of subjects with an overactive immune system, such as autoimmune diseases.

[0025] Unless otherwise specifically specified, the antibodies of the present invention are preferably bispecific antibodies. The bispecific antibodies preferably bind to at least human CD3. In addition, the bispecific antibodies preferably bind to at least surface molecules that are preferentially expressed on human tumor cells. In preferred embodiments, the bispecific antibodies bind to BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, PD-L1, FOLR1, FOLR3, HER2, HM1.24, MCSP, or PSMA. In more preferred embodiments, the bispecific antibodies bind to EGFR or CLEC12A. In more preferred embodiments, the multispecific antibodies bind to EGFR and PD-L1.

[0026] The present invention further provides a pharmaceutical composition comprising an antibody according to the present invention.

[0027] Further, antibodies according to the present invention are provided, further comprising labels, preferably labels for in vivo imaging.

[0028] The present invention also provides a method for treating subjects having or at risk of having a tumor, the method comprising administering a bispecific or multispecific antibody according to the present invention to the subject. Furthermore, bispecific or multispecific antibodies according to the present invention are also provided for use in treating subjects having or at risk of having a tumor. The use of antibodies according to the present invention for the preparation of agents for the treatment of subjects having or at risk of having a tumor is further provided. In a preferred embodiment, the tumor is an EGFR or CLEC12A-positive tumor or an EGFR and PD-L1-positive tumor. [Modes for carrying out the invention]

[0029] An "antibody" is a proteinaceous molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen. Such domains originate from or share sequence homology with the variable region of the antibody.

[0030] Antibody binding has different properties, including specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by the binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. It should be noted herein that “specificity” of an antibody refers to its selectivity for a particular antigen, and “affinity” refers to the strength of the interaction between the antigen-binding site of the antibody and the epitope to which it binds. Antibodies are typically composed of basic structural units—each having two heavy chains and two light chains. Antibodies for therapeutic purposes are preferably as close as possible to the natural antibody of the target being treated (e.g., a human antibody in the case of a human target). The antibodies according to the present invention are not limited to any particular format or method of preparation thereof.

[0031] Therefore, as used herein, “binding specificity” refers to the ability of individual antibody binding sites to react with antigenic determinants. Typically, the antibody binding sites of the present invention are located in the variable domain within the Fab portion, which includes the variable domain, and are constructed from the hypervariable regions of the heavy and light chains.

[0032] The antibody of the present invention is preferably an IgG antibody, preferably an IgG1 antibody. Full-length IgG antibodies may be preferred due to their desirable half-life and immunogenicity, and the desire to maintain them in a state close to a complete autologous (human) molecule. IgG1 is dominant in humans based on its long circulating half-life. To prevent or avoid immunogenicity in humans, the bispecific full-length IgG antibody of the present invention is preferably human IgG1.

[0033] A “bispecific antibody” is an antibody described herein in which one variable domain of the antibody binds to a first antigen, while a second variable domain of the antibody binds to a second antigen, and the first and second antigens are not identical. The term “bispecific antibody” also encompasses biparatopic antibodies in which one variable domain of the antibody binds to a first epitope on an antigen, while a second variable domain of the antibody binds to a second epitope on that antigen. The term further includes antibodies in which at least one VH can specifically recognize the first antigen, and a VL paired with at least one VH in the immunoglobulin variable domain can specifically recognize the second antigen. The resulting VH / VL pair binds to either antigen 1 or antigen 2 and is referred to as a "two-in-one antibody," as described, for example, in International Publication Nos. 2008 / 027236, 2010 / 108127, and Schaefer et al. (Cancer Cell 20, 472-486, October 2011). The bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of preparing it. A bispecific antibody is a multispecific antibody.

[0034] The multispecific polymers or antibodies referred to herein include proteinaceous molecules belonging to the immunoglobulin class of proteins containing two or more domains that bind to an epitope on an antigen, wherein such domains originate from or share sequence homology with the variable region of the antibody, and include proteinaceous molecules that bind to three or more antigens known in the art, such as those described in International Publication No. 2019 / 190327.

[0035] An "antigen" is a molecule that can induce an immune response (to produce an antibody) in a host organism and / or can be targeted by an antibody. At the molecular level, an antigen is characterized by its ability to be bound by an antigen-binding site on an antibody. A mixture of antigens can also be considered an "antigen," that is, a tumor cell lysate or a viral particle may sometimes be referred to as an "antigen," while a preparation of such tumor cell lysates or viral particles will be recognized to contain many antigenic determinants. An antigen contains at least one, but often more than one, epitopes. In the case of binding proteins and antibodies disclosed herein, the antigen is typically bound to the cell membrane and located on the extracellular portion of the cell membrane.

[0036] An "epitope" or "antigen determinant" is a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from continuous amino acids or discontinuous amino acids juxtaposed by the tertiary folding of a protein (so-called linear and conformational epitopes, respectively). Epitopes formed from continuous linear amino acids are typically retained upon exposure to a denaturing solvent, while epitopes formed by tertiary folding typically lose their conformation upon treatment with a denaturing solvent. Epitopes may typically contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in their unique spatial conformation.

[0037] The term “heavy chain” or “immunoglobulin heavy chain” includes the constant region sequence of an immunoglobulin heavy chain from any organism, and unless otherwise specified, includes the heavy chain variable domain. The term heavy chain variable domain includes three heavy chain CDRs and four FR regions unless otherwise specified. Heavy chain fragments include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain (from N-terminus to C-terminus) followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional heavy chain fragment includes a fragment containing at least one CDR capable of specifically recognizing an antigen.

[0038] The term "light chain" refers to the variable domain of an immunoglobulin light chain from any organism, or V L (or their functional fragments, and immunoglobulin constant domains, or C L , or including sequences of their functional fragments. Unless otherwise specified, the term “light chain” may include light chains selected from human kappa, lambda, and combinations thereof. Light chain variable (V L The ) domain typically contains three light chain CDRs and four framework (FR) regions unless otherwise specified. Generally, a full-length light chain contains FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. L The light chain includes a domain and a constant light chain domain. Examples of light chains that can be used in the present invention include those that do not selectively bind to epitopes selectively bound by the heavy chain.

[0039] Suitable light chains for use with the antibodies of the present invention include common light chains (cLCs), such as those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or in silico), which do not substantially inhibit the affinity and / or selectivity of the epitope-binding domain of the heavy chain, but are also suitable for pairing with a set of heavy chains. For example, suitable light chains include those derived from transgenic animals such as MeMo®, which have common light chains integrated into their genome, and can be used to generate a large panel of common light chain antibodies that have diversity in their heavy chains and can specifically bind to the antigen upon exposure to the antigen.

[0040] In this invention, the term "common light chain" refers to a light chain that may be identical or may have some differences in amino acid sequence, and the binding specificity of the antibody of this invention is not affected; that is, the differences do not substantially affect the formation of the functional binding domain.

[0041] For example, within the definition of common chain as used herein, it is possible to prepare or discover variable chains that are not identical but are still functionally equivalent (e.g., by introducing and testing conservative amino acid changes, or amino acid changes in regions that do not contribute to, or only partially contribute to, binding specificity when paired with a congeneral chain). Thus, such variants can also bind to different congeneral chains and form functional antigen-binding domains. Therefore, as used herein, the term “common light chain” refers to a light chain that may be identical or have some amino acid sequence differences while maintaining the binding specificity of the antibody obtained after pairing with a heavy chain. Combinations of a particular common light chain with such functionally equivalent variants are encompassed by the term “common light chain”. For a detailed explanation of the use of common light chains, see International Publications 2004 / 009618 and 2009 / 157771.

[0042] "Fab" refers to a binding domain containing a variable region, typically a binding domain containing a pairing heavy-chain variable region and a light-chain variable region. Fab may include a constant-region domain containing CH1 and VH domains that pair with a constant-state light domain (CL) and a VL domain. Such pairing may occur, for example, as a covalent bond via a disulfide bridge in the CH1 and CL domains.

[0043] A "single-chain variable fragment" (scFv) refers to a binding domain containing a VH domain and a VL domain, which are linked via a linker, such as a peptide linker, for example, an amino acid with a length of approximately 10 to 25.

[0044] The terms "full-length IgG" or "full-length antibody" according to the present invention are defined as containing essentially complete IgG, but not necessarily possessing all the functions of complete IgG. To avoid misunderstanding, full-length IgG contains two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be broken down into domains denoted as CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens via the variable region domain contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domain, mainly the Fc portion. Full-length antibodies according to the present invention include IgG molecules in which mutations may exist to provide desired properties. Full-length IgG must not have any substantial deletions of any part of the region. However, IgG molecules with one or more amino acid residues deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG". For example, such an IgG molecule may have 1 to 10 amino acid residues deleted, preferably in the non-CDR region, and the deleted amino acids are not essential for the binding specificity of IgG.

[0045] In this specification, when referring to nucleic acid or amino acid sequences, “percent (%) identity” is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after the sequences have been aligned for optimal comparison purposes. To optimize the alignment between two sequences, gaps may be introduced in either of the two sequences being compared. Such alignment may be performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over shorter lengths, e.g., about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between two sequences across the reported alignment region.

[0046] The comparison of sequences and the measurement of the degree of sequence identity between two sequences can be achieved using mathematical algorithms. Those skilled in the art will recognize that several different computer programs are available for aligning two sequences and measuring the degree of identity between them (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0047] For the purposes of the present invention and the sequences described herein, the percentage sequence identity between two nucleic acid sequences can be determined using the AlignX application of Vector NTI Program Advance 10.5.2 software with default settings, which include a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22: 4673~4680), a swgapdnarnt score matrix, a gap-opening penalty of 15, and a gap-extension penalty of 6.66. Amino acid sequences can be aligned using the AlignX application of Vector NTI Program Advance 11.5.2 software with default settings, which include a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, 1994), a bloomum62mt2 score matrix, a gap-opening penalty of 10, and a gap-extension penalty of 0.1.

[0048] The term “super-cluster” or “supercluster” as used herein refers to a group of clones, and the binding domain that a group of clones can produce based on the use of the same VH V gene segment and having at least 70% sequence identity and the same HCDR3 length in HCDR3.

[0049] Accordingly, in a preferred embodiment, the present invention provides a “super-cluster” or “supercluster” comprising a binding domain that can be produced by a group of clones, based on the use of the same VH V gene segment and having at least 70% sequence identity and the same HCDR3 length in HCDR3. In a preferred embodiment, the sequence identity is 80%, more preferably 90%, and most preferably 95%, provided that the HCDR3 sequence Clones containing nucleic acids encoding DGGYSYGPYWYFDL and DHRGYGDYEGGGFDY, clones containing nucleic acid sequences encoding HCDR2 sequences GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG, and SIIPIFGTITYAQKFQG are excluded, or VH sequences QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, and A clone containing a nucleic acid sequence encoding EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS is Subject to exclusion, or subject to the condition that clones from the group contain nucleic acids encoding HCDR3, which are composed of bispecific antibodies or are designed to be composed of bispecific antibodies.

[0050] The term “super-cluster 1” or “super-cluster 1” as used herein refers to a group of clones, and a binding domain that a group of clones can produce based on the use of the same VH V gene segment (VH1-69), and having at least 70% sequence identity in HCDR3 and the same HCDR3 length as the members of its supercluster. Examples include MF8048, MF8056, MF8057, MF8058, MF8078, and MF8101. In another preferred embodiment, the anti-CD3 antibody herein is based on the use of the same VH V gene segment of VH1-69, and / or having at least 80% identity in HCDR3, more preferably 90%, and most preferably 95% identity in HCDR3 and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibody herein is based on the use of the same VH V gene segment of VH1-69 and / or has at least 80% identity in HCDR3 and the same HCDR3 length as the encoded CDR3 segment RGNWNPFDP, preferably has at least 90% sequence identity in HCDR3 and the same HCDR3 length, more preferably 95% or most preferably 98% identity and the same HCDR3 length, provided that the HCDR2 sequence Clones containing nucleic acids encoding GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG, and SIIPIFGTITYAQKFQG are excluded, or VH sequences are excluded. EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, A clone containing nucleic acid encoding EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS is Subject to exclusion, or subject to the condition that clones from such group consist of a bispecific antibody or a nucleic acid encoding HCDR3 designed to consist of a bispecific antibody. The term “super-cluster 3” or “super-cluster 3” as used herein refers to a group of clones, and a binding domain that a group of clones may produce based on the use of the same VH V gene segment (VH3-23), and having at least 70% sequence identity in HCDR3 and the same HCDR3 length as the members of its supercluster. Examples include MF8397 and MF8562. In another preferred embodiment, the anti-CD3 antibody as used herein is based on the use of the same VH V gene segment of VH3-23, and / or having at least 80% identity in HCDR3, more preferably 90%, most preferably 95% identity in HCDR3 and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibody herein preferably has at least 90% sequence identity and the same HCDR3 length, more preferably 95% or most preferably 98% identity and the same HCDR3 length, based on the use of the same VH V gene segment of VH3-23 and / or at least 80% identity in HCDR3 and having the same HCDR3 length as the encoded CDR3 segment DGGYSYGPYWYFDL, provided that clones containing nucleic acids encoding the HCDR3 sequence DGGYSYGPYWYFDL are excluded, or the VH sequence A clone containing nucleic acids that encode QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS is, Subject to exclusion, or subject to the condition that clones from the group contain nucleic acids encoding HCDR3, which are composed of bispecific antibodies or are designed to be composed of bispecific antibodies.

[0051] The term “super-cluster 4” or “super-cluster 4” as used herein refers to a group of clones, and a binding domain that a group of clones can produce based on the use of the same VH V gene segment (VH3-9), and having at least 70% sequence identity in HCDR3 and the same HCDR3 length as the members of its supercluster. Examples include MF8508, MF8998, MF10401, and MF10428. In another preferred embodiment, the anti-CD3 antibody as used herein is based on the use of the same VH V gene segment of VH3-9, and / or having at least 80% identity in HCDR3, more preferably 90%, and most preferably 95% identity in HCDR3 and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibody herein preferably has at least 90% sequence identity and the same HCDR3 length, more preferably 95% or most preferably 98% identity and the same HCDR3 length, based on the use of the same VH V gene segment of VH3-9 and / or at least 80% identity in HCDR3 and having the same HCDR3 length as the encoded CDR3 segment DHRGYGDYEGGGFDY, but provided that clones containing nucleic acids encoding the HCDR3 sequence DHRGYGDYEGGGFDY are excluded, or the VH sequence A clone containing nucleic acids encoding EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS is Subject to exclusion, or subject to the condition that clones from the group contain nucleic acids encoding HCDR3, which are composed of bispecific antibodies or are designed to be composed of bispecific antibodies.

[0052] The term “super-cluster 7” or “super-cluster 7” as used herein refers to a group of clones, and a binding domain that a group of clones can produce based on the use of the same VH V gene segment (VH5-51), and having at least 70% sequence identity in HCDR3 and the same HCDR3 length as the members of its supercluster. Examples include MF9249 and MF9267. In another preferred embodiment, the anti-CD3 antibody as used herein is based on the use of the same VH V gene segment of VH5-51, and / or having at least 80% identity in HCDR3, more preferably 90%, most preferably 95% identity in HCDR3 and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibody herein preferably has at least 90% sequence identity and the same HCDR3 length, more preferably 95% or most preferably 98% identity and the same HCDR3 length, based on the use of the same VH V gene segment of VH5-51 and / or at least 80% identity in HCDR3 and having the same HCDR3 length compared to the encoded CDR3 segment HIRYFDWSEDYHYYLDV.

[0053] The present invention V gene segment VH1-69, or The present invention further provides a bispecific antibody containing a variable domain having VH encoded by a variant of the V gene segment VH1-69 that has at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment. This VH is, HCDR3 for MF8048, MF8056, MF8057, MF8058, MF8078, or MF8101, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0054] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0055] In some embodiments, the bispecific antibody does not contain VH encoded by V gene segment VH1-69, or VH encoded by a variant of V gene segment VH1-69 having the HCDR2 sequence GFIPVLGTANYAQKFQG, or GIIPLFGTITYAQKFQG or SIIPIFGTITYAQKFQG.

[0056] In some embodiments, the bispecific antibody is VH encoded by the V gene segments VH1-69, or the VH sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, or VH is not encoded by a variant of the V gene segment VH1-69 that has EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS.

[0057] The present invention V gene segment VH3-23, or The present invention further provides a bispecific antibody containing a variable domain having VH encoded by a variant of the V gene segment VH2-23 that exhibits at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment. This VH is MF8397 or MF8562 HCDR3, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0058] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90%, more preferably at least 93%, and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0059] In some embodiments, the bispecific antibody does not contain VH encoded by the V gene segment VH3-23, or VH encoded by a variant of the V gene segment VH3-23 having the HCDR3 sequence DGGYSYGPYWYFDL.

[0060] In some embodiments, the bispecific antibody does not contain VH encoded by the V gene segment VH3-23, or VH encoded by a variant of the V gene segment VH3-23 having the VH sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS.

[0061] The present invention V gene segment VH3-9, or The present invention further provides a bispecific antibody containing a variable domain having VH encoded by a variant of the V gene segment VH3-9 that has at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment. This VH is, MF8508, MF8998, MF1041, or MF10428 HCDR3, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0062] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0063] In some embodiments, the bispecific antibody does not contain VH encoded by the V gene segment VH3-9, or VH encoded by a variant of the V gene segment VH3-9 having the HCDR3 sequence DHRGYGDYEGGGFDY.

[0064] In some embodiments, the bispecific antibody does not contain VH encoded by the V gene segment VH3-9, or VH encoded by a variant of the V gene segment VH3-9 having the VH sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS.

[0065] The present invention V gene segment VH5-51, or The present invention further provides a bispecific antibody containing a variable domain having VH encoded by a variant of the V gene segment VH5-51 that has at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment. This VH is, HCDR3 for MF9249 or MF9267, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0066] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0067] The bispecific antibodies provided by the present invention as defined herein are preferably not bispecific antibodies containing a CD3-binding variable domain as defined in PCT / NL2019 / 050199.

[0068] The present invention V gene segment VH1-69, or Further providing VH encoded by variants of the V gene segment VH1-69 containing at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment, This VH is, HCDR3 for MF8048, MF8056, MF8057, MF8058, MF8078, or MF8101, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0069] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0070] In some embodiments, the VH is not the VH encoded by the V gene segment VH1-69, nor is it the VH encoded by a variant of the V gene segment VH1-69 having the HCDR2 sequence GFIPVLGTANYAQKFQG, or GIIPLFGTITYAQKFQG or SIIPIFGTITYAQKFQG.

[0071] In some embodiments, the VH is not the VH encoded by the V gene segment VH1-69, or the VH sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, or This is not VH encoded by a variant of the V gene segment VH1-69 having EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS.

[0072] The present invention V gene segment VH3-23, or Further providing is a VH encoded by a variant of the V gene segment VH2-23 containing at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment, This VH is, MF8397 or MF8562 HCDR3, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0073] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90%, more preferably at least 93%, and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0074] In some embodiments, the VH is not the VH encoded by the V gene segment VH3-23, nor is it the VH encoded by a variant of the V gene segment VH3-23 having the HCDR3 sequence DGGYSYGPYWYFDL.

[0075] In some embodiments, the VH is not the VH encoded by the V gene segment VH3-23, nor is it the VH encoded by a variant of the V gene segment VH3-23 having the VH sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS.

[0076] The present invention V gene segment VH3-9, or Further providing VH encoded by a variant of the V gene segment VH3-9 containing at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment, This VH is, HCDR3 for MF8508, MF8998, MF10401, or MF10428, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0077] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0078] In some embodiments, the VH is not VH encoded by the V gene segment VH3-9, nor is it VH encoded by a variant of the V gene segment VH3-9 having the HCDR3 sequence DHRGYGDYEGGGFDY.

[0079] In some embodiments, the VH is not the VH encoded by the V gene segment VH3-9, nor is it the VH encoded by the V gene segment VH3-9 having the VH sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS.

[0080] The present invention V gene segment VH5-51, or Further providing VH encoded by a variant of the V gene segment VH5-51 containing at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of the V gene segment, This VH is, HCDR3 for MF9249 or MF9267, Or further comprising a variant of the HCDR3 having at least 70% sequence identity with the HCDR3 and the same length as the HCDR3.

[0081] In a preferred embodiment, the variant of HCDR3 has the same length as HCDR3 and at least 80% sequence identity with HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity with respect to the sequence of HCDR3.

[0082] The VHs provided by the present invention as defined herein are preferably not VHs of CD3-binding variable domains as defined in PCT / NL2019 / 050199.

[0083] Furthermore, an antigen-binding protein or antibody, preferably a bispecific antibody, is provided, wherein the CDR has 70%, preferably 80%, and more preferably 90% identity with the claimed CDR. In a preferred embodiment, the antigen-binding protein or antibody is a bispecific antibody comprising a CDR having at most 2, preferably at most 1, and more preferably at most 0 amino acid residue mutations, insertions, substitutions, deletions, or additions with respect to the claimed CDR.

[0084] Antigen binding by antibodies is typically mediated through specific three-dimensional structures of both the antibody's complementary region and the antigen and variable domains, allowing these two structures to bind precisely together (a lock-and-key analogy) as opposed to the random, nonspecific adhesion of antibodies. Antibodies typically recognize epitopes of antigens, and such epitopes may similarly be present in other proteins. Antibodies according to the present invention that bind to CD3 or CLEC12A may similarly recognize other proteins if such other proteins contain the same epitope. Therefore, the term “binding” does not exclude the binding of antibodies to one or more other proteins containing the same epitope. The heavy / light chain combination that binds to CD3 in the antibodies of the present invention does not bind to other proteins on the cell membrane after birth, preferably in adult humans. The heavy / light chain combination that binds to CLEC12A, EGFR, PD-L1, or the tumor cell antigens of the present invention does not bind to other proteins on the cell membrane after birth, preferably in adult humans. A suitable tumor antigen-specific arm is disclosed in PCT / NL2019 / 050199.

[0085] "Multiple" means two or more.

[0086] The “variants” of antibodies described herein may include functional portions, derivatives, and / or analogs of antibodies. These include antibody mimetic compounds, monobodies, and aptamers.

[0087] The variants typically maintain the binding specificity of the antibody, for example, the specificity of a bispecific antibody. The variants may be binding domains, polymers, functional parts of an antibody, or derivatives, as described herein.

[0088] The functional portion of a binding domain, polymer, or antibody described herein is a portion that includes a variable domain that binds to the same target as such binding domain, polymer, or antibody.

[0089] The functional derivatives of antibodies described herein are proteins comprising a variable domain that binds to one target and a variable domain that binds to another target, linked by a linking region. The variable domains may be such variable domains or variable domain-like molecules, such as a single-chain Fv(scFv) fragment containing VH and VL linked together via a Fab fragment or linker. Antibody variable domains or antibody variable domain-like molecules can be linked to each other in different ways. Various linker and carrier structures capable of binding to one, two, or more variable domains are described. Antigen-binding proteins described herein are proteins comprising at least one such variable domain. In the case of bispecific or multispecific antigen-binding proteins, such proteins comprise two or more variable domains that bind to at least two different targets. The variable domains are linked to each other via a linking region, which is typically a stretch of 0 to 15, preferably 3 to 12, and more preferably about 5 to 8 amino acid residues. Another example of a variable domain-like molecule is a so-called single-domain antibody fragment. A single-domain antibody fragment (sdAb) is an antibody fragment that has a single monomeric variable antibody region. Like the whole antibody, it can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, a single-domain antibody fragment is much smaller than a typical antibody (150-160 kDa) which consists of two heavy-chain proteins and two light chains, and even smaller than a Fab fragment (approximately 50 kDa, one light chain and half a heavy chain) and a single-chain variable fragment (approximately 25 kDa, two variable regions, one from the light chain and one from the heavy chain). Single-domain antibodies themselves are never much smaller than normal antibodies (typically 90-100 kDa). Single-domain antibody fragments can be genetically engineered from heavy-chain antibodies found in camels, and these are called VHH fragments (nanobodies®). Some fish species also possess heavy-chain-only antibodies (IgNAR, "immunoglobulin neoantigen receptor") from which single-domain antibody fragments called VNAR fragments can be obtained.An alternative approach is to split dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. While most studies on single-domain antibodies currently rely on heavy-chain variable domains, it has been shown that nanobodies derived from the light chain can also bind to target epitopes. Other non-limiting examples of variable domain-like molecules include VHH, human domain antibodies (dAb), and unibodies. The preferred functional portion is the portion containing variable domains, including heavy-chain and light-chain variable regions. Non-limiting examples of such variable domains are F(ab)-fragments and single-chain Fv fragments. The bispecificity format of variable domain(s) binding includes, for example, human serum albumin (HSA) bound to two different scFvs; two different scFvs bound together via a dimerization motif; or a bispecificity mini-antibody containing a self-associating secondary structure such as a helical bundle or coiled coil to result in the dimerization of scFv fragments (Morrison (2007) Nat. Biotechnol 25:1233-34). Examples of suitable HSA linkers and methods for binding scFvs to the linker are described in International Publication No. 2009 / 126920.

[0090] Functional derivatives may be antibody mimes, polypeptides, aptamers, or combinations thereof. These proteins or aptamers typically bind to one target. The proteins of the present invention bind to two or more targets. It should be understood that any combination of these antibodies, antibody mimes, polypeptides, and aptamers can be linked together by methods known in the art. For example, in some embodiments, the binding molecule of the present invention is a conjugate or fusion protein.

[0091] Antibody mimetic molecules are polypeptides that, like antibodies, can specifically bind to antigens but are structurally unrelated to antibodies. Antibody mimetic molecules are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa. Non-limiting examples of antibody mimetic molecules include afibody molecules (typically based on the Z domain of protein A), affilins (typically based on gamma-B crystals or ubiquitin), affimers (typically based on cystatins), afitins (typically based on Sac7d from Sulfolobus acidocaldarius), alphabodies (typically based on triple-helix coiled coils), anticarin (typically based on lipocalin), avimers (typically based on the A domain of various membrane receptors), DARPin (typically based on the ankyrin repeat motif), finomers (typically based on the SH3 domain of Fyn7), Kunitz domain peptides (typically based on the Kunitz domain of various protease inhibitors), and monobodies (typically based on the type III domain of fibronectin).

[0092] A monobody is a synthetic binding protein constructed using a fibronectin type III domain (FN3) as a molecular scaffold. Monobodies are an alternative to antibodies for producing target-binding proteins.

[0093] Monobodies and other antibody mimetic compounds are typically generated from combinatorial libraries in which the scaffold portion is diversified using directed evolution techniques such as molecular display and phage display, mRNA display, and yeast surface display.

[0094] Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers are typically created by selecting from a large pool of random sequences, although naturally occurring aptamers also exist within riboswitches. As macromolecules, aptamers can be used for both basic research and clinical purposes.

[0095] Throughout this specification and the appended claims, the words “comprise,” “include,” and “having,” as well as variations such as “comprises,” “comprising,” “includes,” and “including,” should be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically listed, where permitted by the context.

[0096] The articles "a" and "an" are used herein to refer to one or more (i.e., one or at least one) grammatical objects of the article. For example, "element" may mean one or more elements.

[0097] The antibody of the present invention is preferably a bispecific antibody or a multispecific antibody. The bispecific antibody or multispecific antibody preferably binds to at least human CD3.

[0098] The antigen-binding protein or antibody of the present invention is preferably a bispecific or multispecific antigen-binding protein or antibody. The bispecific or multispecific antigen-binding protein or antibody preferably binds to at least human CD3, and more preferably to at least a surface molecule expressed on human tumor cells. In preferred embodiments, the bispecific or multispecific antigen-binding protein or antibody binds to BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, PD-L1, FOLR1, FOLR3, HER2, HM1.24, MCSP, or PSMA. In particularly preferred embodiments, the bispecific antibody binds to CLEC12A. In particularly preferred embodiments, the multispecific antibody binds to CD3, PD-L1, and EGFR.

[0099] As used herein, the term "CLEC12A" refers to member A of the C-type lectin domain family 12. CLEC12A is a type C lectin protein CLL-1; MICL; dendritic cell-associated lectin 2; type C lectin superfamily; myeloinhibitory type C lectin-like receptor; type C lectin-like molecule-1; DCAL2; CLL1; type C lectin-like molecule 1; DCAL-2; Killer cell lectin-like receptor subfamily L, member 1 (KLRL1); CD371 (differentiation group 371) (Bakker A. et al. Cancer Res. 2004, 64. p8843 50; GenBank(trademark) access number: AY547296; Zhang W. et al. GenBank(trademark) access number: AF247788; AS Marshall, et al. J Biol Chem 2004, 279. p14792-802; GenBank(trademark) access number: AY498550; Y. Han It is also referred to as (et al. Blood 2004, 104, p2858 66; H. Floyd, et al. GenBank™ access number: AY426759; CHChen, et al. Blood 2006, 107, p1459 67). It is also referred to as Id; HGNC: 31713; Entrez Gene: 160364; Ensembl: ENSG00000172322; OMIM: 612088; UniProtKB: Q5QGZ9.

[0100] CLEC12A is an antigen expressed in leukemic blast cells and leukemic stem cells in acute myeloid leukemia (AML), such as CD34-negative or CD34-low expressing leukemic stem cells (a side population) (ABBakker et al. Cancer Res 2004, 64, p8443 50; Van Rhenen et al. 2007 Blood 110:2659; Moshaver et al. 2008 Stem Cells 26:3059), and is also an antigen expressed in myelodysplastic syndrome (MDS) (Bakker et al. 2004 and Toff-Peterson et al., Br.J. Haematol. 175(3):393-401, 2016). In other respects, CLEC12A expression is thought to be limited to hematopoietic cells, particularly myeloid cells in peripheral blood and bone marrow, namely granulocytes, monocytes, and dendritic cell precursors. More importantly, CLEC12A is not present in normal hematopoietic stem cells. In this specification, when CLEC12A is referred to, it refers to human CLEC12A (Sequence ID 1; Figure 19) unless otherwise specified.

[0101] The term "CLEC12A" refers to all variants (such as splices and mutations) and their isoforms mentioned herein that retain a bone marrow expression profile (both surface expression level and mRNA level), as described, for example, in Bakker et al. Cancer Res 2004, 64, pp. 8443-50 and Marshall 2004-J Biol Chem 279(15), pp. 14792-802. Accession numbers are primarily provided as a further identification method, although the actual sequence of the protein may change due to mutations in the coding gene, such as those occurring in some cancers.

[0102] The term "CD3" (differentiation group 3) refers to a protein complex composed of the CD3γ chain (SwissProt P09693), CD3δ chain (SwissProt P04234), CD3ε chain (SwissProt P07766), and CD3ζ chain homodimer (SwissProt P20963). CD3ε is known under various aliases, some of which are "CD3e molecule, epsilon (CD3-TCR complex)"; "CD3e antigen, epsilon polypeptide (TiT3 complex)"; T cell surface antigen T3 / Leu-4 epsilon chain; T3E; T cell antigen receptor complex, epsilon subunit of T3; CD3e antigen; CD3-epsilon 3; IMD18; TCRE. The IDs for the CD3E gene are HGNC:1674;Entrez Gene:916:Ensembl:ENSG00000198851;OMIM:186830, and UniProtKB:P07766. These chains associate with the T cell receptor (TCR) and ζ chain to form the TCR complex, which can generate activation signals in T lymphocytes through mitotic signaling. CD3 is expressed on T cells and NK T cells. In this specification, when CD3 is referred to, it refers to human CD3 (SEQ ID NOs: 2-5; Figure 20) unless otherwise specified.

[0103] BCMA is the tumor necrosis factor receptor superfamily, member 17 (TNFRSF17); TNFRSF13A2; B cell maturation antigen; BCM; B cell maturation factor; B cell maturation protein; also known as CD269 or CD269 antigen. Id:HGNC:11913;Entrez Gene:608;Ensembl:ENSG00000048462;OMIM:109545;UniProtKB:Q02223.

[0104] CD19 is also known as the CD19 molecule; T cell surface antigen Leu-12; CD19 antigen; CVID3 differentiation antigen CD19; B4; B lymphocyte surface antigen B4; B lymphocyte antigen CD19. Its ID is HGNC:1633; Entrez Gene:930; Ensembl:ENSG00000177455; OMIM:107265; UniProtKB:P15391.

[0105] CD20 is also known as transmembrane 4-domain subfamily A member 1 (MS4A1); MS4A2; CD20; S7; leukocyte surface antigen Leu-16; B lymphocyte antigen CD20; Bp35; B lymphocyte cell surface antigen B1; CD20 antigen; CD20 receptor; CVID5; B lymphocyte surface antigen B1; B1; transmembrane 4-domain subfamily A member 1; LEU-16. Id; HGNC: 7315; Entrez Gene: 931; Ensembl: ENSG00000156738; OMIM: 112210; UniProtKB: P11836.

[0106] CD30 is also known as member 8 (TNFRSF8) of the tumor necrosis factor receptor superfamily; Ki-1 antigen; CD30: Ki-1; D1S166E; cytokine receptor CD30; lymphocyte activating antigen CD30; tumor necrosis factor receptor superfamily member 8, CD30L receptor; and CD30 antigen. Its ID is HGNC: 11923; Entrez Gene: 943; Ensembl: ENSG00000120949; OMIM: 153243; UniProtKB: P28908.

[0107] CD33 is also known as the CD33 molecule; SIGLEC-3; CD33 antigen (Gp67); bone marrow surface antigen CD33; sialic acid-binding Ig-like lectin 3; Siglec-3; SIGLEC3; CD33 antigen and gp67. Id; HGNC:1659; Entrez Gene:945; Ensembl:ENSG00000105383; OMIM:159590; UniProtKB:P20138.

[0108] CD38 is also referred to as the CD38 molecule; T10; CD38 antigen (P45); CADPr hydrolase 1; ADP-ribosyl cyclase 1; ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase; NAD(+) nucleosidase; EC3.2.2.5; cyclic ADP-ribose hydrolase 1; and CD38 antigen. Its ID is HGNC:1667; Entrez Gene:952; Ensembl:ENSG00000004468; OMIM:107270; UniProtKB:P28907.

[0109] CD44 is also known as the CD44 molecule (Indian blood group); IN; MDU2; CD44 antigen (homing function and Indian blood group system); MDU3; CDW44; MIC4; CSPG8; chondroitin sulfate proteoglycan 8; HCELL; hematopoietic cell E and L-selective ligand; MC56; extracellular matrix receptor III; Pgp1; heparan sulfate proteoglycan; cell surface glycoprotein CD44; hyaluronic acid receptor; epican; phagocyte glycoprotein 1; homing function and Indian blood group system; ECMR-III; CDw44; HUTCH-I; epican; LHR; PGP-1; CD44 antigen; PGP-I; CP90 lymphocyte homing / adhesion receptor; phagocyte glycoprotein I; and Hermes antigen. Id;HGNC:1681;Entrez Gene:960;Ensembl:ENSG00000026508;OMIM:107269;UniProtKB:P16070.

[0110] CD123 is also known as cell division cycle 123; cell division cycle 123 homolog; C10orf7; cell division cycle protein 123 homolog; D123; protein D123; HT-1080; CCEP123; PZ32; CEP89; cell division cycle 123 homolog (budding yeast (S. Cerevisiae)); FLJ14640; chromosome 10 open reading frame 7. Id; HGNC: 16827; Entrez Gene: 8872: Ensembl: ENSG00000151465; OMIM: 615470; UniProtKB: O75794.

[0111] CD138 is also known as syndecan 1 (SCD1); CD138; SDC; heparan sulfate proteoglycan fibroblast growth factor receptor; syndecan proteoglycan 1; syndecan; SYND1; syndecan-1; and CD138 antigen. Its ID is HGNC:10658; Entrez Gene:6382; Ensembl:ENSG00000115884; OMIM:186355; UniProtKB:P18827.

[0112] CEA is also known as carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5); fetal fecal antigen 100; CD66e; carcinoembryonic antigen; or CD66e antigen. Its ID is HGNC:1817; Entrez Gene:1048; Ensembl:ENSG00000105388; OMIM:114890; UniProtKB:P06731.

[0113] EGFR is also known as the epidermal growth factor receptor; erythroblastic leukemia virus (V-Erb-B) oncogene homolog (avian); ERBB1; PIG61; proto-oncogene C-ErbB-1; avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog; receptor tyrosine protein kinase ErbB-1; cell proliferation inhibitor protein 40; cell proliferation inducer protein 61; HER1; mENA; EC2.7.10.1; EC2.7.10; epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog). Id; HGNC:3236; Entrez Gene:1956; Ensembl:ENSG00000146648; OMIM:131550; UniProtKB:P00533.

[0114] EGFRvIII is a common variant of EGFR (Oncogene.2013 May 23;32(21):2670-81.doi:10.1038 / onc.2012.280.Epub 2012 Jul 16).

[0115] Delta-like 3 (DLL3) is also known as Delta-like 3; Drosophila delta homolog 3; Delta 3; Delta (Drosophila)-like 3; and SCDO1. The IDs for DLL3 are HGNC:2909;Entrez Gene:10683;Ensembl:ENSG00000090932;OMIM:602768, and UniProtKB:Q9NYJ7.

[0116] LGR5 is a leucine-rich repeat-containing G protein-coupled receptor 5. Alternative names for this gene or protein include leucine-rich repeat-containing G protein-coupled receptor 5, leucine-rich repeat-containing G protein-coupled receptor 5; G protein-coupled receptor HG38; G protein-coupled receptor 49; G protein-coupled receptor 67; GPR67; GPR49; orphan G protein-coupled receptor HG38, G protein-coupled receptor 49; GPR49; Hg38, and FEX. The proteins or antibodies of the present invention that bind to LGR5 bind to human LGR5. The LGR5-binding proteins or antibodies of the present invention may also bind to such orthologues due to sequence and tertiary structure similarities between human and other mammalian orthologues, but may not necessarily. The database accession numbers for the human LGR5 protein and the gene encoding it are (NC_000012.12;NT_029419.13;NC_018923.2;NP_001264155.1;NP_001264156.1;NP_003658.1).

[0117] MSLN, or mesoserine, is also known as metserine, pre-pro megakaryocyte-enhancing factor, CAK1 antigen, MPF, soluble MPF mesoserine-associated protein, megakaryocyte growth factor, and SMRP. The IDs for MSLN are HGNC:7371;Entrez Gene:10232;Ensembl:ENSG00000102854;OMIM:601051;UniProtKB:Q13421.

[0118] Folate receptor 1 is also known as FOLR1; folate receptor 1; ovarian tumor-associated antigen MOv18; adult folate-binding protein; folate receptor, adult; KB cell FBP; FR-alpha; FOLR; FBP: folate-binding protein, and folate receptor 1. The ID for FOLR1 is HGNC:3791; Entrez Gene:2348; Ensembl:ENSG00000110195; OMIM:136430; UniProtKB:P15328.

[0119] Folate receptor 3 is also known as FOLR3; folate receptor 3 (gamma); FR-gamma; folate receptor 3; gamma-HFR; and FR-G. The IDs for FOLR3 are HGNC:3795; Entrez Gene:2352; Ensembl:ENSG00000110203; OMIM:602469, and UniProtKB:P41439.

[0120] EPCAM is also known as epithelial cell adhesion molecule; EGP40; M4S1; ESA; MIC18; KS1 / 4; tumor-associated calcium signaling transducer 1; MK-1; TACSTD1; human epithelial glycoprotein-2; TROP1; membrane component, chromosome 4, surface marker (35kD glycoprotein); adenocarcinoma-associated antigen; EGP; cell surface glycoprotein Trop-1; Ep-CAM; epithelial glycoprotein 314; GA733-2; major gastrointestinal tumor-associated protein GA733-2; M1S2; EGP314; CD326 antigen; KSA; epithelial cell surface antigen; DIAR5; epithelial glycoprotein; HNPCC8; hEGP314; antigen identified by monoclonal antibody AUA1; KS 1 / 4 antigen; EGP-2; and ACSTD1. Id;HGNC:11529;Entrez Gene:4072, Ensembl:ENSG00000119888;OMIM:185535;UniProtKB:P16422.

[0121] HER2 is a homolog of the V-Erb-B2 erythroblastic leukemia virus oncogene 2; ERBB2; CD340; NGL, HER-2; HER-2 / neu2NEU2; TKR1; neuroblastoma / glioblastoma-derived oncogene homolog; C-Erb B2 / Neu protein; metastatic lymph node gene 19 protein; Herstatin; prostate cancer gene C-ErbB-2; neuroblastoma / glioblastoma-derived oncogene homolog; prostate cancer gene Neu; receptor tyrosine protein kinase ErbB-2; tyrosine kinase-type cell surface receptor HER2; V-Erb-B2 erythroblastic leukemia virus oncogene homolog 2, neuroblastoma / glioblastoma-derived oncogene homolog; MLN 19; MLN 19;p185erbB2;CD340 antigen;EC2.7.10.1;EC2.7.10;Also known as V-Erb-B2 erythroblastic leukemia virus oncogene homolog 2 (neuroplasmosis / glioblastoma-derived oncogene homolog). Id: HGNC:3430;Entrez Gene:2064;Ensembl:ENSG00000141736;OMIM:164870;UniProtKB:P04626.

[0122] HM1.24 is also known as BST2; bone marrow stromal cell antigen 2; TETHERIN; BST-2; bone marrow stromal cell antigen 2; HM1.24 antigen; Tetherin; CD317; CD317 antigen; NPC-A-7. Id; HGNC:1119; Entrez Gene:684; Ensembl:ENSG00000130303; OMIM:600534; UniProtKB:Q10589.

[0123] MCSP is also known as testicular mitochondria-associated cysteine-rich protein (SMCP); MCSP; MCS; mitochondrial sheath selenoprotein; HSMCSGEN1; or testicular mitochondria-associated cysteine-rich protein. Id;HGNC:6962;Entrez Gene:4184, Ensembl:ENSG00000163206;OMIM:601148;UniProtKB:P49901.

[0124] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing the immune response during certain events such as pregnancy, tissue allogeneic transplantation, autoimmune diseases, and other disease conditions such as hepatitis. Binding of PD-L1 to PD-1 or B7.1(CD80) transmits an inhibitory signal, which reduces the proliferation of PD-1-expressing T cells. PD-1 is thought to be able to regulate the accumulation of exogenous antigen-specific T cells via apoptosis. PD-L1 is expressed by various cancer cells, and its expression is thought to be at least partially involved in weakening the immune response against cancer cells. PD-L1 is a member of the B7 family of proteins and is known by various other names, such as CD274 molecule; CD274 antigen; B7 homolog 1; PDCD1 ligand 1; PDCD1LG1; PDCD1L1; B7H1; PDL1; programmed cell death 1 ligand 1; programmed cell death ligand 1; B7-H1; and B7-H. External IDs for CD274 are HGNC:17635; Entrez Gene:29126; Ensembl:ENSG00000120217; OMIM:605402; UniProtKB:Q9NZQ7.

[0125] PSMA is also known as folate hydrolase (prostate-specific membrane antigen) 1; FOLH1; NAALAD1; FOLH; mGCP; glutamate carboxypeptidase II; N-acetylated-alpha-linked acid dipeptidase I; PSM; NAALADaseI; PSMA; EC3.4.17.21; glutamate carboxylase II; GCP2; cell proliferation inhibitor gene 27 protein; NAALAdase; folyl poly-gamma-glutamate carboxypeptidase; glutamate carboxypeptidase 2; membrane glutamate carboxypeptidase; N-acetylated-alpha-linked acid dipeptidase 1; pteroyl poly-gamma-glutamate carboxypeptidase; prostate-specific membrane antigen variant F; FGCP; folate hydrolase 1; GCPII; and prostate-specific membrane antigen. Id;HGNC:3788;Entrez Gene:2346, Ensembl:ENSG00000086205;OMIM:600934;UniProtKB:Q04609.

[0126] PSMA, also known as PSMA1, is a proteasome (prosome, macropine) subunit, alpha type, and should not be confused with type 1.

[0127] Accession numbers are primarily assigned to provide further methods for identifying targets, and the actual sequence of the bound protein may be altered due to mutations in the coding gene, such as those occurring in some cancers. The antigen-binding site binds to various variants of its own, such as those expressed by some antigen-positive immune or tumor cells.

[0128] In this specification, when referring to genes or proteins, this reference preferably refers to the human form of the gene or protein. In this specification, when referring to genes or proteins, natural genes or proteins, as well as variants of genes or proteins that may be detected in tumors, cancers, etc., preferably variants of genes or proteins that may be detected in human tumors, cancers, etc.

[0129] The bispecific or multispecific antibodies of the present invention preferably bind to human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA proteins, or their variants. The antigen-binding heavy / light chain combination preferably binds to the extracellular portion of the antigen. The bispecific antibodies of the present invention preferably bind to human CLEC12A or its variants. Preferred bispecific antibodies of the present invention bind to human CD3 and human CLEC12A or their variants. In preferred embodiments, the multispecific antibodies bind to CD3, PD-L1, and EGFR.

[0130] HGNC stands for the HUGO Gene nomenclature committee. The number following the abbreviation is an accession number that allows information about genes and proteins encoded by those genes to be retrieved from the HGNC database. Entrez genes provide accession numbers or gene IDs that allow information about genes or proteins encoded by those genes to be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensemble provides accession numbers that allow information about genes or proteins encoded by those genes to be retrieved from the Ensemble database. Ensemble is a joint project between EMBL-EBII and the Wellcome Trust Sanger Institute to develop a software system that generates and maintains automated annotations on selected eukaryotic genomes.

[0131] The present invention provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is as follows: Amino acid sequence: CDR1:SFGIS;CDR2:GFIPVLGTANYAQKFQG;CDR3:RGNWNPFDP or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the array, Includes CDR1, CDR2, and CDR3 (where X1 = K or R and X2 = L or I).

[0132] In a preferred embodiment, X1 = K and X2 = L. In another preferred embodiment, X1 = R and X2 = I.

[0133] The present invention provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1:SKTLTIS;CDR2:GIIPIFGSITYAQKFQD;CDR3:RGNWNPFDP;or Amino acid sequence: CDR1:GSGIS;CDR2:GFIPFFGSANYAQKFRD;CDR3:RGNWNPX 13 DP; (In the array, X 13 Includes CDR1, CDR2, and CDR3 (which are either L or F).

[0134] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1:RX3WIG;CDR2:IIYPGDSDTRYSPSFQG;CDR3:X4IRYFX5WSEDYHYYX6DV; (In the array, Includes CDR1, CDR2, and CDR3 (where X3=F or Y, X4=H or N, X5=D or V, and X6=L or M).

[0135] In a preferred embodiment, X3=F; X4=H, X5=D, and X6=L, or X3=Y, X4=N, X5=V, and X6=M.

[0136] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: Includes CDR1:SYALS;CDR2:GISGSGRTTWYADSVKG, CDR3:DGGYSYGPYWYFDL, CDR1, CDR2, and CDR3.

[0137] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: Includes CDR1:SYALS;CDR2:AISGSGRTTWYADSVKG, CDR3:DGGYTYGPYWYFDL, CDR1, CDR2, and CDR3.

[0138] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: Includes CDR1:DYTMH;CDR2:DISWSSGSIGYADSVKG, CDR3:DHRGYGDYEGGGFDY, CDR1, CDR2, and CDR3.

[0139] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1:DYTMH, CDR2:DISWSX7GX8X9X 10 YADSVKG, CDR3:DHX 11 GYGDYEGGGFDX 12 (In the array, X7 = S or G X8 = S or T, X9 = I or T, X 10 =G or Y, X 11 = R or M, X 12 =H or Y, Preferably, X7, X8, X9 and X 10 is S, S, I and G, or G, S, I and Y, or S, T, T and G, preferably X 11 and X 12are R and H, or R and Y, or M and Y, more preferably X7, X8, X9, X 10 , X 11 and X 12 These are S, S, I, G, R and H, or G, S, I, Y, R and Y, or S, T, T, G, M and Y, or, in other words, preferably X7, X8, X9 and X 10 are S, S, I and G, and X 11 and X 12 These are R and H, or X7, X8, X9 and X 10 are G, S, I and Y, and X 11 and X 12 These are R and Y, or X7, X8, X9 and X 10 are S, T, T and G, and X 11 and X 12 This includes CDR1, CDR2, and CDR3, which are M and Y.

[0140] In a preferred embodiment, the light chain variable region has 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof, as shown in Figure 11A, IgVκ1-39 * Includes the amino acid sequence of the 01 gene segment. IgVκ1-39 * The amino acid sequence of 01 is shown in Figure 11A. IgVκ1-39 is a shortened form of the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin variable kappa 1-39, IGKV139, and IGKV1-39. The external IDs for this gene are HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. The preferred amino acid sequence of IgVκ1-39 is shown in Figure 11A. This lists the sequences of the V region. The V region can be combined with one of the five J regions. Figures 11B and 11C describe two preferred sequences for IgVκ1-39 combined with the J region. The conjugated sequences are shown as IGKV1-39 / jk1 and IGKV1-39 / jk5, with the alternative name IgVκ1-39 *01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * It is 01 (according to the naming convention of the IMGT database on imgt.org on the World Wide Web).

[0141] IgVκ1-39 including the light chain variable region * 01 is preferably a germline sequence. IGJκ containing a light chain variable region. * 01 or / IGJκ5 * It is even more preferable that 01 is a germline sequence. In a preferred embodiment, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0142] In a preferred embodiment, the light chain variable region is germline IgVκ1-39 * Includes 01. In a preferred embodiment, the light chain variable region is kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * Includes 01. In a preferred embodiment, IgVκ1-39 * 01 / IGJκ1 * The value is 01. The light chain variable region is preferably germline kappa light chain IgVκ1-39. * 01 / IGJκ1 * 01 or germline kappa light chain IgVκ1-39 * 01 / IGJκ5 * 01, preferably germline IgVκ1-39 * 01 / IGJκ1 * Includes 01.

[0143] Mature B cells that produce antibodies with light chains often produce light chains that have one or more mutations in their germline sequence, i.e., the normal sequence found in the organism's non-lymphoid cells. The processes involved in these mutations are often called somatic (hyper)mutations. The resulting light chains are called affinity-matured light chains. Such light chains are germline IgVκ1-39 *When derived from sequence 01, IgVκ1-39 * It is a light chain derived from 01. In this specification, the term "IgVκ1-39" is used. * "01" is IgVκ1-39 * The light chain will contain a light chain derived from 01, and mutations introduced by somatic cell hypermutation can also be artificially introduced in the laboratory. In the laboratory, other mutations or mutations can also be introduced into the light chain without necessarily completely but essentially affecting the properties of the light chain. The light chain will contain at least IgVκ1-39 if it contains the sequence shown in Figure 11A, Figure 11D, or Figure 11E, which has 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof. * 01 is a light chain. In a preferred embodiment, IgVκ1-39 * The 01 light chain is a light chain containing the sequence shown in Figure 11A, Figure 11B, or Figure 11C, having mutations, insertions, deletions, substitutions, additions, or combinations thereof of 0-9, 0-8, 0-7, 0-6, 0-5, or 0-4 amino acids. In a preferred embodiment, IgVκ1-39 * The 01 light chain is a light chain containing the sequence shown in Figure 11A, Figure 11B, or Figure 11C, having 0 to 5, preferably 0 to 4, more preferably 0 to 3, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof. In a preferred embodiment, IgVκ1-39 * The 01 light chain is a light chain containing the sequence shown in Figure 11A, Figure 11B, or Figure 11C, having 0 to 2, more preferably 0 to 1, and most preferably 0 amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof. In a preferred embodiment, IgVκ1-39 * The 01 light chain is a light chain containing the sequence shown in Figure 11A or Figure 11B, having mutations, insertions, deletions, substitutions, additions, or combinations thereof of the aforementioned amino acids. In a preferred embodiment, the light chain contains the sequence in Figure 11B.

[0144] The light chain preferably includes a common light chain variable region. The common light chain variable region preferably includes an IgVκ1-39 light chain variable region. The light chain variable region preferably includes germline IgVκ1-39* 01 is a variable region. The light chain variable region is kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01 is preferably included. The light chain variable region is the germline kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01 is preferably included. The light chain variable region preferably has 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof, and the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK.

[0145] The light chain variable region preferably includes CDR1, CDR2, and CDR3 regions containing the amino acid sequence CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, i.e., the CDR of IGKV1-39 (according to IMGT). Amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are preferably not present in the CDR3 region of the light chain variable region, and preferably not in the CDR1 or CDR2 regions of the light chain variable region. In a preferred embodiment, the light chain variable region does not contain deletions, additions, mutations, or insertions to the indicated sequence. In this embodiment, the light chain variable region may have 0 to 5 amino acid substitutions to the indicated amino acid sequence. Amino acid substitutions are preferably conservative amino acid substitutions. The CDR1, CDR2, and CDR3 of the light chain of the antibody of the present invention preferably each contain the amino acid sequence CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, i.e., the CDR of IGKV1-39 (according to IMGT).

[0146] The antigen-binding protein is preferably an antibody, preferably a bispecific or multispecific antibody. The antibody preferably includes a common light chain comprising a common light chain variable region as defined herein and a light chain constant region as defined herein.

[0147] The antibodies of the present invention are preferably bispecific antibodies, as described above. A “bispecific antibody” is an antibody described herein in which one domain of the antibody binds to a first antigen, while a second domain of the antibody binds to a second antigen, and the first and second antigens are not the same. The term “bispecific antibody” also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first epitope on an antigen, and a second VH / VL combination binds to a second epitope. This term further includes antibodies in which VH can specifically recognize the first antigen, and VL, paired with VH in the immunoglobulin variable region, can specifically recognize the second antigen. The resulting VH / VL pair binds to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in International Publication Nos. 2008 / 027236, 2010 / 108127, and Schaefer et al (Cancer Cell 20, 472-486, October 2011). The bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of preparing them.

[0148] A bispecific antibody preferably has a combination of one heavy chain variable region / light chain variable region (VH / VL) that binds to CD3, and a second VH / VL combination that binds to an antigen other than the antigen on CD3. In a preferred embodiment, the antigen is a tumor antigen. In a preferred embodiment, the VL in the first VH / VL combination is similar to the VL in the second VH / VL combination. In a more preferred embodiment, the VL in the first and second VH / VL combinations are identical. In a preferred embodiment, the bispecific antibody is a full-length antibody having a combination of one heavy chain / light chain (H / L) that binds to CD3, and a combination of one H / L chain that binds to another antigen, preferably a tumor antigen. In a preferred embodiment, the light chain in the first H / L chain combination is similar to the light chain in the second H / L chain combination. In a more preferred embodiment, the light chains in the combination of the first and second H / L chains are identical; that is, similar or identical human light chains are so-called "common light chains" that can bind to different heavy chains to form antibodies having a functional antigen-binding domain. In a preferred embodiment, the light chain in the combination of the first H / L chains includes a light chain variable region similar to the light chain variable region in the combination of the second H / L chain. In a more preferred embodiment, the light chain variably regions in the combination of the first and second H / L chains are identical; that is, similar or identical human light chain variably regions are so-called "common light chain variably regions" that can bind to different heavy chain variably regions to form antibodies having a functional antigen-binding domain. It is preferable that the light chain containing the common light chain variably region is a common light chain.

[0149] The common light chain of the bispecific antibody is preferably the IgVκ1-39 light chain as described above herein.

[0150] The present invention also provides alternative bispecificity formats, such as those described in Spiess, C. et al. (Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol.Immunol. (2015) http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003). Bispecificity antibody formats other than conventional antibodies having two H / L combinations have at least a variable domain comprising the heavy chain variable region and the light chain variable region of the present invention. This variable domain may be ligated to a single-chain Fv fragment, monobody, VH, and Fab fragment that provides a second binding activity.

[0151] In the bispecific antibodies of the present invention, the light chain in the CD3-binding H / L chain combination is preferably the same as the light chain in the H / L chain combination that can bind to antigens other than CD3, preferably tumor antigens. In a more preferred embodiment, the light chains in the H / L chain combinations are identical, i.e., the human light chain is a so-called "common light chain" that can bind to different heavy chains to form antibodies having a functional antigen-binding domain. Preferably, the common light chain has a germline sequence. The preferred germline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield, and solubility. The preferred germline light chain is IgVκ1-39, preferably a reconstituted germline human κ light chain IgVκ1-39*01 / IGJκ1*01, or a fragment or functional equivalent thereof (i.e., the same IgVκ1-39 gene fragment but a different IGJκ gene fragment) (nomenclature according to the IMGT database on imgt.org on the World Wide Web).

[0152] As used herein, the term “abnormal cells” includes tumor cells, more specifically, preleukemic cells such as cells that cause myelodysplastic syndrome (MDS), and tumor cells of hematological origin, including leukemic cells such as acute myeloid leukemia (AML) tumor cells or chronic myeloid leukemia (CML) cells.

[0153] As used herein, the terms “immune effector cells” or “effector cells” refer to cells within the natural repertoire of cells in the mammalian immune system that can be activated to affect the viability of target cells. Immune effector cells include lymphoid cells, e.g., natural killer (NK) cells, T cells including cytotoxic T cells, or B cells, and myeloid cells, e.g., monocytes or macrophages, dendritic cells, and neutrophil granulocytes. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophil granulocytes. Recruitment of effector cells to abnormal cells means bringing immune effector cells into close proximity to abnormal target cells, which can then initiate direct or indirect death of the abnormal cells.

[0154] As used herein, the terms “subject” and “patient” are used synonymously and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs (for example, human patients, including those suffering from cancer).

[0155] As used herein, the terms “treat,” “treating,” and “treatment” refer to any type of intervention or procedure performed, or administration of an activator or combination of activators, aimed at reversing, alleviating, restoring, inhibiting, slowing, or preventing the progression, development, severity, or relapse of any disease-related symptoms, complications, conditions, or biochemical signs.

[0156] As used herein, “effective treatment” or “positive treatment response” refers to a beneficial effect, such as a treatment that results in the recovery of at least one symptom of a disease or disorder, such as cancer. Beneficial effects can result in an improved state beyond baseline, such as an improvement beyond the measurement or observation made before the initiation of therapy with this method. Beneficial effects can result in a state that slows, stabilizes, stops, or reverses the progression of cancer in a subject at any clinical stage, for example, as demonstrated by a reduction or elimination of clinical or diagnostic symptoms of the disease, or a reduction or elimination of cancer markers. Effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or inhibit tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0157] The term “therapeutic dose” refers to the amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic outcome. The outcome may be a reduction, recovery, remission, decrease, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease, or other desired changes in the ecosystem. In some embodiments, the therapeutic dose is sufficient to delay tumor development. In some embodiments, the therapeutic dose is sufficient to prevent or delay tumor recurrence. The therapeutic dose may be administered in one or more doses. A therapeutic dose of an agent or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, block, slow, or halt cancer cell infiltration into peripheral organs to some extent; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. For example, "therapeutic dose" is the amount of CLEC12A / CD3 bispecific antibody that results in a reduction of cancer (e.g., a reduction in the number of cancer cells) or a slowing of the progression of cancers such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

[0158] The present invention also provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Including EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0159] Further provided are an antigen-binding protein, preferably an antibody, that binds to human CD3 and includes an antibody-variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Including EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0160] Also provided are an antigen-binding protein that binds to human CD3, preferably an antibody, which includes an antibody-variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS includes, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0161] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, Includes EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, or EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0162] Amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are preferably not present in the CDR3 region of the heavy chain variable region, and preferably not in the CDR1 and / or CDR2 regions of the heavy chain variable region. In a preferred embodiment, the heavy chain variable region does not contain deletions, additions or mutations or insertions to the indicated sequence. In one embodiment, the heavy chain variable region may have 0 to 10, preferably 0 to 5, amino acid substitutions to the indicated amino acid sequence. In a preferred embodiment, the heavy chain variable region includes, at positions other than the CDR, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and preferably 0, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof to the indicated amino acid sequence. A combination of insertions, additions, deletions, or substitutions is a claimed combination if the aligned sequence does not differ at more than 10 positions (preferably 5 or less). A gap in one of the aligned sequences corresponds to the same number of amino acids skipped in the other sequence. Amino acid substitutions, if any, are preferably conservative.

[0163] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises the amino acid sequence MF8057, MF8058, or MF8078 as shown in Figure 13.

[0164] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises the amino acid sequence MF8397 as shown in Figure 13.

[0165] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises the amino acid sequence MF8508 as shown in Figure 13.

[0166] The present invention further provides an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises the amino acid sequence MF9249 or MF9267 as shown in Figure 13.

[0167] The light chain preferably includes the CDR1, CDR2, and CDR3 regions as defined elsewhere in this specification. It preferably includes the common light chain variable region as defined elsewhere in this specification, and preferably the common light chain. The bispecific antibody preferably further includes a combination of heavy and light chains that binds to another antigen, preferably a tumor antigen. The light chain of the heavy and light chain combination that binds to another antigen is preferably the common light chain as defined elsewhere in this specification. The heavy chain of the heavy and light chain combination that binds to another antigen preferably has the amino acid sequence: MF8233(EGFR) The heavy chain variable region includes QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQGTLVTVSS (having 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than CDR), or MF4327(CLEC12A) It contains a heavy chain variable region including QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGTTGDWFDYWGQGTLVTVSS (including mutations, insertions, deletions, substitutions, additions, or combinations thereof of 0 to 10, preferably 0 to 5, amino acids).

[0168] Variable domains that bind to CLEC12A having a heavy chain variable region and a common light chain region as defined herein are described in particular in International Publication No. 2014 / 051433 and International Publication No. 2017 / 010874, which are specifically referred to for the purposes of this specification and are incorporated herein by reference. The heavy chain variable region of a heavy chain / light chain combination that binds to human EGFR or CLEC12A may have 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof with respect to the indicated amino acid sequence. In preferred embodiments, the heavy chain variable region includes 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the indicated amino acid sequence. The combination of insertions, additions, deletions, or substitutions is the claimed combination if the aligned sequence does not differ at more than 5 positions. A gap in one of the aligned sequences corresponds to the same number of amino acids skipped in the other sequence.

[0169] It is preferable that amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof do not occur or are absent at the heavy-chain-light-chain bonding interface.

[0170] When amino acids change at the H / L chain interaction interface, it is preferable that the corresponding amino acids in the other chain also change to accommodate the change. The insertion or addition of amino acids is preferably not accompanied by the insertion or addition of proline.

[0171] The addition of amino acids can, in principle, be considered the same as insertion. Adding an amino acid to one of the ends of a polypeptide chain may, in some cases, not be considered an insertion, but rather a strict addition (extension). In this invention, both addition within the chain and addition to one of the ends are considered insertions.

[0172] Amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are preferably not present in the CDR3 region of the heavy chain variable region, and preferably not in the CDR1 or CDR2 region of the heavy chain variable region. In a preferred embodiment, the heavy chain variable region does not contain deletions, additions, mutations, or insertions to the indicated sequence. In this embodiment, the heavy chain variable region may have 0 to 5 amino acid substitutions to the indicated amino acid sequence. The amino acid substitutions are preferably conservative amino acid substitutions. The CD3-binding VH CDR1, CDR2, and CDR3 of the present invention preferably include combinations of CD3-binding VH CDR1, CD2, and CDR3 shown in Figure 13, preferably combinations of CDR1, CD2, and CDR3 of one VH from MF8057, MF8058, MF8078, MF8397, MF8508, MF9249, or MF9267.

[0173] The constant region of the antibody of the present invention, including bispecific or multispecific antibodies, is preferably a human constant region. This constant region may contain one or more, preferably 10 or fewer, preferably 5 or fewer, amino acids that are different from the constant region of naturally occurring human antibodies. The variable domains of the various antibodies prepared herein are derived from a human antibody variable domain library. Therefore, these variable domains are human. The unique CDR region may be derived from humans, synthetically, or from another organism. The antibody or bispecific antibody of the present invention is preferably a human or humanized antibody. Suitable heavy chain constant regions are non-limitingly illustrated in Table 12.

[0174] In this technical field, various methods exist for producing antibodies. Bispecific antibodies are typically produced by cells that express nucleic acids encoding antibodies. Suitable cells for antibody production are hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. In a particularly preferred embodiment, the cell is a CHO cell.

[0175] Various institutions and companies have developed cell lines for the large-scale production of antibodies, for example, for clinical use. Non-limiting examples of such cell lines include CHO cells, NS0 cells, or PER.C6 cells. These cells are also used for other purposes, such as protein production. Cell lines developed for the industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. In a preferred embodiment, the present invention provides an industrial cell line for producing the antibodies of the present invention.

[0176] In one embodiment, the present invention provides cells comprising antibodies and / or nucleic acids according to the present invention. These cells are preferably animal cells, more preferably mammalian cells, more preferably primate cells, and most preferably human cells. For the purposes of the present invention, suitable cells are any cells that may, and preferably, contain, antibodies and / or nucleic acids according to the present invention.

[0177] The present invention further provides cells comprising the antibody according to the present invention. Preferably, the cells (typically in vitro, isolated, or recombinant cells) produce the antibody. In a preferred embodiment, the cells are hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER.C6 cells. In a particularly preferred embodiment, the cells are CHO cells. Cell cultures comprising the cells according to the present invention are further provided. Various institutions and companies have developed cell lines for the large-scale production of antibodies, for example, for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. These cells are also used for other purposes, such as protein production. Cell lines developed for the industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, in a preferred embodiment, the present invention provides the use of cell lines developed for the large-scale production of antibodies for the production of the antibody of the present invention. The present invention further provides cells for producing antibodies comprising nucleic acid molecules encoding the VH, VL, and / or heavy and light chains of the claimed antibody. Preferably, the nucleic acid molecule is one that codes for the VH identified in Figure 13, and is a nucleic acid molecule that codes for the VH identified by the digit 4327, or by the digit 8233, or a combination thereof.

[0178] The present invention further provides a method for producing antibodies, comprising culturing the cells of the present invention and collecting antibodies from the culture. Preferably, the cells are cultured in serum-free medium. Preferably, the cells are adapted for suspension growth. Furthermore, antibodies that can be obtained by the method for producing antibodies according to the present invention are provided. Preferably, the antibodies are purified from the culture medium. Preferably, the antibodies are purified by affinity.

[0179] The cells of the present invention are, for example, hybridoma cell lines, CHO cells, 293F cells, NS0 cells, or any other cell type known to be suitable for antibody production for clinical purposes. In a particularly preferred embodiment, the cells are human cells. Preferably, cells transformed with the adenovirus E1 region or its functional equivalent. A preferred example of such a cell line is the PER.C6 cell line or its equivalent. In a particularly preferred embodiment, the cells are CHO cells or their variants. Preferably, variants utilizing a glutamine synthetase (GS) vector system for antibody expression.

[0180] The present invention further provides a method for producing antibodies, comprising culturing the cells of the present invention and collecting antibodies from the culture. Preferably, the cells are cultured in serum-free medium. Preferably, the cells are adapted for suspension growth. Furthermore, antibodies that can be obtained by the method for producing antibodies according to the present invention are provided. Preferably, the antibodies are purified from the culture medium. Preferably, the antibodies are purified by affinity.

[0181] Bispecific antibodies are also typically produced by cells expressing nucleic acids that encode antibodies. In this case, the cells express different light and heavy chains that constitute the bispecific antibody. To achieve this, the cells express two different heavy chains and at least one light chain. Since unmodified heavy chains can pair with each other to form dimers, such cells typically produce two monospecific antibodies (homodimers) in addition to a bispecific antibody (heterodimer). This principle also applies to unmodified heavy chains comprising a first heavy chain with one heavy chain variable region and a second heavy chain with at least two heavy chain variable regions, so that cells expressing these two heavy chains produce monospecific antibodies (homodimers of pairings of two first heavy chains), tetravalent antibodies (homodimers of pairings of two second heavy chains), and triplicate antibodies (heterodimers of first and second heavy chains). The number of possible heavy / light chain combinations in the produced antibody increases when the cell expresses two or more light chains. To reduce the number of different antibody species (different heavy and light chain combinations) produced, the aforementioned "common light chain" is preferred.

[0182] Antibody-producing cells expressing a common light chain and two equal amounts of heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies each (i.e., identical heavy-light chain combinations). Alternatively, in the example above relating to a first heavy chain with one variable region and a second heavy chain with two variable regions, the two heavy chains typically produce 50% triplicity, 25% monospecificity, and 25% quadruplespecificity.

[0183] Several methods have been disclosed to favor the production of bispecific antibodies or, conversely, monospecific antibodies, and these methods can be further used to favor the production of multispecific antibodies. In the present invention, it is preferable that cells prefer the production of bispecific antibodies to the production of corresponding monospecific antibodies. Such is typically achieved by modifying the constant regions of the heavy chains so that they are more favorable to heterodimerization (i.e., dimerization with the heavy chain of another heavy / light chain combination) than to homodimerization. In a preferred embodiment, the bispecific antibody of the present invention comprises two different immunoglobulin heavy chains having compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. This compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. Various methods have been described in the art in which such heterodimerization of heavy chains can be achieved, including their use in "knob-into-hole" bispecific antibodies.

[0184] Methods and means for producing bispecific antibodies using compatible heterodimerizing domains are disclosed in U.S. Patent Application No. 13 / 866,747 (currently issued as U.S. Patent No. 9,248,181), U.S. Patent Application No. 14 / 081,848 (currently issued as U.S. Patent No. 9,358,286), and PCT / NL2013 / 050294 (published as International Publication No. 2013 / 157954; incorporated herein by reference). These means and methods can be advantageously employed in the present invention. Specifically, preferred mutations for producing essentially bispecific full-length IgG molecules only are amino acid substitutions L351K and T366K (according to EU numbering) in the first CH3 domain ("KK-mutant" heavy chain) and amino acid substitutions L351D and L368E in the second domain ("DE-mutant" heavy chain), or vice versa. The preferential pairing of DE- and KK- variants to form heterodimers (so-called "DEKK" bispecific molecules) has been previously demonstrated in our U.S. Patents No. 9,248,181 and 9,358,286, and International Publication No. 2013 / 157954. Homodimerization of DE- variant heavy chains (DEDE homodimers) or KK- variant heavy chains (KKKK homodimers) rarely occurs due to the strong repulsive force between charged residues at the CH3-CH3 interface between identical heavy chains. In one embodiment, a heavy / light chain combination containing a variable domain that binds to CD3 includes the KK variant of the heavy chain. In this embodiment, a heavy / light chain combination containing a variable domain that binds to an antigen other than CD3 includes the DE variant of the heavy chain. In a preferred embodiment, the antigen other than CD3 is CLEC12A. In a preferred embodiment, the VH of the variable domain that binds to CLEC12A is MF4327, as shown in Figure 13.

[0185] Some antibodies are modified in the CH2 / lower hinge region, for example, to reduce Fc receptor interaction or C1q binding. In some embodiments, the antibody of the present invention is an IgG antibody having a mutated CH2 and / or lower hinge domain so as to reduce the interaction between the bispecific IgG antibody and the Fc gamma receptor. Such a mutated CH2 and / or lower hinge domain preferably includes amino acid substitutions at positions 235 and / or 236 (according to EU numbering), preferably L235G substitutions and / or G236R substitutions.

[0186] Alternatively, some antibodies may be modified, for example, to enhance Fc receptor interaction or C1q binding. Such modifications may be preferable in embodiments targeting autoimmune adaptations.

[0187] The present invention further provides a method for treating a subject, comprising administering the antigen-binding protein of the present invention, preferably the antibody of the present invention, to a subject in need thereof. The present invention further provides the antigen-binding protein of the present invention, preferably the antibody of the present invention, for use in treating a subject in need thereof. The subject preferably has cells to be removed from the body. The cells may be abnormal immune cells or cancer cells that induce an autoimmune response. Variable domains suitable for this purpose are, in particular, those having a common light chain and VHs of MF9249 and MF8397. These preferably have low affinity and low cytotoxic activity but are functional under autoimmune response conditions. Variable domains suitable for this purpose are, in particular, those having a common light chain and VHs of MF9267, MF8057, MF8058, MF8078 and MF8508. These variable domains have suitable affinity and suitable cytotoxic activity for anticancer purposes.

[0188] Furthermore, the invention described herein includes an antigen-binding protein or antibody having a high-affinity variable domain with high cell-killing activity that can be administered locally or expressed locally and includes a binding domain MF8078. The invention further provides a method of treating a subject, including administering to a subject in need thereof the antigen-binding protein of the invention, preferably the antibody of the invention, through local administration means known to those skilled in the art, including local treatment for other cancers in another compartment such as a tumor-lysing virus, melanoma or brain.

[0189] The invention further provides the antigen-binding protein of the invention, preferably the antibody of the invention, for use in the treatment of a subject in need thereof, and the antigen-binding protein or antibody has moderate to high affinity and relatively high cytotoxicity such as those described herein. Suitable variable domains for this purpose are, inter alia, those having a common light chain and VH of MF8057, MF8058, MF9267, MF8508 and MF8078.

[0190] The invention further provides a method of treating cancer or cancer risk in a subject, including administering to a subject in need thereof an antigen-binding protein, preferably an antibody, that binds to human CD3 and includes an antibody variable domain comprising a heavy chain variable region and a light chain variable region, and the heavy chain variable region has the following amino acid sequence: CDR1: SFGIS CDR2: GFIPVLGTANYAQKFQG CDR3: RGNWNPFDP, or amino acid sequence: CDR_{1}: SX_{1}TFTIS, CDR_{2}: GIIPX_{2}FGTITYAQKFQG, CDR_{3}: RGNWNPFDP, (In the sequence, X_{1}=K or R, X_{2}=L or I) and includes CDR1, CDR2 and CDR3.

[0191] In a preferred embodiment, X1 = K and X2 = L. In another preferred embodiment, X1 = R and X2 = I.

[0192] The present invention further provides antigen-binding proteins, preferably antibodies, for use in the treatment of subjects requiring such use, and these antigen-binding proteins or antibodies have moderate to high affinity and relatively high cytotoxicity, such as those described herein. Variable domains suitable for this purpose include, in particular, those having a common light chain and VHs of MF8048, MF8056, and MF8101.

[0193] The present invention further provides a method for treating cancer or the risk of cancer in a subject, comprising administering an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, to a subject in need thereof, wherein the heavy-chain variable region has the following amino acid sequence: CDR1:SKTLTIS, CDR2:GIIPIFGSITYAQKFQD, CDR3:RGNWNPFDP, or Amino acid sequence: CDR1: GSGIS, CDR2:GFIPFFGSANYAQKFRD, CDR3:RGNWNPX 13 DP (In the array, X 13 Includes CDR1, CDR2, and CDR3 (aa CDR1, CDR2, and CDR3) which are either L or F.

[0194] The present invention further provides a method for treating cancer or the risk of cancer in a subject, comprising administering an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, to a subject in need thereof, wherein the heavy-chain variable region comprises the following amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Including EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0195] The present invention further provides a method for treating cancer or cancer risk in a subject, which comprises administering to the subject an antigen-binding protein, preferably an antibody, that binds to human CD3 and comprises an antibody variable domain containing a heavy chain variable region and a light chain variable region. The heavy chain variable region has the following amino acid sequence: CDR1: RX3WIG, CDR2: IIYPGDSDTRYSPSFQG, CDR3: X4IRYFX5WSEDYHYYX6DV (In the sequence, X3 = F or Y, X4 = H or N, X5 = D or V, X6 = L or M), and comprises CDR1, CDR2 and CDR3.

[0196] In one embodiment 、 X3 = F, X4 = H, X5 = D, and X6 = L. In a further embodiment, X3 = Y, X4 = N, X5 = V, and X6 = M).

[0197] The present invention further provides a method for treating cancer or cancer risk in a subject, which comprises administering to the subject an antigen-binding protein, preferably an antibody, that binds to human CD3 and comprises an antibody variable domain containing a heavy chain variable region and a light chain variable region. The heavy chain variable region has the following amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, It has mutations, insertions, deletions, substitutions, additions, or combinations thereof of 0 to 10, preferably 0 to 5 amino acids at one or more positions other than the CDR.

[0198] The present invention further provides a method for treating cancer or cancer risk in a subject, which comprises administering to the subject a human CD3-binding antigen-binding protein, preferably an antibody, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: SYALS, CDR2: GISGSGRTTWYADSVKG, CDR3: DGGYSYGPYWYFDL, and comprises CDR1, CDR2 and CDR3.

[0199] The present invention further provides a method for treating cancer or cancer risk in a subject, which comprises administering to the subject a human CD3-binding antigen-binding protein, preferably an antibody, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: SYALS, CDR2: AISGSGRTTWYADSVKG, CDR3: DGGYTYGPYWYFDL, and comprises CDR1, CDR2 and CDR3.

[0200] The present invention further provides a method for treating cancer or cancer risk in a subject, which comprises administering to the subject a human CD3-binding antigen-binding protein, preferably an antibody, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or The molecule contains QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS and has 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than CDR.

[0201] The present invention further provides a method for treating cancer or the risk of cancer in a subject, comprising administering an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, to a subject in need thereof, wherein the heavy-chain variable region has the following amino acid sequence: CDR1:DYTMH, CDR2:DISWSSGSIGYADSVKG, Includes CDR1, CDR2, and CDR3, which contain CDR3:DHRGYGDYEGGGFDY.

[0202] The present invention further provides a method for treating cancer or the risk of cancer in a subject, comprising administering an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, to a subject in need thereof, wherein the heavy-chain variable region has the following amino acid sequence: CDR1:DYTMH, CDR2:DISWSX7GX8X9X 10 YADSVKG, CDR3:DHX 11 GYGDYEGGGFDX 12 (In the array, X7 = S or G X8 = S or T, X9 = I or T, X 10 =G or Y, X 11 = R or M, X 12 =H or Y, Preferably, X7, X8, X9 and X 10 is S, S, I and G, or G, S, I and Y, or S, T, T and G, preferably X 11 and X 12 are R and H, or R and Y, or M and Y, more preferably X7, X8, X9, X 10 , X 11 and X 12 These are S, S, I, G, R and H, or G, S, I, Y, R and Y, or S, T, T, G, M and Y, or, in other words, preferably X7, X8, X9 and X 10 are S, S, I and G, and X 11 and X 12 These are R and H, or X7, X8, X9 and X 10 are G, S, I and Y, and X 11 and X 12 These are R and Y, or X7, X8, X9 and X 10 are S, T, T and G, and X 11 and X 12 This includes CDR1, CDR2, and CDR3, which are M and Y.

[0203] The present invention further provides a method for treating cancer or the risk of cancer in a subject, comprising administering an antigen-binding protein, preferably an antibody, that binds to human CD3, comprising an antibody-variable domain including a heavy-chain variable region and a light-chain variable region, to a subject in need thereof, wherein the heavy-chain variable region comprises the following amino acid sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, or Including EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0204] In this specification, the antigen-binding protein, preferably an antibody, in the treatment described above preferably includes a heavy-light chain (H / L) combination that binds to the tumor antigen.

[0205] The antibody is preferably human or humanized. Preferably, the antibody comprises two different immunoglobulin heavy chains having compatible heterodimerization domains. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. The bispecific antibody is an IgG antibody having a mutated CH2 and / or lower hinge domain for cancer immunotherapy applications where the interaction between the bispecific or multispecific IgG antibody and the Fc gamma receptor is reduced. The mutated CH2 and / or lower hinge domain preferably includes amino acid substitutions at positions 235 and / or 236 (according to EU numbering), preferably L235G substitutions and / or G236R substitutions. Alternatively, for autoimmune applications, the interaction with the bispecific or multispecific Fc gamma receptor is enhanced, or ADCC and CDC are enhanced by modifications to the CH2 and / or CH3 domains. For example, by manipulating the Fc region that binds to a more selective activating receptor (by introducing amino acid substitutions), antibodies with a greater ability to mediate the desired cytotoxic activity by a CD3-targeted binding arm for the treatment of anti-cancer mabs or autoimmune-related diseases can be produced. For example, a reported technique for enhancing the ADCC of an antibody is afucosylation. (See Junttila, TT, K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11):4481-4489). Thus, a bispecific antibody according to the present invention is further provided, which is afucosylated.Alternatively or additionally, several other strategies, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), have been reported to be used to achieve ADCC enhancement, all of which seek to improve Fc binding to low-affinity activated FcγRIIIa and / or reduce binding to low-affinity inhibitor FcγRIIb.

[0206] The antibody preferably contains a common light chain.

[0207] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence: CDR1:SFGIS CDR2:GFIPVLGTANYAQKFQG CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the array, X1 = K or R, Includes CDR1, CDR2, and CDR3 (where X2 = L or I).

[0208] In a preferred embodiment, X1 = K and X2 = L. In another preferred embodiment, X1 = R and X2 = I.

[0209] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence: CDR1:SKTLTIS, CDR2:GIIPIFGSITYAQKFQD, CDR3:RGNWNPFDP, or Amino acid sequence: CDR1: GSGIS, CDR2:GFIPFFGSANYAQKFRD, CDR3:RGNWNPX 13 DP (In the array, X 13 Includes CDR1, CDR2, and CDR3 (which are either L or F).

[0210] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Including EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0211] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence: CDR1: RX3WIG, CDR2:IIYPGDSDTRYSPSFQG, CDR3:X4IRYFX5WSEDYHYYX6DV (In the array, X3 = F or Y, X4 = H or N, X5 = D or V Includes CDR1, CDR2, and CDR3 (where X6 = L or M).

[0212] In one embodiment, 、 X3=F, X4=H, X5=D, and X6=L. In a further embodiment, X3=Y, X4=N, X5=V, and X6=M.

[0213] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Including EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0214] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence: CDR1: SYALS, CDR2:GISGSGRTTWYADSVKG, Includes CDR1, CDR2, and CDR3, which contain CDR3:DGGYSYGPYWYFDL.

[0215] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the antibody variable domain comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises an amino acid sequence: CDR1: SYALS, CDR2:AISGSGRTTWYADSVKG, Includes CDR1, CDR2, and CDR3, which include CDR3:DGGYTYGPYWYFDL.

[0216] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS includes, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0217] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence: CDR1:DYTMH, CDR2:DISWSSGSIGYADSVKG, Includes CDR1, CDR2, and CDR3, which contain CDR3:DHRGYGDYEGGGFDY.

[0218] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the antibody variable domain comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises an amino acid sequence: CDR1:DYTMH, CDR2:DISWSX7GX8X9X 10 YADSVKG, CDR3:DHX 11 GYGDYEGGGFDX 12 (In the array, X7 = S or G X8 = S or T, X9 = I or T, X 10 =G or Y, X 11 = R or M, X 12 =H or Y, Preferably, X7, X8, X9 and X 10 is S, S, I and G, or G, S, I and Y, or S, T, T and G, preferably X 11 and X 12 are R and H, or R and Y, or M and Y, more preferably X7, X8, X9, X 10 , X 11 and X 12These are S, S, I, G, R and H, or G, S, I, Y, R and Y, or S, T, T, G, M and Y, or, in other words, preferably X7, X8, X9 and X 10 are S, S, I and G, and X 11 and X 12 These are R and H, or X7, X8, X9 and X 10 are G, S, I and Y, and X 11 and X 12 These are R and Y, or X7, X8, X9 and X 10 are S, T, T and G, and X 11 and X 12 This includes CDR1, CDR2, and CDR3, which are M and Y.

[0219] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, which comprises a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, each of which comprises a different heavy chain variable region and a common light chain variable region, the heavy chain variable region of the variable domain that binds to human CD3 having an amino acid sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, or Including EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0220] In one embodiment, the heavy chain variable region of the variable domain that binds to the tumor antigen preferably comprises the amino acid sequence MF8233(EGFR)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQGTLVTVSS, At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0221] In another embodiment, the heavy chain variable region of the variable domain that binds to the tumor antigen is preferably, MF4327(CLEC12A) The amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVPOSTRDTSTSTVYMELSSLRSEDTAVYYCAKGTTGDWFDYWGQGTLVTVSS is included. At one or more positions other than CDR, there are 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof.

[0222] The present invention further provides antibodies or derivatives thereof, or pharmaceutical compositions thereof, for use in the treatment of subjects requiring such treatment. For the treatment of subjects with or at risk of developing tumors, the antibody is preferably a bispecific antibody of the present invention. Preferably, the CD3-conjugated antibody comprises a heavy / light chain combination that binds to a tumor antigen.

[0223] CD3 / tumor antigen bispecific antibodies and pharmaceutical compositions comprising such bispecific antibodies are provided for use in the treatment of solid tumors or hematological malignancies. Preferred solid tumors are epithelial in origin and include gynecological cancers such as ovarian tumors and endometrial tumors, prostate cancer, brain cancer, or any other solid tumor.

[0224] The present invention provides a CD3 / tumor antigen bispecific antibody or derivative thereof, or a pharmaceutical composition containing such a bispecific antibody or derivative thereof, for use in the treatment of various myeloid-origin leukemias and preleukemias, and in the treatment of B-cell lymphomas. Diseases that can be treated according to the present invention include myeloid leukemias or preleukemias such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), and Hodgkin lymphoma and most non-Hodgkin lymphomas. B-ALL, T-ALL, and mantle cell lymphoma are also targets for treatment with the antibodies of the present invention. Accordingly, the present invention provides a bispecific full-length IgG antibody according to the present invention for use as a pharmaceutical in the treatment of myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), multiple myeloma (MM), or preferably acute myeloid leukemia (AML). The use of the bispecific IgG antibody according to the present invention in the preparation of agents for the treatment or prevention of MDS, CML, MM, or preferably AML is also provided.

[0225] The amount of antibody according to the present invention to be administered to a patient is typically within the therapeutic window, meaning that a sufficient amount is used to obtain a therapeutic effect, but that amount does not exceed a threshold that would result in unacceptable side effects. The smaller the amount of antibody required to obtain the desired therapeutic effect, the larger the therapeutic window will typically be. Therefore, antibodies according to the present invention that exert a sufficient therapeutic effect at low doses are preferred.

[0226] Approximately 30,000 patients are diagnosed with AML annually in Europe and the United States. The majority of these patients are over 60 years of age. Advanced age is a major negative determinant of prognosis in AML, with a long-term survival rate (5 years) of approximately 10% for elderly AML patients treated intensively. Disease progression is observed within 3 years in almost all patients who achieve remission during induction chemotherapy. Current post-remission treatments have shown limited value, if any, in elderly patients with AML. Thus, a significant burden of residual resistant leukemia remains, and the surviving subpopulation of drug-resistant leukemia cells rapidly develops relapses. In efforts to induce and maintain complete remission, a novel class of drugs with entirely different modes of action is needed to target these chemotherapy-unresponsive AML tumor cells. While complete remission (CR) can be achieved in over 50% of elderly AML patients and approximately 80% of younger patients with a combination of several intensive chemotherapy regimens, increasing response or survival rates remains a major investigational challenge. A recently published network meta-analysis of 65 randomized clinical trials (15,110 patients) in elderly patients with AML showed that the majority of adjusted inductive regimens had an efficacy profile equivalent to or worse than the conventional 3+7 inductive regimen using daunorubicin and cytarabine. This standard treatment for AML is associated with high morbidity and even mortality. The majority of patients in complete remission (CR) relapse due to residual leukemic stem cells after chemotherapy. Further dose increases are limited due to unacceptable toxicity. Therefore, there is an urgent need for new treatment modes, preferably with less toxicity, particularly in elderly patients with AML.

[0227] Treatment of chemotherapy-ineffective AML can be achieved by redirecting T cells from the patient's own immune system to AML tumor cells, and then using bispecific antibodies to perform tumor-targeted activation of the T cells. This process is also known as the so-called "T cell engagement approach." In this way, the patient's immune system is strengthened and retargeted to attack and eradicate AML tumor cells. For example, the CD3xCLEC12A bispecific IgG antibody efficiently redirects T cells towards AML tumor cells, thereby inducing lysis of the AML tumor cells. [Brief explanation of the drawing]

[0228] [Figure 1]Evaluation of functional activity: This is a T cell cytotoxicity assay using BxPC3 target cells during treatment with EGFR×CD3 bispecific antibodies. Each bispecific antibody contains a CD3-binding domain including a heavy chain variable region specified by the MF number, and an EGFR-binding domain including the heavy chain variable region MF8233. These variable regions pair with a common light chain to form an EGFR×CD3 bispecific antibody. Affinity for BxPC3 lysis (HPB-ALL binding) is measured. The specific antibodies of the present invention show relatively low levels of binding to HPB-ALL cells, indicating that the CD3-binding domain of the antibody binds to human CD3 with relatively low affinity. It is clear that the relatively low affinity does not necessarily inhibit the cytotoxicity of tumor antigen-mediated T cells of BxPC3 cells (BxPC3 lysis, vertical axis). The bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently lyse BxPC3 cells, whereas the bispecific antibodies MF8233×MF8397 and MF8233×MF9249, while exhibiting similar binding, do not do so efficiently. Furthermore, in a comparison of bispecific antibodies, the bispecific antibody MF8233×MF6955 binds to HPB-ALL (i.e., human CD3) with higher affinity, but does not lyse BxPC3 as efficiently as the bispecific antibodies MF8233×MF8508 and MF8233×MF8057, which bind to CD3 to a relatively lower degree. MF6955 is a heavy chain variable region that combines with the common light chain and is used as a comparative reference sequence, corresponding to H1H7232B(1129)VH in U.S. Patent Application Publication 2014 / 0088295(A1). MF6955 and other bispecific antibodies containing the same EGFR-binding domain separately exhibit higher affinity for human CD3 and more efficient killing than MF9267. In contrast, other antibodies incorporating the CD3-binding domain of the present invention, such as MF8058, have nearly the same binding activity as MF6955, but demonstrate more efficient killing of BxPC3 cells, such as MF8233×MF8058. Other bispecific antibodies containing the CD3-binding domain of the present invention that can bind to CD3, such as MF8233×MF8078, exhibit relatively high binding and more efficient killing, which are useful for specific applications described herein.In contrast, other bispecific antibodies containing the binding domain of the present invention that can bind to CD3, such as MF8233×MF9249 and MF8233×MF8397, exhibit relatively low affinity and low toxicity, but are useful for the alternative applications described herein. [Figure 2] These are antibody titration curves showing the ability to induce T cell-mediated killing % of BxPC3 target cells compared to the case without an antibody control. Curves for antibodies MF8233×MF8078, MF8233×MF8397, and MF8233×MF8508 are shown. [Figure 3] This is a summary of titration curve data for various bispecific antibodies in T cell cytotoxicity by BxPC3-targeted cells. The CD3 Fab column shows the MF number of the CD3-binding arm. The EGFR arm has the indicated MF8233 number. This column shows the supercluster number for setting up variants based on the same VH gene segment. The column showing CD3 binding reflects the results of HBP-ALL binding experiments. Results of two independent cytotoxicity assays to determine the ability of BxPC3-targeted cells to induce T cell-mediated lysis are shown. [Figure 4] This describes T cell activation in a T cell cytotoxicity assay on BxPC3-targeted cells on CD8+ T cells due to expression. The antibody titration curves present various supercluster numbers exhibiting variants of the CD3-binding domain. The other arm of the bispecific antibody possesses the heavy chain-binding domain of MF8233. For comparison, bispecific antibodies MF8233×MF6955 and MF8233×MF6964 were also tested, with MF6955 and MF6964 being heavy chain variable regions combined with a common light chain and used as comparative reference sequences, corresponding to H1H7232B(1129)VH and HH7241B(1145) respectively in U.S. Patent Application Publication 2014 / 0088295(A1). [Figure 5]This is a summary of titration curve data for various antibodies in a cytotoxicity assay of T cell-activated T cells using BxPC3-targeted cells. The MF nr. column indicates the MF number of the CD3-binding domain. The EGFR-binding domain has the MF8233 number shown. Supercluster information for various CD3-binding domain sequences is shown in the "Supercluster" column. The column showing CD3 affinity reflects the results of HBP-ALL binding experiments. Results for CD4+ and CD8+ cells are shown for markers CD69 and CD25. The bispecific antibodies shown are examples from a larger pool of bispecific antibodies. [Figure 6] Evaluation of functional activity: This is a T cell cytotoxicity assay using BxPC3 target cells. The assay measures affinity (HPB-ALL binding) versus CD8+ T cell activation, as measured by CD69 expression. The specific antibodies of this invention exhibit relatively low levels of binding to HPB-ALL cells, indicating that the antibody's CD3-binding domain binds to human CD3 with relatively low affinity. Such affinity does not necessarily inhibit tumor antigen-mediated T cell activation, as exemplified by the results of CD8-positive CD69 activation analysis. The bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently activate T cells, whereas the bispecific antibodies MF8233×MF8397 and MF8233×MF9249 do not. Specific CD3-binding domains that do not efficiently bind to these cells also do not activate T cells (see lower left corner). Other CD3-binding domains, MF8508 and MF8057, bind less to HPB-ALL cells than the reference CD3-binding domain MF6955, and activate T cells to a similar degree, for example. Other bispecific antibodies containing the CD3-binding domain of the present invention, such as MF8078, exhibit relatively high binding and high levels of activation for use in specific applications described herein. In contrast, other bispecific antibodies containing the CD3-binding domain of the present invention, such as MF9249 and MF8397, exhibit relatively low affinity and low activation, but are useful for alternative applications described herein. [Figure 7]Functional Evaluation: This is a T-cell cytotoxicity assay using HCT116 target cells. The assay measures affinity for HCT-116 lysis (HPB-ALL binding). The specific bispecific antibodies of the present invention exhibit low levels of binding to HPB-ALL cells, indicating that the antibody's CD3-binding domain binds to human CD3 with relatively low affinity. It is clear that low affinity does not necessarily inhibit tumor antigen-mediated cytolysis of HCT-116 cells (vertical axis). Bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently lyse HCT-116 cells, whereas bispecific antibodies MF8233×MF8397 and MF8233×MF9249 do not lyse cells as efficiently. For comparison, the bispecific antibodies MF8233×MF6955 and MF8233×MF6964 bind to HPB-ALL (i.e., human CD3) with higher affinity than, for example, MF8233×MF8508, MF8233×MF8057, and MF8233×MF9267, but do not lyse HCT-116 cells more efficiently than MF8233×MF8508 or MF8233×MF9267, or significantly exceed MF8233×MF8057 in terms of binding difference, as presented herein. Other bispecific antibodies containing the binding domain of the present invention capable of binding to CD3, such as MF8078 for use in specific applications described herein, exhibit relatively high binding and high levels of killing. In contrast, other bispecific antibodies containing the binding domain of the present invention capable of binding to CD3, such as MF9249 and MF8397, exhibit relatively low affinity and low killing, but are useful for alternative applications described herein. [Figure 8] This is an antibody titration curve in a T-cell cytotoxicity assay using HCT-116 target cells, showing the percentage of HCT-116 cells killed compared to a control without antibody. Curves for various bispecific antibodies are shown. [Figure 9]This is a summary of titration curve data for various antibodies in a T cell cytotoxicity assay using HCT-116 target cells. The CD3 Fab column shows the MF number of the CD3-binding domain. The EGFR-binding domain has the MF number 8233 shown. This column shows the number of superclusters for setting up variants based on the same VH gene segment. The column showing CD3 binding reflects the results of HBP-ALL binding experiments. The lysis rate of HCT-116 cells and the EC50 value (ng / mL) for lysis are shown in the next column. The bispecific antibodies shown are examples from a larger pool of bispecific antibodies. [Figure 10] Figures 10A and 10B show schematic diagrams of the MV1624 expression vector and the MV1625 expression vector, respectively. [Figure 11A] This is the common light chain used in single-specificity and bispecificity IgG. It is the common light chain amino acid sequence. [Figure 11B] This is the common light chain used in single-specific and bispecific IgG. The DNA sequence and translated version of the common light chain variable domain are shown (IGKV1-39 / jk1). [Figure 11C] This is the common light chain used in single-specific and bispecific IgG. It includes the DNA sequence and translated version of the common light chain constant region. [Figure 11D] This is a common light chain used in monospecific and bispecific IgG. It is a translation of the IGKV1-39 / jk5 common light chain variable domain. [Figure 11E] This is the common light chain used in monospecific and bispecific IgG. It is the V region IGKV1-39A. [Figure 11F] These are the common light chains used in monospecific and bispecific IgG. These are the common light chains CDR1, CDR2, and CDR3. [Figure 12A] This is the IgG heavy chain for the generation of bispecific molecules. It is the CH1 region. [Figure 12B] This is the IgG heavy chain for the generation of bispecific molecules. It is the hinge region. [Figure 12C] This is the IgG heavy chain for generating bispecific molecules. It is the CH2 region. [Figure 12D] This is an IgG heavy chain for the generation of bispecific molecules. It is a CH2 containing L235G and G236R silencing substitutions. [Figure 12E] This is an IgG heavy chain for the generation of bispecific molecules. It is a CH3 domain containing substituted L351K and T366K(KK). [Figure 12F] This is an IgG heavy chain for the generation of bispecific molecules. It is a CH3 domain containing substituted L351D and L368E(DE). [Figure 13-1] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-2] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-3] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-4] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-5] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-6] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-7] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-8] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-9] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-10] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-11]These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-12] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-13] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-14] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-15] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-16] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-17] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-18] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-19] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-20] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-21] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-22] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-23] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 13-24] These are various DNA coding sequences and amino acid sequences of the heavy chain variable region and its portions as described herein. [Figure 14A] This involves further characterization of clones from Supercluster 1 compared to clones MF8057 and MF8058. It also includes the binding of selected MF clones to HPB-ALL human cells expressing the human CD3-TCR complex in a FACS assay. [Figure 14B] This is a further characterization of clones from supercluster 1 compared to clones MF8057 and MF8058. It is an HCT-116 cell-mediated T cell cytotoxicity assay showing the percentage of HCT-116 cell killing. [Figure 14C] This involves further characterization of clones from Supercluster 1 compared to clones MF8057 and MF8058. It also includes quantification of the activation markers CD25 and CD69 in FACS, which indicate T cell activation. [Figure 14D] This involves further characterization of clones from Supercluster 1 compared to clones MF8057 and MF8058. It also includes quantification of the activation markers CD25 and CD69 in FACS, which indicate T cell activation. [Figure 14E] This involves further characterization of clones from Supercluster 1 compared to clones MF8057 and MF8058. It also includes quantification of the activation markers CD25 and CD69 in FACS, which indicate T cell activation. [Figure 14F] Further characterization of clones from Supercluster 1 compared to clones MF8057 and MF8058. Cytokine production in the supernatant from cytotoxic assays. [Figure 14G] Further characterization of clones from Supercluster 1 compared to clones MF8057 and MF8058. Cytokine production in the supernatant from cytotoxic assays. [Figure 15A] This involves characterizing clones from Supercluster 4. It also involves the binding of selected MF clones to HPB-ALL human cells. [Figure 15B]This is a characterization of clones from Supercluster 4. It is a T-cell cytotoxicity assay using BxPC3 cells, showing the percentage of BXP3 cells killed. [Figure 15C] This involves characterization of clones from Supercluster 4. It also includes cytokine production in the supernatant from cytotoxic assays. [Figure 15D] This involves characterization of clones from Supercluster 4. It also includes cytokine production in the supernatant from cytotoxic assays. [Figure 15E] This involves characterization of clones from Supercluster 4. It also includes cytokine production in the supernatant from cytotoxic assays. [Figure 16A] This is an evaluation of CD3 functional activity. It is the X-axis affinity (HPB-ALL) for HCT-116 lysis on the Y-axis for further cloning from supercluster 1 (MF8048, MF8101, MF8056), supercluster 3 (MF8562), and supercluster 4 (MF8998). [Figure 16B] This is an evaluation of CD3 functional activity. The antibodies belong to Supercluster 1 and Supercluster 4, which exhibit similar activity in cytotoxic assays and at different binding affinities. [Figure 16C] This is an evaluation of CD3 functional activity. The antibodies belong to Supercluster 1 and Supercluster 3, exhibiting similar binding affinity and differential lysis activity. [Figure 17] This describes the activity of CD3 Fab MF8998 and MF8058 in the bispecific CD3×EGFR format. [Figure 18] This is FACS binding data for a large panel of CD3-specific IgG. Binding to the CD3δε-Fc antigen was determined for antibodies MF5196, MF6955, and MF6964 using BIAcore™, while FACS binding data for HPB-ALL cells is shown for the rest of the clone. [Figure 19-1] This is the nucleotide sequence of human CLEC12A. [Figure 19-2]This is the nucleotide sequence of human CLEC12A. [Figure 19-3] This is the nucleotide sequence of human CLEC12A. [Figure 19-4] This is the nucleotide sequence of human CLEC12A. [Figure 19-5] This is the nucleotide sequence of human CLEC12A. [Figure 19-6] This is the nucleotide sequence of human CLEC12A. [Figure 19-7] This is the nucleotide sequence of human CLEC12A. [Figure 19-8] This is the nucleotide sequence of human CLEC12A. [Figure 19-9] This is the nucleotide sequence of human CLEC12A. [Figure 19-10] This is the nucleotide sequence of human CLEC12A. [Figure 19-11] This is the nucleotide sequence of human CLEC12A. [Figure 19-12] This is the nucleotide sequence of human CLEC12A. [Figure 20] These are the amino acid sequences of the human CD3γ-,δ-,ε-, andζ-chains.

[0229] The following examples illustrate the present invention. [Examples]

[0230] cell line BxPC3 is a human pancreatic cancer cell line.

[0231] HCT-116 is a human colon cancer cell line.

[0232] CD3-mediated immunization of Memo(registered trademark) mice To produce human antibodies that bind to CD3, transgenic mice of the human common light chain and human heavy chain (HC) minilocules (including selected human V gene segments, all human D and all human J) (see International Publication No. 2009 / 157771 incorporated herein by reference) were immunized with TCR / CD3-containing lipoparticles (Intergral Molecular). These mice are referred to as "MeMo®" mice. Specific heavy chain variable regions or trivalent polymers having the sequences disclosed herein can be prepared by any means known to those skilled in the art.

[0233] MeMo® mice were immunized with human 5D5M TCR / CD3 derived from Hek293T containing lipoparticles, and subsequently immunized with human T cells to generate an anti-TCR / CD3 immune response and an anti-TCR / CD3 antibody panel.

[0234] Lipoparticles allow for the direct enrichment of structurally intact membrane proteins from the cell surface, enabling the manipulation of these complex proteins as soluble, high-concentration proteins for antibody immunization and screening.

[0235] The lipoparticles used for immunization in this study contain the 5D5M TCRαβ combination. A vector containing the 5D5M TCRαβ combination was synthesized and cloned, and used to generate lipoparticles containing this TCR / CD3 combination by transient transfection into HEK293T cells (Intergral Molecular).

[0236] 5D5M TCRα MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVMDSNYQLIWGAGTKLIIKPDIQNPDP AVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 5D5M TCRβ MRIRLLCCVAFSLLWAGPVIAGITQAPTSQILAAGRRMTLRCTQDMRHNAMYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDGYSVSRANTDDFPLTLASAVPSQTSVYFCASSEAGGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEIS HTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0237] MeMo(registered trademark) mice were used for immunization with TCR / CD3 lipoparticles and primary human T cells.

[0238] The immunization schedule included time points at days 35, 56, 77, and 98. Antigen-specific Ig serum titers were determined by ELISA using QTG-derived 3SDX TCR / CD3-positive and negative lipoparticles with anti-mouse IgG detection, and by ELISA using CD3c5E-Fc fusion protein as a positive control. Reactivity was observed in serum collected on day 35 to determine which mice expressed the relevant anti-TCR / CD3 response.

[0239] Lymphatic tissue for antibody discovery was collected from all immunized mice and stored as follows: The titer is either 1 / 300 in human TCR / CD3 (in ELISA using lipo particles), or The titers were less than 1 / 300 and greater than 1 / 100 in human TCR / CD3, and did not increase during the final booster immunization.

[0240] Primary immunization using lipoparticles (Priming immunization) To prime the humoral immune response to TCR / CD3 in MeMo® mice, lipoparticles containing the human 5D5M TCRαβ combination were used for immunization. The lipoparticles were used with first and second injection Gerbu adjuvants.

[0241] Booster immunization using polyclonal T cells Mice were immunized by subcutaneous injection of a cell suspension. The first booster immunization (day 28) contained a mixture of cells and an adjuvant in PBS, and all subsequent injections consisted solely of cells in PBS. Mice expressing a serum IgG titer of 1 / 300 against human TCR / CD3 (measured by ELISA using lipoparticles) on day 35 received additional cell injections on days 42, 43, and 44. Mice that failed to meet these criteria received booster immunization with cells (days 42 and 49). All subsequent immunizations were administered as subcutaneous injections of cells in PBS. After final immunization, the mice were sacrificed and serum, spleen, and left inguinal lymph node were collected.

[0242] Screening of serum from immunized mice during ELISA Provisional serum IgG titers were screened using ELISA with TCR / CD3-containing lipoparticles and "null" lipoparticles. Serum IgG titers were measured using anti-mouse IgG staining because this staining method has been shown to be the most sensitive.

[0243] Generation of an "immune" phage antibody repertoire by RT-PCR cloning of the VH gene Inguinal lymph nodes from successfully immunized mice were used to construct a repertoire of "immune" phage antibodies. RNA was extracted from lymphoid tissue using Trizol LS, and 1 μg of total RNA was used in an RT reaction with IgG-CH1 specific primers. The resulting cDNA was then used to amplify a polyclonal pool of VH-coding cDNA using our own developed VH-specific primers, essentially as described in Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581-97). To present the Fab fragment on the phage, the resulting PCR products were then cloned in a phagemide vector, as described in de Haard et al. (J Biol Chem. 1999 Jun 25;274(26):18218-30), except that the light chain was the same for all antibodies and encoded by the vector. After ligation, E. coli TG1 bacteria were transformed using phagemid, and the transformed bacteria were seeded onto LB-agar plates containing ampicillin and glucose. All phage libraries contained >10e6 transformants and had an insert frequency of >80%. After overnight growth, the bacteria were collected and used to prepare phages according to an established protocol (de Haard et al., J Biol Chem. 1999 Jun 25;274(26):18218-30).

[0244] Selection of phages possessing Fab fragments that specifically bind to human CD3. Phage libraries were rescued according to a standardized procedure (J Mol Biol, 1991 Dec 5;222(3):581-97; J Biol Chem. 1999 Jun 25;274(26):18218-30), and phages were selected in one or more rounds of selection of an immunophage antibody repertoire. In the first round, recombinant CD3 protein was coated onto wells of maxisorp® ELISA plates or NUNC immunotubes, while in the second round, either recombinant CD3 protein or cells overexpressing human CD3 protein were used. The maxisorp® ELISA plates or immunotubes were blocked with 4% ELK. The phage antibody libraries were also blocked with 4% ELK, and human IgG was overused to deplete the Fc region binder before adding the phage libraries to the coated antigens.

[0245] The phage library and coated proteins were incubated at room temperature for 2 hours under shaking conditions. The plates or tubes were then washed with 0.05% Tween-20 in PBS, followed by 5-10 washes with PBS. The bound phages were eluted using 50 mM glycine (pH 2.2), added to E. coli TG-1, and incubated at 37°C for phage infection.

[0246] Next, infected bacteria were seeded onto agar plates containing ampicillin and glucose and incubated overnight at 37°C. After the first round of selection, colonies were scraped from the plates, combined, rescued, and amplified to prepare an enriched first-round phage pool for the synthetic repertoire. For the "immune" repertoire, single clones were screened for target binding after the first round of phage selection.

[0247] Antibody cloning and production The bispecific antibodies used herein typically differ from each other only in specific amino acid sequences of one or both of the heavy chain variable regions of the variable domains. The heavy chain variable regions were prepared by cloning them into expression vectors for the expression of the heavy and light chains. Methods for producing bispecific antibodies are known in the art.

[0248] In short, the DNA encoding the heavy chain variable region of the CD3-targeting variable domain was cloned into the MV1624 vector (see Figure 10A) encoding KK residues (L351K, T366K) in the CH3 region for the generation of IgG heavy chain heterodimers (International Publication Nos. 2013 / 157954 and 2013 / 157953). The Fc constant region contains a mutation in CH2 to silence the Fc effector function. The DNA encoding the heavy chain variable region replaces the stuffer region in the construct. Prior to the variable region is the encoded HC signal peptide (not shown). The DNA encoding the heavy chain variable region of the EGFR-targeting variable domain was cloned into vector MV1625 (Figure 10B), which expresses the second double chain of the base antibody portion of a bispecific antibody having the L351D-L368E mutation in the CH3 region (International Publication Nos. 2013 / 157954 and 2013 / 157953). The DNA encoding the heavy chain variable region replaces the stuffer region in the construct. Prior to the variable region is the encoded HC signal peptide (not shown). Both constructs also contain an expression cassette for the expression of the IGKV1-39 / jk1 light chain. Expression of the two heavy chains together with the light chain described above results in the production of a bispecific antibody.

[0249] 293-F cells were used for the expression of the designed antibody in a 24-well plate format. Two days before transfection, the 293-F cell stock was divided in 1:1 ratio with 293-F culture medium and incubated overnight at 37°C and 8% CO2 with an orbital shaking speed of 155 rpm. The cells were divided into 5 × 10⁶ cells the day before transfection. 5The cells were diluted to a density of cells / mL. 4 mL of the cell suspension was seeded into a 24-deep-well plate covered with a permeable seal and incubated overnight at 37°C and 8% CO2 with an orbital shaking rate of 285 rpm. On the day of transfection, 4.8 mL of 293-F culture medium was mixed with 240 μg of polyethyleneimine (PEI) linear (MW25,000). For each IgG produced, 200 μL of the 293F culture medium-PEI mixture was added to 8 μL of DNA (4 μL of DNA encoding each heavy chain in the case of an IgG heterodimer). The mixture was incubated at room temperature for 20 minutes before gently adding it to the cells. The day after transfection, penicillin-streptomycin (Pen Strep) diluted in 500 μL of 293F medium was added to each well. The plates were incubated at 37°C and 8% CO2 at an orbital shaking rate of 285 rpm until retrieved 7 days after transfection. The plates were centrifuged for 5 minutes in 500 g of supernatant containing IgG, filtered through a 10-12 μm meltblown polypropylene filter plate, and stored at -20°C before purification.

[0250] Purification of antibodies from culture supernatant The culture medium containing the antibody is collected and centrifuged to remove cell debris. Next, Protein A Sepharose beads are added to the culture medium. The culture medium and Protein A Sepharose beads are incubated with the antibody to bind to them.

[0251] After incubation, the beads are isolated from the culture medium and washed with a vacuum filter. Antibodies are eluted from the beads by incubation with elution buffer.

[0252] Optionally, replace / desalt the buffer solution of the purified IgG.

[0253] buffer exchange To desalt the purified antibody, the antibody fraction is centrifuged using a filter plate or filter column. The plate or column is centrifuged to reduce the volume of the antibody fraction. Subsequently, PBS or the required buffer is added to the fraction to replace the buffer with the low-salt buffer. Optionally, this centrifugation step followed by the addition of buffer can be repeated to further desalt the antibody storage buffer.

[0254] Antibody tumor antigen-specific T cell activation and lysis of BxPC3 cells or HTC-116 cells. In cytotoxic assays, the ability of specific CD3 × tumor antigen bispecific IgG combinations to induce tumor antigen-specific T cell activation and lysis of tumor antigen-positive target cells was tested. Effector cells were resting T cells derived from healthy donors, and target cells were BxPC3 cells or HTC-116 cells.

[0255] Using Ficoll and EasySep human T cell isolation kits according to standard procedures, quiescent T cells were isolated from whole blood of healthy donors, and T cell purity of over 95% was confirmed with anti-CD3 antibody using flow cytometry analysis, followed by cryopreservation. For cytotoxicity assays, cryopreserved T cells were thawed and used if their viability after thawing, as measured by standard trypan blue staining, exceeded 90%. In short, the cytotoxicity assay involved co-culturing thawed quiescent T cells and BxPC3 or HCT116 target cells at a 5:1 E:T ratio for 48 hours. Antibodies were tested within a dilution range. CD3 monospecific antibodies and EGFR monospecific antibodies, as well as unrelated IgG1 isotype control mAbs, were included in the assay as controls (e.g., antibodies that bind to CD3 and another antigen such as tetanus toxin (TT)). T cell activation was quantified using flow cytometry; CD8 T cells were gated based on CD8 expression, and their activation status was analyzed by subsequently measuring CD69 expression on the T cells. Target cell lysis was determined by measuring the percentage of viable cells by measuring ATP levels assessed by CellTiterGlo (Promega). ATP levels, measured by luminescence using an Envision microplate reader, yielded relative luminescence (RLU) values, which were analyzed using GraphPad Prism.

[0256] The target cell solubility for each sample was calculated as follows: % lethality = (100 - (RLU sample / RLU without IgG) × 100).

[0257] In this assay, the bispecific antibody has two binding domains. One of the binding domains targets EGFR, and the other targets CD3. Both binding domains have the same (common) light chain variable region (VL) and different heavy chain variable regions (VH). The EGFR-targeting binding domain has a VH with the amino acid sequence MF8233. The CD3-targeting binding domain has a VH with the amino acid sequence of one of the MFs shown for CD3. The bispecific antibody contains a mutation within CH2 to silence the Fc effector function.

[0258] The antibody MF8233×MF8397 induced upregulation of CD69 (Figures 4-6) and CD25 (Figures 4-6) to CD4 and CD8 T cells, as determined after 48 hours of co-culture at a 5:1 E±T ratio. T cell-mediated lysis was measured after 48 hours.

[0259] Characterization of CD3 bispecific antibodies Candidate EGFR / CD3 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed CD3 on HPB-ALL cells (DSMZ, ACC483) can be evaluated by flow cytometry (following the FACS procedure previously described in International Publication No. 2014 / 051433). In one embodiment, binding of a candidate EGFR / CD3 bispecific antibody to CD3 on HPB ALL cells is demonstrated by flow cytometry performed according to a standard procedure known in the art. Binding to cell-expressed CD3 can be confirmed using CHO cells transfected with CD3δ / ε or CD3γ / ε. The binding of candidate bispecific IgG1 to EGFR can be determined using CHO cells transfected with BxPC3 and HCT-116, as well as an EGFR expression construct. CD3 monospecific antibodies and EGFR monospecific antibodies, as well as unrelated IgG1 isotype control mAbs, are included in the assay as controls (e.g., antibodies that bind to CD3 and another antigen such as tetanus toxin (TT)).

[0260] Further clone generation from superclusters 1, 3, and 4 From immunophage library screening (as described in the section "Selection of phages possessing Fab fragments that specifically bind to human CD3"), additional clones were characterized by possessing Fab fragments that specifically bind to human CD3. Further clones were identified from Supercluster 1, including MF8048, MF8101, and MF8056. Additional clones were identified from Superclusters 3 and 4, including MF8562 from Supercluster 3 and MF8998 from Supercluster 4.

[0261] Further novel clones were identified from Supercluster 4 using next-generation sequencing (NGS) analysis. NGS was performed on the VH gene pool present in MeMo® mice used to generate the anti-CD3 panel. For this purpose, sequence datasets obtained from different mice were compared with MF sequences belonging to MF in Supercluster 4. This led to the identification of sequence variant clones MF10401 and MF10428 belonging to Supercluster 4. Regarding the different sequences, several distinct mutations were found in HCDR1 and HCDR2.

[0262] The VH sequences of all additional clones from superclusters 1, 3, and 4 were cloned into the MV1624(DM-KK) vector and expressed in a CD3×EGFR bispecific format for further characterization, as described in the section "Antibody Cloning and Production" above.

[0263] Further characterization of clones from Superclusters 1 and 4 Further clones from Supercluster 1 were characterized for their functional activity in a bispecific format. The EGFR-binding domain of the bispecific CD3×EGFR antibodies has the amino acid sequence encoded by MF8233. As a control, these CD3 clones were also tested with another antigen (e.g., tetanus toxin) having the amino acid sequence encoded by MF1337. Reference MFs from Supercluster 1 (MF8057 and MF8058) were included to directly compare the affinity of the sequence variants with that of already characterized MF clones from Supercluster 1, according to the "Antibody tumor antigen-specific T cell activation and lysis of BxPC3 cells or HTC-116 cells" and "Characterization of CD3 bispecific antibodies" described above. Binding affinity to HPB-All cells expressing the human CD3-TCR complex was assessed using flow cytometry (Figures 14A and 18), and assays were performed for T cell activation and lysis of tumor antigen-positive target cells (HCT-116) in cytotoxic assays (Figures 14B-E). No target cell lysis was observed with the bispecific antibody with the MF1337 control arm. Target cell lysis was observed in a dose-dependent manner for the different CD3 clones tested. Low target cell lysis was observed for MF8048. Expression levels of the activation markers CD69 and CD25 on CD4 and CD8 T cells were measured by FACS staining to assess T cell activation. Dose-dependent T cell activation was observed for all clones, and no T cell activation was observed in the negative control. Finally, cytokine production of IFN-γ and TNF-α was measured in the supernatant derived from the EGFR × CD3 cytotoxicity assay using HCT-116 cells after 48 hours using the Luminex® assay (eBiosciences®) according to standard manufacturer instructions (Figure 14F-G).

[0264] To further characterize clones belonging to Supercluster 4, binding affinity to HPB-ALL cells was determined by FACS (Figure 15). A monovalent antibody with two identical MF1337 arms specific to PG1337 and tetanus toxin was used as a negative control. For cytotoxicity assays, the activity of MF8998 was tested using HCT-116 and BxPC3 cells as target cells, and the activity of MF10401 and MF10428 was tested using BxPC3 target cells. A CD3×TAA bispecific antibody with known high activity was included as a positive control. Target cell lysis was quantified using cell viability measurements. Cytokine levels of IL-6, IFN-γ, and TNF-α were measured using the Luminex® assay with the supernatant from the cytotoxicity assays.

[0265] Therefore, it was found that the three supercluster 4 clones tested exhibited different binding but similar lytic activity. Although the lytic activity of these clones was similar, a reduction in cytokine production was observed. [Table 1] [Table 2] [Table 3]

[0266] All further identified MFs were observed to be functional, as assessed by cytotoxic assays. Next, graphs were plotted between lysis on the Y-axis and binding affinity on the X-axis (Figure 16A) to understand the intra- and overall relationships between lysis and affinity in superclusters. Overall, a diverse panel of anti-CD3 Fabs consisting of multiple superclusters was generated, covering a wide range of affinities. Interestingly, clones exhibiting similar activity but different CD3 binding were identified in superclusters 1 and 4 (Figure 16B). Comparison of superclusters 1 and 3 revealed clones exhibiting similar CD3 binding and different activity (Figure 16C). [Table 4]

[0267] Characterization of CD3 antigen As described above, two clones from supercluster 1 and supercluster 4, namely MF8058 and MF8998, respectively, were found to have similar lytic activity in a bispecific format with clone MF8233 as a Fab arm binding to EGFR as a tumor cell antigen (Figure 17). In this experiment, lytic activity against HCT-116 cells was measured as described herein. MF9257×MF8233 was used as a positive control and MF9257×MF1337 as a negative control. As can be seen from Figure 17 and Table 5, dose-dependent high killing rates were observed for multiple test bispecific antibodies. [Table 5]

[0268] binding affinity As described in the section “Characterization of CD3 Bispecific Antibodies,” the binding affinity of additional CD3 clones was analyzed by FACS on HPB-ALL cells expressing human CD3. The affinity of MF6955 and MF6964 to CD3 was measured by surface plasmon resonance (SPR) technique using BIAcore® T100. Anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat. Nr. 555784) was conjugated to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). CD3×TAA bispecific antibody was then captured on this sensor surface. Recombinant purified human CD3δε-Fc antigen was then flowed onto the sensor surface in concentration ranges for measuring on and off rates. After each cycle, the sensor surface was regenerated by a pulse of HCl to recapture the CD3×TAA bispecific antibody. From the resulting sensorgrams, the on and off rates were determined using BIA evaluation software. Figure 18 illustrates the binding affinity ranges of the generated CD3 panel.

Claims

1. An antigen-binding protein that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: SFGIS, CDR2: GFIPVLGTANYAQKFQG, CDR3: RGNWNPFDP, or Amino acid sequence: CDR1: SKTFTIS, CDR2:GIIPLFGTITYAQKFQG, CDR3: RGNWNPFDP, or CDR1: SRTFTIS, CDR2: SIIPIFGTITYAQKFQG, CDR3: RGNWNPFDP Includes CDR1, CDR2 and CDR3, and, The aforementioned light chain variable region has the following amino acid sequence: CDR1: QSISSY, CDR2: AAS, CDR3:QQSYSTP Antigen-binding proteins including CDR1, CDR2, and CDR3.

2. An antigen-binding protein that binds to human CD3, comprising an antibody-variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following amino acid sequence: CDR1: SKTLTIS, CDR2: GIIPIFGSITYAQKFQD, CDR3: RGNWNPFDP Includes CDR1, CDR2 and CDR3, and, The aforementioned light chain variable region has the following amino acid sequence: CDR1: QSISSY, CDR2: AAS, CDR3:QQSYSTP Antigen-binding proteins including CDR1, CDR2, and CDR3.

3. The antibody comprises a variable domain including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is an amino acid sequence EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFRSFGISWVRQAPGQGLEWMGGGFIPVLGTTANYAQKFQGRVTIIADKSTNTAYMELSSLRRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSVKVSSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITITADKSTNTAFFMELSSLRSEDTAMYYYCTRRGNWNPFDPWGQGTLVTVSS, or EVQLVQSGSELKKPGSSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITATADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS includes, At one or more positions other than the CDR, there are insertions, deletions, substitutions, additions, or combinations thereof of 0 to 10 amino acids, and The light chain variable region is an amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK, or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL Including EIK, The antigen-binding protein according to claim 1 or 2, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than the CDR.

4. The heavy chain variable region is an amino acid sequence EVQLVQSGAEVKKPGSSSVKVSCKASGGGTFRSFGISWVRQAPGQGLEWMGGGFIPVLGTTANYAQKFQGRVTIIADKSTNTAYMELSSLRRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSVKVSSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITITADKSTNTAFFMELSSLRSEDTAMYYYCTRRGNWNPFDPWGQGTLVTVSS, or EVQLVQSGSELKKPGSSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITATADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS includes, At one or more positions other than the CDR, there are insertions, deletions, substitutions, additions, or combinations thereof of 0 to 5 amino acids, and The light chain variable region is an amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK, or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL Including EIK, The antigen-binding protein according to claim 1 or 2, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than the CDR.

5. An antigen-binding protein according to any one of claims 1 to 4, which is an antibody.

6. An antigen-binding protein according to any one of claims 1 to 4, which is a bispecific antibody.

7. The antigen-binding protein according to claim 6, wherein the bispecific antibody comprises the H / L chain combination described in any one of claims 1 to 4 and the H / L chain combination that binds to a tumor antigen.

8. The antigen-binding protein according to claim 7, wherein the H / L chain combination that binds to the tumor antigen binds to human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA protein, or a variant thereof.

9. The antigen-binding protein according to any one of claims 5 to 8, wherein the antibody is human or a humanized antibody.

10. The antigen-binding protein according to any one of claims 6 to 9, wherein the bispecific antibody comprises two different immunoglobulin heavy chains having compatible heterodimerization domains.

11. The antigen-binding protein according to claim 10, wherein the compatible heterodimer domain is a compatible immunoglobulin heavy chain CH3 heterodimer domain.

12. The antigen-binding protein according to any one of claims 6 to 11, wherein the bispecific antibody is an IgG antibody and has a mutated CH2 and / or lower hinge domain such that the interaction between the bispecific IgG antibody and the Fc gamma receptor is reduced.

13. The antigen-binding protein according to claim 12, wherein the mutated CH2 and / or lower hinge domain includes an amino acid substitution at position 235 and / or position 236 (according to EU numbering).

14. The antigen-binding protein according to claim 13, wherein the mutated CH2 and / or lower hinge domain comprises an L235G substitution and / or a G236R substitution.

15. The antigen-binding protein according to any one of claims 6 to 14, wherein the bispecific antibody comprises a common light chain.

16. An antigen-binding protein according to any one of claims 1 to 15, for use in the treatment of a subject requiring such treatment.

17. The antigen-binding protein according to claim 16, wherein the subject has cancer or is being treated for cancer.

18. The antigen-binding protein according to claim 16 or 17, wherein the treatment comprises local administration and / or local release of the antigen-binding protein according to claim 1 or 2.

Images