CD3 binding molecules

Abstract

The problem to be solved by the present invention is to provide an antibody, preferably a bispecific or multispecific antibody, for the treatment of tumors.SOLUTION: The present invention relates to antigen-binding proteins that bind to human CD3, wherein the heavy chain variable region comprises the following amino acid sequences: CDR1: SFGISCDR2: GFIPVLGTANYAQKFQGCDR3: RGNWNPFDP or the amino acid sequences: CDR1: SX1TFTIS, CDR2: GIIPX2FGTITYAQKFQG, CDR3: RGNWNPFDP, wherein = K or R and = L or I. The present invention relates to antigen-binding proteins that bind to human, wherein the heavy chain variable region comprises the following amino acid sequences:::: RGNWNPFDP or the amino acid sequences: = K or R and = L or I. The present invention relates to antigen-binding proteins that bind to human. X1 CDR3 X2 CDR2 CDR1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies, in particular to the field of therapeutic antibodies. The antibodies can be used for human therapy. More specifically, the present invention relates to antibodies, preferably bispecific or multispecific antibodies, for the treatment of tumors. [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, to treat transplant rejection due to their immunosuppressive properties. Antibodies that are bispecific for CD3 on T cells and for surface target antigens on cancer cells can link any type of T cell to cancer cells, regardless of T cell receptor specificity, costimulation, or peptide antigen presentation. Such bispecific T cell-engaging antibodies show great promise in the treatment of various cancers and tumor growths.

[0003] It is an object of the present invention to provide new antibodies having essentially, but not necessarily completely, CD3 binding properties with improved properties, with relatively low affinity accompanied by higher cytotoxicity, which are suitable for immuno-oncology applications for engaging T cells and effector cells, and conversely, new antibodies having relatively high affinity CD3 binding properties accompanied by lower cytotoxicity, which are suitable for autoimmune applications for downregulating T cells and effector cells.A further object of the present invention is to provide T cell-engaging CD3 binding proteins and antibodies with the above properties, which bind to at least one additional membrane-bound molecule. Summary of the Invention

[0004] The present invention provides an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SFGIS CDR2:GFIPVLGTANYAQKFQG CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the sequence, X1=K or R, 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence: CDR1:SKTLTIS, CDR2:GIIPIFGSITYAQKFQD, CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:GSGIS, CDR2:GFIPFFGSANYAQKFRD, CDR3:RGNWNPX 13 DP (In the sequence, X 13 =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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Contains EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0007] Further provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 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).

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

[0009] The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Includes EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0010] Further provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SYALS, CDR2:GISGSGRTTWYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DGGYSYGPYWYFDL.

[0011] Further provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SYALS, CDR2:AISGSGRTTWYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DGGYTYGPYWYFDL.

[0012] Further provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or Contains QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0013] Also provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:DYTMH, CDR2:DISWSSGSIGYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DHRGYGDYEGGGFDY.

[0014] Also provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 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).

[0015] In one embodiment, X7, X8, X9 and X 10 are S, S, I and G, and X 11 and X 12 are R and H. In another embodiment, X, X, X and X 10 are G, S, I and Y, and X 11 and X 12 are R and Y. In another embodiment, X, X, X and X 10 are S, T, T and G, and X 11 and X 12 are M and Y.

[0016] Further provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, or EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, or EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, or Contains EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0017] The light chain variable region in the antigen-binding protein, preferably an antibody, of the present invention preferably comprises a consensus light chain variable region. The consensus light chain variable region preferably comprises an IgVκ1-39 light chain variable region. The light chain variable region preferably comprises a germline IgVκ1-39 * The light chain variable region is the kappa light chain IgVκ1-39. * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01. In one embodiment, the light chain variable region comprises the human germline kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39* 01 / IGJκ5 * 01. The light chain variable region preferably comprises the amino acid sequence Includes 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 a combination thereof.

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

[0019] The antibody preferably comprises a heavy / light chain combination that binds to human CD3 as described herein and a heavy / light chain combination that binds to a tumor antigen. The heavy / light chain combination that binds to a 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 antibodies, bispecific antibodies or multispecific antibodies of the invention are preferably human or humanized antibodies.

[0021] Bispecific or multispecific antibodies preferably comprise two different immunoglobulin heavy chains with compatible heterodimerization domains, which are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains.

[0022] Bispecific or multispecific antibodies are IgG antibodies with mutated CH2 and / or lower hinge domains such that the interaction of the bispecific or multispecific IgG antibody with Fc gamma receptors is reduced. The mutated CH2 and / or lower hinge domain preferably comprises 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 or multispecific antibodies preferably comprise a common light chain.

[0024] The present invention further provides an antigen-binding protein or antibody as provided herein for use in treating a subject in need thereof. The subject preferably has cancer or is 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 0-10 amino acid substitutions, mutations, insertions, additions, or deletions, are preferred for treatment, particularly for treatments involving local administration and / or local release of the antigen-binding protein or antibody. Antigen-binding proteins or antibodies having the CDR and / or VH sequences of MF9249, MF9267, or MF8397, or variants thereof having 0-10 amino acid substitutions, mutations, insertions, additions, or deletions, are preferred for treatment of subjects with an overactive immune system, such as an autoimmune disease.

[0025] The antibodies of the present invention are preferably bispecific antibodies unless otherwise specifically specified. Bispecific antibodies preferably bind to at least human CD3. Additionally, bispecific antibodies preferably bind to at least a surface molecule preferentially expressed on human tumor cells. In a preferred embodiment, the bispecific 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 a more preferred embodiment, the bispecific antibody binds to EGFR or CLEC12A. In a more preferred embodiment, the multispecific antibody binds to EGFR and PD-L1.

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

[0027] There is further provided an antibody according to the invention further comprising a label, preferably a label for in vivo imaging.

[0028] The present invention also provides a method for treating a subject 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. Also provided is a bispecific or multispecific antibody according to the present invention for use in treating a subject having or at risk of having a tumor. Further provided is the use of an antibody of the present invention for the preparation of a medicament for treating a subject having or at risk of having a tumor. In a preferred embodiment, the tumor is an EGFR- or CLEC12A-positive tumor or an EGFR- and PD-L1-positive tumor. DETAILED DESCRIPTION OF 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 being derived from or sharing sequence homology with the variable regions of the antibody.

[0030] Antibody binding has different properties, including specificity and affinity. Specificity determines which antigen or epitope thereof is specifically bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. It is important to note that, as used herein, the "specificity" of an antibody refers to its selectivity for a particular antigen, while "affinity" refers to the strength of the interaction between the antibody's antigen-binding site and the epitope it binds to. Antibodies typically consist of basic structural units—each with two heavy chains and two light chains. Therapeutic antibodies are preferably as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies in the case of a human subject). The antibodies of the present invention are not limited to any particular format or method of preparation.

[0031] Thus, as used herein, "binding specificity" refers to the ability of an individual antibody binding site to react with an antigenic determinant. Typically, the binding sites of the antibodies of the present invention are located in the variable domains of the Fab portion, which comprises the variable domains, and are constructed from the hypervariable regions of the heavy and light chains.

[0032] The antibodies of the present invention are preferably IgG antibodies, preferably IgG1 antibodies. Full-length IgG antibodies may be preferred due to their favorable half-life and the desire to remain close to the complete autologous (human) molecule for immunogenicity reasons. IgG1 is preferred due to its long circulating half-life in humans. To prevent or avoid immunogenicity in humans, the bispecific full-length IgG antibodies according to the present invention are 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, where 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 a first antigen and a VL paired with at least one VH in an immunoglobulin variable domain can specifically recognize a second antigen. The resulting VH / VL pairs bind to either antigen 1 or antigen 2 and are called "two-in-one antibodies" and are described, for example, in WO 2008 / 027236, WO 2010 / 108127, and Schaefer et al. (Cancer Cell 20, 472-486, October 2011). Bispecific antibodies according to the present invention are not limited to any particular bispecific format or method for preparing them. Bispecific antibodies are multispecific antibodies.

[0034] Multispecific multimers or antibodies as referred to herein encompass proteinaceous molecules belonging to the immunoglobulin class of proteins that contain two or more domains that bind to epitopes on antigens, such domains being derived from or sharing sequence homology with the variable regions of antibodies, and include proteinaceous molecules that bind to three or more antigens known in the art, such as those described in WO 2019 / 190327.

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

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

[0037] The term "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain constant region sequences from any organism, and includes a heavy chain variable domain unless otherwise specified. The term heavy chain variable domain includes three heavy chain CDRs and four FR regions unless otherwise specified. Fragments of heavy chains 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, hinge, CH2 domain, and CH3 domain. Functional fragments of heavy chains include fragments containing at least one CDR capable of specifically recognizing an antigen.

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

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

[0040] The term "common light chain" according to the present invention refers to light chains which may be identical or which may have some amino acid sequence differences, wherein the binding specificity of the antibody of the present invention is not affected, i.e. the differences do not substantially affect the formation of a functional binding region.

[0041] For example, it is possible to prepare or discover variable chains that are not identical but are still functionally equivalent within the definition of a common chain used herein (e.g., by introducing and testing conservative amino acid changes, amino acid changes in regions that do not contribute, or only partially contribute, to binding specificity when paired with a cognate chain, etc.). Thus, such variants are also capable of combining different cognate chains to form functional antigen-binding domains. Thus, as used herein, the term "common light chain" refers to a light chain that may be identical or may have some amino acid sequence differences while retaining the binding specificity of the resulting antibody after pairing with a heavy chain. The combination of a particular common light chain and such a functionally equivalent variant is encompassed by the term "common light chain." For a detailed description of the use of common light chains, see International Publication Nos. 2004 / 009618 and 2009 / 157771.

[0042] "Fab" refers to a binding domain comprising a variable region, typically a binding domain comprising paired heavy and light chain variable regions. Fab may comprise constant region domains, including CH1 and VH domains paired with a constant light domain (CL) and VL domain. Such pairing may occur, for example, as a covalent bond via disulfide bridges in the CH1 and CL domains.

[0043] "Single-chain variable fragment" (scFv) refers to a binding domain comprising a VH domain and a VL domain connected via a linker, eg, a peptide linker, eg, about 10 to about 25 amino acids in length.

[0044] The term "full-length IgG" or "full-length antibody" according to the present invention is defined as including essentially intact IgG, but does not necessarily have all the functions of intact IgG. For the avoidance of doubt, full-length IgG comprises 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 designated CH1, CH2, CH3, VH, CL, and VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domains, primarily the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations that provide desired properties may be present. Full-length IgGs need not have substantial deletions of any of the regions. However, IgG molecules in which one or several amino acid residues have been deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG." For example, such IgG molecules can have deletions of 1 to 10 amino acid residues, preferably in non-CDR regions, where the deleted amino acids are not essential for the binding specificity of the IgG.

[0045] As used herein, 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 the residues in a selected sequence after aligning the sequences for optimal comparison purposes. Gaps may be introduced into either of the two sequences being compared to optimize alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over shorter lengths, such as about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0046] Comparison of sequences and determination of the percentage of sequence identity between two sequences can be achieved using a mathematical algorithm.Those skilled in the art will recognize that several different computer programs are available for aligning two sequences and determining the identity between two sequences (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 purposes of the present invention and the sequences described herein, percent 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 utilize a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22:4673-4680), a swgapdnarnt scoring 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 utilize a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, 1994), a blosum62mt2 scoring 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 domains that they can produce based on using the same VH V gene segment and having at least 70% sequence identity in the HCDR3 and the same HCDR3 length.

[0049] Thus, in a preferred embodiment, the present invention provides a "super-cluster" or "supercluster" comprising a group of clones and the binding domains that can be produced by the group of clones based on using the same VH V gene segment and having at least 70% sequence identity in the HCDR3 and the same HCDR3 length. In a preferred embodiment, the sequence identity is 80%, more preferably 90%, and most preferably 95% sequence identity, provided that the HCDR3 sequence clones containing nucleic acids encoding DGGYSYGPYWYFDL and DHRGYGDYEGGGFDY, clones containing nucleic acid sequences encoding the HCDR2 sequences GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG, and SIIPIFGTITYAQKFQG, or VH sequences, QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, and A clone containing a nucleic acid sequence encoding EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS was provided that clones from the group are excluded or contain nucleic acid encoding an HCDR3 that is comprised by or designed to be comprised by a bispecific antibody.

[0050] The term "super-cluster 1" or "supercluster 1," as used herein, refers to a group of clones and the binding domains 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 members of that supercluster. Examples include MF8048, MF8056, MF8057, MF8058, MF8078, and MF8101. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment, VH1-69, and / or having at least 80% identity in HCDR3, more preferably 90% identity in HCDR3, and most preferably 95% identity in HCDR3, and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment of VH1-69 and / or have at least 80% identity in HCDR3 and the same HCDR3 length compared to the encoded CDR3 segment RGNWNPFDP, preferably 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, with the proviso that the HCDR2 sequence provided that clones containing nucleic acids encoding GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG and SIIPIFGTITYAQKFQG are excluded, or EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, A clone containing nucleic acid encoding EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS was provided that clones from the group contain nucleic acids encoding HCDR3s that are comprised by or designed to be comprised in bispecific antibodies. The term "super-cluster 3" or "supercluster 3" herein refers to a group of clones and the binding domains that can be produced by the group of clones 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 members of the supercluster. Examples include MF8397 and MF8562. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment (VH3-23) and / or having at least 80% identity in HCDR3, more preferably 90% identity in HCDR3, and most preferably 95% identity in HCDR3, and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment of VH3-23 and / or have at least 80% identity in HCDR3 and the same HCDR3 length compared to the encoded CDR3 segment DGGYSYGPYWYFDL, preferably 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, with the proviso that clones containing nucleic acids encoding the HCDR3 sequence DGGYSYGPYWYFDL are excluded, or the VH sequence A clone containing nucleic acid encoding QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS was prepared using the method of provided that clones from the group are excluded or contain nucleic acid encoding an HCDR3 that is comprised by or designed to be comprised by a bispecific antibody.

[0051] The term "super-cluster 4" or "supercluster 4," as used herein, refers to a group of clones and the binding domains that a group of clones can produce based on using the same VH V gene segment (VH3-9) and having at least 70% sequence identity in HCDR3 and the same HCDR3 length as members of that supercluster. Examples include MF8508, MF8998, MF10401, and MF10428. In another preferred embodiment, the anti-CD3 antibodies herein are based on using the same VH V gene segment, VH3-9, and / or having at least 80% identity in HCDR3, more preferably 90% identity in HCDR3, and most preferably 95% identity in HCDR3, and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment of VH3-9 and / or have at least 80% identity in HCDR3 and the same HCDR3 length compared to the encoded CDR3 segment DHRGYGDYEGGGFDY, preferably 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, with the proviso that clones containing nucleic acids encoding the HCDR3 sequence DHRGYGDYEGGGFDY are excluded, or the VH sequence A clone containing nucleic acid encoding EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS was provided that clones from the group are excluded or contain nucleic acid encoding an HCDR3 that is comprised by or designed to be comprised by a bispecific antibody.

[0052] The term "super-cluster 7" or "supercluster 7" as used herein refers to a group of clones and the binding domains that can be produced by a group of clones 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 members of that supercluster. Examples include MF9249 and MF9267. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment, VH5-51, and / or having at least 80% identity in HCDR3, more preferably 90% identity in HCDR3, and most preferably 95% identity in HCDR3, and the same HCDR3 length. In another preferred embodiment, the anti-CD3 antibodies herein are based on using the same VH V gene segment of VH5-51 and / or have at least 80% identity in HCDR3 and the same HCDR3 length compared to the encoded CDR3 segment HIRYFDWSEDYHYYLDV, preferably have 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.

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

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

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

[0056] In some embodiments, the bispecific antibody comprises a VH encoded by the V gene segment VH1-69, or the VH sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, or It does not have a VH encoded by a variant of V gene segment VH1-69 having EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS.

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

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

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

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

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

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

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

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

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

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

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

[0068] The present invention provides V gene segment VH1-69, or Further provided is a VH encoded by a variant of the V gene segment VH1-69 comprising at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity to the sequence of said V gene segment; This VH is HCDR3 of MF8048, MF8056, MF8057, MF8058, MF8078 or MF8101, or a variant of said HCDR3 that has at least 70% sequence identity with said HCDR3 and the same length as said HCDR3.

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

[0070] In some embodiments, the VH is not a VH encoded by the V gene segment VH1-69, or 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 a VH encoded by the V gene segment VH1-69, or alternatively, is a VH sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, or QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, or and not the VH encoded by the variant of V gene segment VH1-69 having EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS.

[0072] The present invention provides V gene segment VH3-23, or Further provided is a VH encoded by a variant of the V gene segment VH2-23 comprising at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity to the sequence of said V gene segment; This VH is HCDR3 of MF8397 or MF8562, or a variant of said HCDR3 that has at least 70% sequence identity with said HCDR3 and the same length as said HCDR3.

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

[0074] In some embodiments, the VH is not a VH encoded by V gene segment VH3-23 or a VH encoded by a variant of V gene segment VH3-23 having the HCDR3 sequence DGGYSYGPYWYFDL.

[0075] In some embodiments, the VH is not a VH encoded by the V gene segment VH3-23 or a variant of the V gene segment VH3-23 having the VH sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS.

[0076] The present invention provides V gene segment VH3-9, or Further provided is a VH encoded by a variant of V gene segment VH3-9 comprising at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity to the sequence of said V gene segment; This VH is HCDR3 of MF8508, MF8998, MF10401, or MF10428, or a variant of said HCDR3 that has at least 70% sequence identity with said HCDR3 and the same length as said HCDR3.

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

[0078] In some embodiments, the VH is not a VH encoded by V gene segment VH3-9 or a VH encoded by a variant of V gene segment VH3-9 having the HCDR3 sequence DHRGYGDYEGGGFDY.

[0079] In some embodiments, the VH is not a VH encoded by the V gene segment VH3-9, or is not a VH encoded by the V gene segment VH3-9 having the VH sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS.

[0080] The present invention provides V gene segment VH5-51, or Further provided is a VH encoded by a variant of the V gene segment VH5-51 comprising at least 70%, preferably at least 80%, more preferably at least 90%, and more preferably at least 95% sequence identity to the sequence of said V gene segment; This VH is HCDR3 of MF9249 or MF9267, or a variant of said HCDR3 that has at least 70% sequence identity with said HCDR3 and the same length as said HCDR3.

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

[0082] The VH provided by the invention as defined herein is preferably not the VH of the CD3-binding variable domain defined in PCT / NL2019 / 050199.

[0083] Also provided are antigen binding proteins or antibodies, preferably bispecific antibodies, wherein the CDRs have 70%, preferably 80%, more preferably 90% identity to the claimed CDRs. In a preferred embodiment, the antigen binding protein or antibody is a bispecific antibody comprising a CDR with up to two, preferably up to one, and more preferably up to zero amino acid residue mutations, insertions, substitutions, deletions, or additions to the claimed CDRs.

[0084] Antigen binding by an antibody is typically mediated through the specific three-dimensional structure of both the antibody's complementary region and the antigen and variable domains, allowing these two structures to bind together precisely (an interaction similar to a lock and key) as opposed to random, nonspecific adhesion of the antibody. Antibodies typically recognize an epitope of the antigen, and such epitopes may also exist in other proteins, and antibodies of the present invention that bind to CD3 or CLEC12A may recognize other proteins as well, even if such other proteins contain the same epitope. Thus, the term "binding" does not exclude the antibody's binding to another protein or proteins containing the same epitope. Heavy chain / light chain combinations in the antibodies of the present invention that bind to CD3 do not bind to other proteins on the membrane of cells in vivo, preferably in adult humans. Heavy chain / light chain combinations that bind to CLEC12A, EGFR, PD-L1, or tumor cell antigens of the present invention do not bind to other proteins on the membrane of cells in vivo, preferably in adult humans. Suitable tumor antigen-specific arms are disclosed in PCT / NL2019 / 050199.

[0085] "Plurality" means two or more.

[0086] "Variants" of antibodies described herein may include functional portions, derivatives and / or analogs of antibodies, including antibody mimetics, monobodies and aptamers.

[0087] The variants typically maintain the binding specificity of the antibody, e.g., the specificity of a bispecific antibody. The variants may be functional portions or derivatives of binding domains, multimers, or antibodies as described herein.

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

[0089] A functional derivative of an antibody described herein is a protein 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 domain may be such a variable domain or a variable domain-like molecule, such as a Fab fragment or a single-chain Fv (scFv) fragment comprising a VH and a VL linked together via a 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 linking one, two, or more variable domains have been described. The antigen-binding proteins described herein are proteins comprising at least one of such variable domains. In the case of bispecific or multispecific antigen-binding proteins, such proteins comprise two or more variable domains, at least two of which bind to different targets. The variable domains are linked to each other via a linking moiety, 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 containing a single monomeric variable antibody region. Like whole antibodies, it can selectively bind to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibody fragments are much smaller than typical antibodies (150–160 kDa), which are composed of two heavy chain proteins and two light chains. They are also smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable regions, one from the light chain and one from the heavy chain). Single-domain antibodies themselves are not much smaller than normal antibodies (typically 90–100 kDa). Single-domain antibody fragments can be engineered from heavy-chain antibodies found in camels; these are called VHH fragments (nanobodies®). Some fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin new antigen receptor") from which single domain antibody fragments called VNAR fragments can be derived.An alternative approach is to split the dimeric variable domain from common immunoglobulin G (IgG) from humans or mice into monomers. Most research into single-domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to be capable of binding to target epitopes. Other non-limiting examples of variable domain-like molecules are VHHs, human domain antibodies (dAbs), and unibodies. Preferred functional moieties are those comprising variable domains comprising heavy and light chain variable regions. Non-limiting examples of such variable domains are F(ab)-fragments and single-chain Fv fragments. Bispecific formats for variable domain(-like) linkages include, for example, human serum albumin (HSA) linked to two different scFvs; two different scFvs linked together via a dimerization motif; or bispecific miniantibodies containing self-associating secondary structures such as helix bundles or coiled coils to induce dimerization of the scFv fragments (Morrison (2007) Nat. Biotechnol 25:1233-34). Examples of suitable HSA linkers and methods for linking scFvs to the linker are described in WO 2009 / 126920.

[0090] The functional derivative may be an antibody mimic, a polypeptide, an aptamer, or a combination 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 mimics, polypeptides, and aptamers can be linked together by methods known in the art. For example, in some embodiments, the binding molecules of the present invention are conjugates or fusion proteins.

[0091] Antibody mimetics are polypeptides that, like antibodies, can specifically bind to antigens but are not structurally related to antibodies. Antibody mimetics are typically artificial peptides or proteins with a molar mass of approximately 3-20 kDa. Non-limiting examples of antibody mimetics are affibody molecules (typically based on the Z domain of protein A), affilins (typically based on gamma-B crystalloid or ubiquitin), affimers (typically based on cystatins), affitins (typically based on Sac7d from Sulfolobus acidocaldarius), alphabodies (typically based on triple-helical coiled-coils), anticalins (typically based on lipocalins), avimers (typically based on the A domains of various membrane receptors), DARPins (typically based on ankyrin repeat motifs), fynomers (typically based on the SH3 domain of Fyn7), kunitz domain peptides (typically based on the Kunitz domains of various protease inhibitors), and monobodies (typically based on the type III domain of fibronectin).

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

[0093] Monobodies and other antibody mimics are typically generated from combinatorial libraries in which portions of the scaffold are 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 usually generated by selection from a large pool of random sequences, although natural aptamers also exist in 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," are to be interpreted inclusively, that is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

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

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

[0098] The antigen-binding proteins or antibodies of the present invention are preferably bispecific or multispecific antigen-binding proteins or antibodies. The bispecific or multispecific antigen-binding proteins or antibodies preferably bind to at least human CD3, and more preferably bind to at least a surface molecule expressed on a human tumor cell. In a preferred embodiment, the bispecific or multispecific antigen-binding proteins or 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 a particularly preferred embodiment, the bispecific antibody binds to CLEC12A. In a particularly preferred embodiment, the multispecific antibody binds to CD3, PD-L1, and EGFR.

[0099] As used herein, the term "CLEC12A" refers to C-type lectin domain family 12 member A. CLEC12A is a member of the C-type lectin protein CLL-1; MICL; dendritic cell-associated lectin 2; C-type lectin superfamily; myeloid inhibitory C-type lectin-like receptor; C-type lectin-like molecule-1; DCAL2; CLL1; C-type lectin-like molecule 1; DCAL-2; killer cell lectin-like receptor subfamily L, member 1 (KLRL1); CD371 (cluster of differentiation 371) (Bakker A. et al. Cancer Res. 2004, 64, p8843-50; GenBank™ Accession No.: AY547296; Zhang W. et al. GenBank™ Accession No.: AF247788; A.S. Marshall, et al. J Biol Chem 2004, 279, p14792-802; GenBank™ Accession No.: AY498550; Y. Han et al. Blood 2004, 104, p2858 66; H. Floyd, et al. GenBank™ Accession Number: AY426759; C. H. Chen, et al. Blood 2006, 107, p1459 67). Also referred to as Id; HGNC: 31713; Entrez Gene: 160364; Ensembl: ENSG00000172322; OMIM: 612088; UniProtKB: Q5QGZ9.

[0100] CLEC12A is an antigen expressed on leukemic blast cells and leukemic stem cells, such as CD34-negative or CD34-low expressing leukemic stem cells (side populations), in acute myeloid leukemia (AML) (A.B. Bakker et al. Cancer Res 2004, 64, p8443 50; Van Rhenen et al. 2007 Blood 110:2659; Moshaver et al. 2008 Stem Cells 26:3059), as well as in myelodysplastic syndromes (MDS) (Bakker et al. 2004 supra and Toff-Peterson et al., Br. J. Haematol. 175(3):393-401, 2016). CLEC12A expression is otherwise thought to be restricted to cells of the hematopoietic lineage, particularly the myeloid lineage, i.e., granulocytes, monocytes, and dendritic cell precursors in peripheral blood and bone marrow. More importantly, CLEC12A is not present in normal hematopoietic stem cells. As used herein, when CLEC12A is mentioned, it refers to human CLEC12A (SEQ ID NO: 1; Figure 19) unless otherwise specified.

[0101] The term "CLEC12A" refers to all variants (such as splices and mutations) and isoforms thereof mentioned herein that retain the myeloid expression profile (both at the surface expression level and at the mRNA level), as described, for example, in Bakker et al. Cancer Res 2004, 64, p8443-50 and Marshall 2004-J Biol Chem 279(15), p14792-802. The accession number is provided primarily as a further means of identification, although the actual sequence of the protein may vary due to mutations in the encoding gene, such as those that occur in some cancers.

[0102] The term "CD3" (cluster of differentiation 3) refers to a protein complex composed of the CD3γ chain (SwissProt P09693), CD3δ chain (SwissProt P04234), CD3ε chain (SwissProt P07766), and the 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; and 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 a TCR complex, which can generate activation signals in T lymphocytes through mitogenic signaling. CD3 is expressed on T cells and NK T cells. When CD3 is mentioned herein, it refers to human CD3 (SEQ ID NOS:2-5; Figure 20) unless otherwise specified.

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

[0104] CD19 is also known as CD19 molecule; T cell surface antigen Leu-12; CD19 antigen; CVID3 differentiation antigen CD19; B4; B lymphocyte surface antigen B4; B lymphocyte antigen CD19. Id; 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 called tumor necrosis factor receptor superfamily, member 8 (TNFRSF8); Ki-1 antigen; CD30: Ki-1; D1S166E; cytokine receptor CD30; lymphocyte activation antigen CD30; tumor necrosis factor receptor superfamily member 8, CD30L receptor; CD30 antigen. Id; HGNC: 11923; Entrez Gene: 943; Ensembl: ENSG00000120949; OMIM: 153243; UniProtKB: P28908.

[0107] CD33 is also known as CD33 molecule; SIGLEC-3; CD33 antigen (Gp67); myeloid 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 known as CD38 molecule; T10; CD38 antigen (P45); CADPr hydrolase 1; ADP-ribosyl cyclase 1; ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase; NAD(+) nucleosidase; EC 3.2.2.5; cyclic ADP-ribose hydrolase 1; CD38 antigen. Id; HGNC: 1667; Entrez Gene: 952; Ensembl: ENSG00000004468; OMIM: 107270; UniProtKB: P28907.

[0109] CD44 is also known as 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; hyaluronan 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 (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; CD138 antigen. Id; HGNC:10658; Entrez Gene:6382; Ensembl:ENSG00000115884; OMIM:186355; UniProtKB:P18827.

[0112] CEA is also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); fetal stool antigen 100; CD66e; carcinoembryonic antigen; and CD66e antigen. Id; HGNC: 1817; Entrez Gene: 1048; Ensembl: ENSG00000105388; OMIM: 114890; UniProtKB: P06731.

[0113] EGFR is also referred to as 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 inhibitory protein 40; cell proliferation inducer protein 61; HER1; mENA; EC 2.7.10.1; EC 2.7.10; 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 referred to as: Delta-like 3; Drosophila Delta homolog 3; Delta 3; Delta (Drosophila)-like 3; and SCDO1. The identities for DLL3 are HGNC:2909; Entrez Gene:10683; Ensembl:ENSG00000090932; OMIM:602768, and UniProtKB:Q9NYJ7.

[0116] LGR5 is leucine-rich repeat-containing G protein-coupled receptor 5. Alternative names for this gene or protein are 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. Proteins or antibodies of the present invention that bind to LGR5 bind to human LGR5. LGR5-binding proteins or antibodies of the present invention will also bind to human and other mammalian orthologs due to sequence and tertiary structure similarities between such orthologs, although this is not necessarily the case. 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 mesothelin is also called metthelin; pre-pro megakaryocyte potentiating factor; CAK1 antigen; MPF; soluble MPF mesothelin-related 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 referred to 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 IDs for FOLR1 are HGNC:3791; Entrez Gene:2348; Ensembl:ENSG00000110195; OMIM:136430; UniProtKB:P15328.

[0119] Folate receptor 3 is also referred to 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 signal transducer 1; MK-1; TACSTD1; human epithelial glycoprotein-2; TROP1; membrane component, chromosome 4, surface marker (35 kD 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; ACSTD1. Id;HGNC:11529;Entrez Gene:4072, Ensembl:ENSG00000119888;OMIM:185535;UniProtKB:P16422.

[0121] HER2 is a member of the V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2; ERBB2; CD340; NGL; HER-2; HER-2 / neu2NEU2; TKR1; neuro / glioblastoma-derived oncogene homolog; C-Erb B2 / Neu protein; metastatic lymph node gene 19 protein; herstatin; prostate oncogene C-ErbB-2; neuroblastoma / glioblastoma-derived oncogene homolog; prostate oncogene Neu; receptor tyrosine protein kinase ErbB-2; tyrosine kinase cell surface receptor HER2; V-Erb-B2 erythroblastic leukemia viral oncogene homolog 2; neuro / glioblastoma-derived oncogene homolog; MLN 19; MLN 19; p185erbB2; CD340 antigen; EC 2.7.10.1; EC 2.7.10; also known as V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2 (neuro / 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 called testicular mitochondrial-associated cysteine-rich protein (SMCP); MCSP; MCS; mitochondrial sheath selenoprotein; HSMCSGEN1; testicular mitochondrial-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 immune responses during certain events, such as pregnancy, tissue allotransplantation, autoimmune diseases, and other disease states, 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 control the accumulation of foreign antigen-specific T cells through apoptosis. PD-L1 is expressed by various cancer cells, and its expression is thought to be at least partially involved in dampening 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; the 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; glutamic acid carboxypeptidase II; N-acetylated-alpha-linked acidic dipeptidase I; PSM; NAALADaseI; PSMA; EC 3.4.17.21; glutamic acid carboxylase II; GCP2; cell proliferation inhibitory gene 27 protein; NAALAdase; folylpoly-gamma-glutamic acid carboxypeptidase; glutamic acid carboxypeptidase 2; membrane glutamic acid carboxypeptidase; N-acetylated-alpha-linked acidic dipeptidase 1; pteroylpoly-gamma-glutamic acid carboxypeptidase; prostate-specific membrane antigen variant F; FGCP; folate hydrolase 1; GCPII; prostate-specific membrane antigen. Id;HGNC:3788;Entrez Gene:2346, Ensembl:ENSG00000086205;OMIM:600934;UniProtKB:Q04609.

[0126] PSMA should not be confused with proteasome (macropain) subunit, alpha type 1, also known as PSMA1.

[0127] The accession number is provided primarily to provide an additional method of target identification; the actual sequence of the bound protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The antigen-binding site binds to various variants thereof, such as those expressed by some antigen-positive immune or tumor cells.

[0128] When referring to a gene or protein herein, the reference preferably refers to the human form of the gene or protein. When referring to a gene or protein herein, the reference refers to the native gene or protein as well as mutant forms of the gene or protein that can be detected in tumors, cancers, etc., preferably mutant forms of the gene or protein that can 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 protein, or variants thereof. The antigen-binding heavy chain / light chain combination preferably binds to the extracellular portion of the antigen. Preferably, the bispecific antibodies of the present invention bind to human CLEC12A or variants thereof. Preferred bispecific antibodies of the present invention bind to human CD3 and human CLEC12A or variants thereof. In a preferred embodiment, the multispecific antibody binds to CD3, PD-L1, and EGFR.

[0130] HGNC stands for HUGO Gene nomenclature committee. The number after the abbreviation is an accession number by which information on the gene and the protein encoded by the gene can be retrieved from the HGNC database. Entrez genes provides accession numbers or gene IDs by which information on the gene or the protein encoded by the gene can be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensemble provides accession numbers by which information on the gene or the protein encoded by the gene can be obtained from the Ensemble database. Ensemble is a collaborative project between EMBL-EBII and the Wellcome Trust Sanger Institute to develop a software system to generate and maintain 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: Amino acid sequence: CDR1:SFGIS;CDR2:GFIPVLGTANYAQKFQG;CDR3:RGNWNPFDP or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the sequence, 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence: CDR1: SKTLTIS; CDR2: GIIPIFGSITYAQKFQD; CDR3: RGNWNPFDP; or Amino acid sequence: CDR1:GSGIS;CDR2:GFIPFFGSANYAQKFRD;CDR3:RGNWNPX 13 DP; (In the sequence, X 13 = or 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence: CDR1:RX3WIG;CDR2:IIYPGDSDTRYSPSFQG;CDR3:X4IRYFX5WSEDYHYYX6DV; (In the sequence, 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence: CDR1: SYALS; CDR2: GISGSGSRTTWYADSVKG, CDR3: DGGYSYGPYWYFDL.

[0137] The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1: SYALS; CDR2: AISGSGRTTWYADSVKG, CDR3: DGGYTYGPYWYFDL.

[0138] The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: It contains CDR1, CDR2 and CDR3, including CDR1: DYTMH; CDR2: DISWSSGSIGYADSVKG, CDR3: DHRGYGDYEGGGFDY.

[0139] The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3, 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 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, Preferably, X7, X8, X9 and X 10 are S, S, I and G, or are G, S, I and Y, or are S, T, T and G, and preferably X 11 and X 12are R and H, or R and Y, or M and Y, and more preferably X7, X8, X9, X 10 , X 11 and X 12 are S, S, I, G, R and H, or are G, S, I, Y, R and Y, or are 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 are R and H, or X7, X8, X9 and X 10 are G, S, I and Y, and X 11 and X 12 are R and Y, or X7, X8, X9 and X 10 are S, T, T and G, and X 11 and X 12 is M and Y).

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

[0141] IgVκ1-39 containing the light chain variable region * Preferably, O1 is a germline sequence. * 01 or / IGJκ5 * It is further preferred 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 * 01. In a preferred embodiment, the light chain variable region comprises the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01. In a preferred embodiment, IgVκ1-39 * 01 / IGJκ1 * 01. The light chain variable region is preferably the 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 undergone one or more mutations relative to the germline sequence, i.e., the normal sequence in non-lymphoid cells of the organism. The process involved in these mutations is often called somatic (hyper)mutation. The resulting light chains are called affinity-matured light chains. Such light chains are derived from the germline IgVκ1-39 *IgVκ1-39 when derived from the 01 sequence * 01. As used herein, the phrase "IgVκ1-39" is used interchangeably with "IgVκ1-39" * 01" is IgVκ1-39 * 11A, 11D, or 11E, with 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof. * 01 light chain. In a preferred embodiment, IgVκ1-39 * A 01 light chain is a light chain comprising the sequence shown in Figure 11A, 11B, or 11C with 0-9, 0-8, 0-7, 0-6, 0-5, or 0-4 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof. * The IgVκ1-39 light chain is a light chain comprising the sequence shown in Figure 11A, 11B, or 11C with 0 to 5, preferably 0 to 4, and more preferably 0 to 3 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof. * A 01 light chain is a light chain comprising the sequence shown in Figure 11A, 11B, or 11C with 0 to 2, more preferably 0 to 1, and most preferably 0 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, IgVκ1-39 * An O1 light chain is a light chain comprising the sequence shown in Figure 11A or 11B with any of the amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof described above. In a preferred embodiment, the light chain comprises the sequence of Figure 11B.

[0144] The light chain preferably comprises a common light chain variable region. The common light chain variable region preferably comprises an IgVκ1-39 light chain variable region. The light chain variable region preferably comprises a germline IgVκ1-39* The light chain variable region is a kappa light chain IgVκ1-39 variable region. * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * Preferably, the light chain variable region comprises the germline kappa light chain IgVκ1-39. * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01. The light chain variable region preferably comprises 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, which has 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0145] The light chain variable region preferably comprises CDR1, CDR2, and CDR3 regions comprising the amino acid sequences CDR1-QSISSY, CDR2-AAS, and CDR3-QQSYSTP, i.e., the CDRs of IGKV1-39 (according to IMGT). Amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are preferably absent in the CDR3 region of the light chain variable region, and preferably absent in the CDR1 or CDR2 regions of the light chain variable region. In a preferred embodiment, the light chain variable region does not comprise deletions, additions, mutations, or insertions relative to the depicted sequence. In this embodiment, the light chain variable region may have 0 to 5 amino acid substitutions relative to the depicted amino acid sequence. The 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 comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, and CDR3-QQSYSTP, i.e., the CDRs of IGKV1-39 (according to IMGT), respectively.

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

[0147] As described above, the antibodies of the present invention are preferably bispecific antibodies. 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, where the first and second antigens are not identical. 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 the VH can specifically recognize a first antigen, and the VL paired with the VH in the immunoglobulin variable region can specifically recognize a 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 WO 2008 / 027236, WO 2010 / 108127, and Schaefer et al. (Cancer Cell 20, 472-486, October 2011). Bispecific antibodies according to the invention are not limited to any particular bispecific format or method for preparing them.

[0148] A bispecific antibody preferably has one heavy chain variable region / light chain variable region (VH / VL) combination that binds to CD3 and a second VH / VL combination that binds to an antigen other than an 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 VLs in the first and second VH / VL combinations are identical. In a preferred embodiment, the bispecific antibody is a full-length antibody having one heavy chain / light chain (H / L) combination that binds to CD3 and one H / L chain combination 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 first and second H / L chain combinations are identical, i.e., similar or identical human light chains are so-called "common light chains" that can bind to different heavy chains to form antibodies with functional antigen-binding domains. In a preferred embodiment, the light chains in the first H / L chain combination comprise a light chain variable region that is similar to the light chain variable region in the second H / L chain combination. In a more preferred embodiment, the light chain variable regions in the first and second H / L chain combinations are identical, i.e., similar or identical human light chain variable regions are so-called "common light chain variable regions" that can bind to different heavy chain variable regions to form antibodies with functional antigen-binding domains. Light chains that comprise a common light chain variable region are preferably common light chains.

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

[0150] The present invention also provides alternative bispecific 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). Bispecific antibody formats that are not conventional antibodies having two H / L combinations have at least a variable domain comprising a heavy chain variable region and a light chain variable region of the present invention. This variable domain may be linked to a single-chain Fv fragment, monobody, VH, or 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 capable of binding to an antigen other than CD3, preferably a tumor antigen. In a more preferred embodiment, the light chains in the H / L chain combination are identical, i.e., the human light chain is a so-called "common light chain," which is a light chain that can bind to a different heavy chain to form an antibody with a functional antigen-binding domain. Preferably, the common light chain has a germline sequence. Preferred germline sequences are light chain variable regions that are frequently used in the human repertoire and have good thermodynamic stability, yield, and solubility. A preferred germline light chain is IgVκ1-39, preferably the rearranged 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 at imgt.org on the World Wide Web).

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

[0153] As used herein, the term "immune effector cell" or "effector cell" refers to a cell within the natural repertoire of cells in a mammalian immune system that can be activated to affect the viability of a target cell. Immune effector cells include lymphoid cells, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, and myeloid lineage cells, such as 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 that the immune effector cells are brought into close proximity with the abnormal target cells, allowing the effector cells to initiate direct or indirect killing of the abnormal cells.

[0154] As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal (e.g., a patient suffering from cancer, such as a human patient), including a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc.

[0155] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or procedure performed, or the administration of an active agent or combination of active agents to a subject, with the goal of reversing, mitigating, ameliorating, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of symptoms, complications, pathology, or biochemical manifestations associated with a disease.

[0156] As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can result in an improved state over a baseline, such as an improvement over measurements or observations made before initiation of therapy according to the method. For example, a beneficial effect can result in a state that slows, stabilizes, stops, or reverses the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease, or a reduction or elimination of markers of cancer. Effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or arrest tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0157] The term "therapeutic amount" refers to an amount of a drug or combination of drugs that provides a desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, remission, reduction, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, or other desired change in biology. In some embodiments, a therapeutic amount is an amount sufficient to slow tumor development. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay tumor recurrence. A therapeutic amount can be administered in one or more administrations. A therapeutic amount of a drug or composition can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, prevent, slow, or stop to some extent cancer cell invasion into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) relieve to some extent one or more symptoms associated with cancer. In one example, a "therapeutic amount" is the amount of CLEC12A / CD3 bispecific antibody that results in a reduction in cancer (e.g., a reduction in cancer cell count) or a slowing of progression of cancer, 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Contains EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0159] Further provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Includes EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0160] Also provided is an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or Contains QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0161] The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, Contains EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, or EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0162] The amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are preferably absent in the CDR3 region of the heavy chain variable region, and preferably absent 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 relative to the sequence shown. In one embodiment, the heavy chain variable region can have 0 to 10, preferably 0 to 5, amino acid substitutions relative to the amino acid sequence shown. In a preferred embodiment, the heavy chain variable region contains 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 at positions other than the CDRs relative to the amino acid sequence shown. A combination of insertions, additions, deletions, or substitutions is a claimed combination if the aligned sequences do not differ at more than 10 positions (preferably 5 or fewer). Gaps in one of the aligned sequences correspond to the same number of amino acids skipped in the other sequence. Amino acid substitutions, if any, are preferably conservative amino acid substitutions.

[0163] The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of 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 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of MF9249 or MF9267 as shown in Figure 13.

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

[0168] Variable domains that bind to CLEC12A having a heavy chain variable region and a common light chain region as defined herein are described, inter alia, in International Publication Nos. WO 2014 / 051433 and WO 2017 / 010874, which are specifically referred to for purposes of this specification and incorporated herein by reference. The heavy chain variable region of a heavy chain / light chain combination that binds to human EGFR or CLEC12A can have 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof relative to the amino acid sequence shown. In preferred embodiments, the heavy chain variable region contains 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, or preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the amino acid sequence shown. A combination of insertions, additions, deletions, or substitutions is a claimed combination if the aligned sequences do 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] Preferably, no amino acid mutations, insertions, deletions, substitutions, additions, or combinations thereof are made / present at the binding interface between the heavy and light chains.

[0170] When an amino acid at the interface of H / L chain interactions is changed, the corresponding amino acid in the other chain is preferably changed to accommodate the change. The insertion or addition of an amino acid preferably does not involve the insertion or addition of a proline.

[0171] The addition of an amino acid can, in principle, be considered the same as an insertion. The addition of an amino acid to one of the termini of a polypeptide chain is sometimes not considered an insertion, but rather a strict addition (extension). In the present invention, both addition within the chain or addition to one of the termini is considered to be an insertion.

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

[0173] The constant regions of the antibodies of the present invention, including bispecific or multispecific antibodies, are preferably human constant regions. These constant regions may contain one or more, preferably no more than 10, and preferably no more than 5, amino acids different from the constant regions of naturally occurring human antibodies. The variable domains of the various antibodies prepared herein are derived from a human antibody variable domain library. These variable domains are therefore human. The unique CDR regions may be of human origin, synthetic, or derived from another organism. The antibodies or bispecific antibodies of the present invention are preferably human or humanized. Suitable heavy chain constant regions are exemplified, but not limited to, in Table 12.

[0174] Various methods for producing antibodies exist in the art. Bispecific antibodies are typically produced by cells expressing nucleic acids encoding the antibodies. Cells suitable for producing antibodies are hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. In a particularly preferred embodiment, the cells are CHO cells.

[0175] Various institutions and companies have developed cell lines for the large-scale production of antibodies, e.g., 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 industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. In a preferred embodiment, the present invention provides industrial cell lines that produce the antibodies of the present invention.

[0176] In one embodiment, the present invention provides a cell comprising an antibody according to the invention and / or a nucleic acid according to the invention. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, and most preferably a human cell. For the purposes of the present invention, a suitable cell is any cell that can comprise, and preferably produce, an antibody according to the invention and / or a nucleic acid according to the invention.

[0177] The present invention further provides a cell comprising an antibody of the present invention. Preferably, the cell (typically an in vitro, isolated, or recombinant cell) produces the antibody. In a preferred embodiment, the cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NSO cell, or a PER.C6 cell. In a particularly preferred embodiment, the cell is a CHO cell. A cell culture comprising a cell of the present invention is also provided. Various institutions and companies have developed cell lines for large-scale production of antibodies, e.g., for clinical use. Non-limiting examples of such cell lines are CHO cells, NSO cells, or PER.C6 cells. These cells are also used for other purposes, such as protein production. Cell lines developed for 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 a cell line developed for large-scale antibody production for the production of an antibody of the present invention. The present invention further provides a cell for producing an antibody, comprising nucleic acid molecules encoding the VH, VL, and / or heavy and light chains of the claimed antibody. Preferably, the nucleic acid molecule encodes a VH identified in Figure 13, is a nucleic acid molecule encoding a VH identified by the number 4327, or identified by the number 8233, or a combination thereof.

[0178] The present invention further provides a method for producing an antibody, comprising culturing the cells of the present invention and harvesting the antibody from the culture. Preferably, the cells are cultured in a serum-free medium. Preferably, the cells are adapted to suspension growth. Further provided is an antibody obtainable by the method for producing an antibody according to the present invention. The antibody is preferably purified from the culture medium. Preferably, the antibody is affinity purified.

[0179] The cells of the present invention may be, for example, hybridoma cell lines, CHO cells, 293F cells, NS0 cells, or any other cell type known for its suitability for producing antibodies for clinical purposes. In a particularly preferred embodiment, the cells are human cells. Preferably, the cells are cells transformed with the adenovirus E1 region or a functional equivalent thereof. 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 variants thereof, preferably variants that utilize the glutamine synthetase (GS) vector system for antibody expression.

[0180] The present invention further provides a method for producing an antibody, comprising culturing the cells of the present invention and harvesting the antibody from the culture. Preferably, the cells are cultured in a serum-free medium. Preferably, the cells are adapted to suspension growth. Further provided is an antibody obtainable by the method for producing an antibody according to the present invention. The antibody is preferably purified from the culture medium. Preferably, the antibody is affinity purified.

[0181] Bispecific antibodies are also typically produced by cells expressing nucleic acids encoding the antibodies. In this case, the cells express different light and heavy chains that make up the bispecific antibody. To achieve this, the cells express two different heavy chains and at least one light chain. Because unmodified heavy chains can pair with each other to form dimers, such cells typically produce two monospecific antibodies (homodimers) in addition to bispecific antibodies (heterodimers). 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, allowing cells expressing these two heavy chains to produce monospecific antibodies (homodimers of pairing of two first heavy chains), tetravalent antibodies (homodimers of pairing of two second heavy chains), and triabodies (heterodimers of first and second heavy chains). The number of possible heavy / light chain combinations in the produced antibodies increases when a cell expresses more than one light chain. 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 equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% each monospecific antibodies (i.e., with identical heavy and light chain combinations). Alternatively, in the above example with a first heavy chain having one variable region and a second heavy chain having two variable regions, the two heavy chains will typically produce 50% trispecific, 25% monospecific, and 25% tetraspecific antibodies.

[0183] Several methods have been published for favoring the production of bispecific antibodies, or vice versa, favoring the production of monospecific antibodies, and these methods can further be used to favor the production of multispecific antibodies. In the present invention, it is preferred that cells prefer the production of bispecific antibodies over the production of the corresponding monospecific antibodies. This is typically achieved by modifying the heavy chain constant regions so that they favor heterodimerization (i.e., dimerization with heavy chains of other heavy chain / light chain combinations) over homodimerization. In a preferred embodiment, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. The art describes various ways in which such heterodimerization of heavy chains can be achieved, including the use of "knob-into-hole" bispecific antibodies.

[0184] U.S. Patent Application No. 13 / 866,747 (now issued as U.S. Patent No. 9,248,181), U.S. Patent Application No. 14 / 081,848 (now issued as U.S. Patent No. 9,358,286), and PCT / NL2013 / 050294 (published as WO 2013 / 157954; incorporated herein by reference) disclose methods and means for producing bispecific antibodies using compatible heterodimerization domains. These means and methods can be advantageously employed in the present invention. Specifically, preferred mutations for producing essentially only bispecific full-length IgG molecules 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-mutant and KK-mutant heavy chains to form heterodimers (so-called "DEKK" bispecific molecules) has previously been demonstrated in our U.S. Patents Nos. 9,248,181 and 9,358,286 and WO 2013 / 157954. Homodimerization of DE-mutant heavy chains (DEDE homodimers) or KK-mutant heavy chains (KKKK homodimers) rarely occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains. In one embodiment, a heavy chain / light chain combination comprising a variable domain that binds to CD3 comprises a KK mutant of the heavy chain. In this embodiment, a heavy chain / light chain combination comprising a variable domain that binds to an antigen other than CD3 comprises a DE mutant 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 FIG. 13.

[0185] Some antibodies are modified in the CH2 / lower hinge region, for example, to reduce Fc receptor interaction or to reduce C1q binding. In some embodiments, the antibodies of the invention are IgG antibodies with mutated CH2 and / or lower hinge domains such that the interaction of the bispecific IgG antibody with Fc gamma receptors is reduced. Such mutated CH2 and / or lower hinge domains preferably comprise amino acid substitutions at positions 235 and / or 236 (according to EU numbering), preferably an L235G substitution and / or a G236R substitution.

[0186] Alternatively, some antibodies are modified, for example, to enhance Fc receptor interactions or to enhance C1q binding. Such modifications may be preferred in embodiments directed to autoimmune indications.

[0187] The present invention further provides a method of treating a subject, comprising administering an antigen-binding protein of the invention, preferably an antibody of the invention, to a subject in need thereof. The present invention further provides an antigen-binding protein of the invention, preferably an antibody of the 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 inducing an autoimmune response or cancer cells, for example. Suitable variable domains for this purpose are, inter alia, those comprising a common light chain and the VHs of MF9249 and MF8397. These preferably have low affinity and low cell-killing activity, but are functional under autoimmune response conditions. Suitable variable domains for this purpose are, inter alia, those comprising a common light chain and the VHs of MF9267, MF8057, MF8058, MF8078, and MF8508. These variable domains have suitable affinity and suitable cell-killing activity for anti-cancer purposes.

[0188] Furthermore, the invention described herein includes antigen binding proteins or antibodies having high affinity variable domains with high cell killing activity, including binding domain MF8078, which may be administered locally or expressed locally. The invention further provides methods of treating a subject comprising administering an antigen binding protein of the invention, preferably an antibody of the invention, to a subject in need thereof via local administration means as would be known to one skilled in the art, including, for example, local treatments for oncolytic viruses, melanoma or other cancers of another compartment such as the brain.

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

[0190] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SFGIS CDR2:GFIPVLGTANYAQKFQG CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the sequence, X1=K or R, X2=L or I).

[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 an antigen binding protein of the invention, preferably an antibody of the invention, for use in treating a subject in need thereof, which 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 comprising the consensus light chain and VH of MF8048, MF8056 and MF8101.

[0193] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SKTLTIS, CDR2:GIIPIFGSITYAQKFQD, CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:GSGIS, CDR2:GFIPFFGSANYAQKFRD, CDR3:RGNWNPX 13 DP (In the sequence, X 13 = or L or F).

[0194] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, the 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 amino acid sequence EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGGFIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWLGGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTRRGNWNPFDPWGQGTLVTVSS, EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWLGSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTRRGNWNPFDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWLGGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCARRGNWNPFDPWGQGTLVTVSS, or Contains EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0195] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 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).

[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 of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, the 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 amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Includes EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0198] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SYALS, CDR2:GISGSGRTTWYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DGGYSYGPYWYFDL.

[0199] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:SYALS, CDR2:AISGSGRTTWYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DGGYTYGPYWYFDL.

[0200] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, the 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 amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or 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 the CDRs.

[0201] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 amino acid sequence: CDR1:DYTMH, CDR2:DISWSSGSIGYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DHRGYGDYEGGGFDY.

[0202] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, 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 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, Preferably, X7, X8, X9 and X 10 are S, S, I and G, or are G, S, I and Y, or are S, T, T and G, and preferably X 11 and X 12 are R and H, or R and Y, or M and Y, and more preferably X7, X8, X9, X 10 , X 11 and X 12 are S, S, I, G, R and H, or are G, S, I, Y, R and Y, or are 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 are R and H, or X7, X8, X9 and X 10 are G, S, I and Y, and X 11 and X 12 are R and Y, or X7, X8, X9 and X 10 are S, T, T and G, and X 11 and X 12 is M and Y).

[0203] The present invention further provides a method of treating cancer or the risk of cancer in a subject comprising administering to a subject in need thereof an antigen binding protein, preferably an antibody, that binds to human CD3, the 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 amino acid sequence EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDHRGYGDYEGGGFDYWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDHRGYGDYEGGGFDHWGQGTLVTVSS, EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSDISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDHMGYGDYEGGGFDYWGQGTLVTVSS, or Contains EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSDISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDHRGYGDYEGGGFDYWGRGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0204] The antigen binding proteins, preferably antibodies, in the treatments set out herein above preferably comprise a heavy-light chain (H / L) combination that binds to a tumor antigen.

[0205] The antibody is preferably a human or humanized antibody. Preferably, the antibody comprises two different immunoglobulin heavy chains with compatible heterodimerization domains. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. The bispecific antibody is an IgG antibody with mutated CH2 and / or lower hinge domains for cancer immunotherapy applications in which the interaction of the bispecific or multispecific IgG antibody with Fc gamma receptors is reduced. The mutated CH2 and / or lower hinge domain preferably comprises an amino acid substitution at positions 235 and / or 236 (according to EU numbering), preferably an L235G substitution and / or a G236R substitution. Alternatively, for autoimmune applications, the interaction of the bispecific or multispecific with Fc gamma receptors is enhanced, or ADCC and CDC are increased by modifications to the CH2 and / or CH3 domains. For example, by engineering the Fc region (by introducing amino acid substitutions) to bind to activating receptors with greater selectivity, antibodies can be generated with greater ability to mediate the cytotoxic activity desired by anti-cancer Mabs or CD3-targeting binding arms for the treatment of autoimmune-related diseases. For example, afucosylation is a reported technique for enhancing the ADCC of antibodies. (See Junttila, T.T., 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, there is further provided a bispecific antibody according to the present invention, wherein the antibody is afucosylated.Alternatively or additionally, several other strategies have been reported to be used to achieve ADCC enhancement, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), all of which seek to improve Fc binding to the low-affinity activating FcγRIIIa and / or reduce binding to the low-affinity inhibitor FcγRIIb.

[0206] The antibodies preferably comprise a common light chain.

[0207] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the amino acid sequence: CDR1:SFGIS CDR2:GFIPVLGTANYAQKFQG CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:SX1TFTIS, CDR2:GIIPX2FGTITYAQKFQG, CDR3:RGNWNPFDP, (In the sequence, X1=K or R, 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, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the amino acid sequence: CDR1:SKTLTIS, CDR2:GIIPIFGSITYAQKFQD, CDR3:RGNWNPFDP, or Amino acid sequence: CDR1:GSGIS, CDR2:GFIPFFGSANYAQKFRD, CDR3:RGNWNPX 13 DP (In the sequence, X 13 = or L or F).

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

[0211] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the 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).

[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, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or Includes EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0214] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the amino acid sequence: CDR1:SYALS, CDR2:GISGSGRTTWYADSVKG, CDR1, CDR2, and CDR3, including CDR3: DGGYSYGPYWYFDL.

[0215] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising 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, wherein the heavy chain variable region has the amino acid sequence: CDR1:SYALS, CDR2:AISGSGRTTWYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DGGYTYGPYWYFDL.

[0216] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the amino acid sequence QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or Contains QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS, At one or more positions other than the CDRs, there are 0 to 10, preferably 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof.

[0217] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 has the amino acid sequence: CDR1:DYTMH, CDR2:DISWSSGSIGYADSVKG, CDR1, CDR2 and CDR3 include CDR3: DHRGYGDYEGGGFDY.

[0218] The present invention further provides a bispecific antigen-binding protein, preferably a bispecific antibody, comprising 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, wherein the heavy chain variable region has the 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, Preferably, X7, X8, X9 and X 10 are S, S, I and G, or are G, S, I and Y, or are S, T, T and G, and preferably X 11 and X 12 are R and H, or R and Y, or M and Y, and more preferably X7, X8, X9, X 10 , X 11 and X 12are S, S, I, G, R and H, or are G, S, I, Y, R and Y, or are 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 are R and H, or X7, X8, X9 and X 10 are G, S, I and Y, and X 11 and X 12 are R and Y, or X7, X8, X9 and X 10 are S, T, T and G, and X 11 and X 12 is M and Y).

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

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

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

[0222] The present invention further provides an antibody or derivative thereof of the present invention, or a pharmaceutical composition of the present invention, for use in treating a subject in need thereof. For treating a subject having or at risk of having a tumor, the antibody is preferably a bispecific antibody of the present invention. Preferably, the CD3-binding antibody comprises a heavy chain / 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 treating solid or hematological tumors. Preferred solid tumors are of epithelial origin and are gynecological cancers such as ovarian and endometrial tumors, prostate cancer, brain cancer, or any other solid tumor.

[0224] The CD3 / tumor antigen bispecific antibody or derivative thereof of the present invention, or a pharmaceutical composition comprising such a bispecific antibody or derivative thereof, is provided for use in the treatment of various leukemias and preleukemias of myeloid origin, as well as in the treatment of B-cell lymphomas. Diseases that can be treated according to the present invention include acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML), as well as myeloid leukemias or preleukemias, such as Hodgkin's lymphoma and most non-Hodgkin's 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). Use of a bispecific IgG antibody according to the present invention in the preparation of a medicament for the treatment or prevention of MDS, CML, MM, or preferably AML is also provided.

[0225] The amount of an antibody according to the present invention to be administered to a patient typically falls within the therapeutic window, meaning that an amount sufficient to achieve a therapeutic effect is used, but that the amount does not exceed a threshold that causes unacceptable side effects. The smaller the amount of antibody required to achieve 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 each year in Europe and the United States. The majority of these patients are aged 60 years or older. Advanced age is the major negative determinant of prognosis in AML, with long-term (5-year) survival rates for intensively treated elderly AML patients being approximately 10%. In almost all patients who achieve remission during induction chemotherapy, disease progression is observed within 3 years. Current postremission treatments have shown limited, if any, value in elderly patients with AML. Consequently, a significant burden of residual resistant leukemia remains, and a surviving subpopulation of drug-resistant leukemia cells rapidly develops relapse. In efforts to induce and maintain complete remission, novel classes of drugs with distinct modes of action to target these chemotherapy-unresponsive AML tumor cells are needed. Complete remission (CR) can be achieved with several intensive chemotherapy combinations in more than 50% of elderly AML patients and approximately 80% of younger patients, but increasing response rates or survival remains a major investigational challenge. A recently published network meta-analysis of 65 randomized clinical trials (15,110 patients) in elderly patients with AML found that the majority of modified induction regimens had comparable or worse efficacy profiles compared with the traditional 3+7 induction regimen using daunorubicin and cytarabine. This standard treatment for AML is associated with high morbidity and even mortality. The majority of patients in CR relapse due to residual leukemic stem cells after chemotherapy. Further dose escalation is limited by unacceptable toxicity. Therefore, there is an urgent need for new treatment modalities, preferably with less toxicity, especially in elderly patients with AML.

[0227] Treatment of AML that does not respond to chemotherapy can be achieved by redirecting T cells from the patient's own immune system to AML tumor cells, followed by tumor-specific activation of the T cells using bispecific antibodies. This process is also known as the "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 toward AML tumor cells, thereby inducing their lysis. [Brief explanation of the drawings]

[0228] [Figure 1]Assessment of functional activity: T cell cytotoxicity assay using BxPC3 target cells upon treatment with EGFRxCD3 bispecific antibodies. Each bispecific antibody contains a CD3-binding domain comprising a heavy chain variable region designated by an MF number and an EGFR-binding domain comprising the heavy chain variable region MF8233. These variable regions are paired with a common light chain to form the EGFRxCD3 bispecific antibody. Affinity for BxPC3 lysis (HPB-ALL binding). Certain antibodies of the present invention exhibit 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. Clearly, the relatively low affinity does not necessarily inhibit tumor antigen-mediated T cell cytotoxicity of BxPC3 cells (BxPC3 lysis, vertical axis). The bispecific antibodies MF8233xMF8508 and MF8233xMF8057 can efficiently lyse BxPC3 cells, whereas the bispecific antibodies MF8233xMF8397 and MF8233xMF9249 have similar binding but do so less efficiently. Furthermore, in a comparison of bispecific antibodies, the bispecific antibody MF8233xMF6955 binds to HPB-ALL (i.e., human CD3) with higher affinity, but lyses BxPC3 less efficiently than the bispecific antibodies MF8233xMF8508 and MF8233xMF8057, which bind to CD3 to a lesser extent. MF6955 is a heavy chain variable region combined with a common light chain and used as a reference sequence for comparison, and corresponds to H1H7232B(1129)VH in U.S. Patent Application Publication No. 2014 / 0088295(A1). MF6955 and a comparative benchmark bispecific antibody having the same separate EGFR-binding domain have higher affinity for human CD3 than MF9267 and exhibit more efficient killing than MF9267. In contrast, other antibodies incorporating a CD3-binding domain of the invention, such as MF8058, have approximately the same binding activity as MF6955 but demonstrate more efficient killing of BxPC3 cells, such as MF8233xMF8058. Other bispecific antibodies comprising a binding domain of the invention capable of binding to CD3 exhibit relatively high binding and more efficient killing, such as MF8233xMF8078, which are useful for certain applications described herein.In contrast, other bispecific antibodies comprising a binding domain of the invention capable of binding to CD3, such as MF8233xMF9249 and MF8233xMF8397, exhibit relatively low affinity and low killing, but are useful for the alternative applications described herein. [Figure 2] 1 is an antibody titration curve showing the ability to induce % T cell-mediated killing of BxPC3 target cells compared to a no antibody control. Curves are shown for antibodies MF8233xMF8078, MF8233xMF8397, and MF8233xMF8508. [Figure 3] This figure summarizes titration curve data for various bispecific antibodies in T cell cytotoxicity with BxPC3 target cells. The CD3 Fab column indicates the MF number of the CD3-binding arm. The EGFR arm has the indicated MF number 8233. This column indicates the supercluster number for a set of variants based on the same VH gene segment. The CD3 binding column reflects the results of the HBP-ALL binding experiment. The results of two independent cytotoxicity assays to determine the ability to induce T cell-mediated lysis of BxPC3 target cells are shown. [Figure 4] Figure 1 shows T cell activation in a T cell cytotoxicity assay using BxPC3 target cells on CD8+ T cells by expression. Figure 2 shows antibody titration curves for various supercluster numbers, representing variants of the CD3-binding domain. The other arm of the bispecific antibody contains the heavy chain-binding domain of MF8233. For comparison, the bispecific antibodies MF8233 x MF6955 and MF8233 x MF6964 were also tested. MF6955 and MF6964 are heavy chain variable regions combined with a common light chain and used as reference sequences, corresponding to H1H7232B(1129)VH and HH7241B(1145), respectively, in U.S. Patent Application Publication No. 2014 / 0088295(A1). [Figure 5]Figure 11 is a summary of titration curve data for various antibodies in a T cell cytotoxicity assay for T cell activation with BxPC3 target cells. The MF nr. column indicates the MF number of the CD3-binding domain. The EGFR-binding domain has the indicated MF number 8233. Supercluster information for various CD3-binding domain sequences is shown in the column "Supercluster." The column showing CD3 affinity reflects the results of HBP-ALL binding experiments. CD4+ and CD8+ cell results are shown for the markers CD69 and CD25. The bispecific antibodies shown are examples from a larger pool of bispecific antibodies. [Figure 6] Assessment of functional activity: T cell cytotoxicity assay using BxPC3 target cells. Affinity (HPB-ALL binding) measured by CD69 expression versus CD8+ T cell activation. Certain antibodies of the present invention exhibit 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. Such affinity does not necessarily inhibit tumor antigen-mediated T cell activation, as exemplified by the results of the CD8+ CD69 activation assay. Bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently activate T cells, whereas bispecific antibodies MF8233×MF8397 and MF8233×MF9249 do not. Certain CD3-binding domains that do not efficiently bind to these cells also do not activate T cells (see bottom left corner). Other CD3-binding domains, MF8508 and MF8057, bind less well to HPB-ALL cells than the reference CD3-binding domain, MF6955, and activate T cells to a similar extent, for example. Other bispecific antibodies comprising a binding domain of the invention capable of binding to CD3 exhibit relatively high binding and high levels of activation, such as MF8078, which is used in certain applications described herein. In contrast, other bispecific antibodies comprising a binding domain of the invention capable of binding to CD3, such as MF9249 and MF8397, exhibit relatively low affinity and activation, but are useful in alternative applications described herein. [Figure 7]Functionality assessment: T cell cytotoxicity assay using HCT116 target cells. Affinity for HCT-116 lysis (HPB-ALL binding). Certain bispecific antibodies of the invention exhibit 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. Clearly, 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. For comparison, the bispecific antibodies MF8233xMF6955 and MF8233xMF6964 bind to HPB-ALL (i.e., human CD3) with higher affinity than, for example, MF8233xMF8508, MF8233xMF8057, and MF8233xMF9267, but do not lyse HCT-116 cells more efficiently than MF8233xMF8508 or MF8233xMF9267 in tests such as those presented herein, or significantly exceed MF8233xMF8057 in terms of binding differences. Other bispecific antibodies comprising binding domains of the invention capable of binding to CD3 exhibit relatively high binding and high levels of killing, such as MF8078, which finds use in certain applications described herein. In contrast, other bispecific antibodies comprising binding domains of the 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] 1 is an antibody titration curve for a T cell cytotoxicity assay using HCT-116 target cells showing the % killing of HCT-116 cells compared to a no antibody control. Curves are shown for various bispecific antibodies. [Figure 9]This figure summarizes titration curve data for various antibodies in a T cell cytotoxicity assay with HCT-116 target cells. The CD3 Fab column indicates the MF number of the CD3-binding domain. The EGFR-binding domain has the indicated MF number 8233. This column indicates the supercluster number for configuring variants based on the same VH gene segment. The CD3 binding column reflects the results of the HBP-ALL binding experiment. The lysis rate and EC50 values ​​(ng / mL) for lysis of HCT-116 cells are shown in the next column. The bispecific antibodies shown are examples from a larger pool of bispecific antibodies. [Figure 10] 10A and 10B present schematic diagrams of the MV1624 and MV1625 expression vectors. [Figure 11A] Figure 1 shows the common light chain used in monospecific and bispecific IgG. Figure 2 shows the common light chain amino acid sequence. [Figure 11B] This is the common light chain used in monospecific and bispecific IgG. This is the DNA sequence and translation of the common light chain variable domain (IGKV1-39 / jk1). [Figure 11C] This is the common light chain used in monospecific and bispecific IgG. This is the DNA sequence and translation of the common light chain constant region. [Figure 11D] This is the 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. The V region is IGKV1-39A. [Figure 11F] Common light chain used in monospecific and bispecific IgG. CDR1, CDR2, and CDR3 of the common light chain. [Figure 12A] IgG heavy chain for the generation of bispecific molecules. CH1 region. [Figure 12B] IgG heavy chain for the generation of bispecific molecules. Hinge region. [Figure 12C] IgG heavy chain for the generation of bispecific molecules. CH2 region. [Figure 12D] IgG heavy chain for the generation of bispecific molecules. CH2 containing L235G and G236R silencing substitutions. [Figure 12E] IgG heavy chain for the generation of bispecific molecules. CH3 domain containing substitutions L351K and T366K (KK). [Figure 12F] IgG heavy chain for the generation of bispecific molecules. CH3 domain containing substitutions L351D and L368E(DE). [Figure 13-1] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-2] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-3] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-4] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-5] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-6] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-7] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-8] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-9] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-10] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-11]Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-12] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-13] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-14] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-15] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-16] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-17] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-18] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-19] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-20] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-21] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-22] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-23] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 13-24] Described herein are various DNA coding sequences and amino acid sequences for heavy chain variable regions and portions thereof. [Figure 14A] Characterization of further clones from supercluster 1 in comparison with clones MF8057 and MF8058. Binding of selected MF clones to HPB-ALL human cells expressing the human CD3-TCR complex in a FACS assay. [Figure 14B] Characterization of further clones from supercluster 1 in comparison to clones MF8057 and MF8058. T cell cytotoxicity assay with HCT-116 cells showing % killing of HCT-116 cells. [Figure 14C] Characterization of further clones from supercluster 1 compared to clones MF8057 and MF8058. Quantification of activation markers CD25 and CD69 by FACS, indicative of T cell activation. [Figure 14D] Characterization of further clones from supercluster 1 compared to clones MF8057 and MF8058. Quantification of activation markers CD25 and CD69 by FACS, indicative of T cell activation. [Figure 14E] Characterization of further clones from supercluster 1 compared to clones MF8057 and MF8058. Quantification of activation markers CD25 and CD69 by FACS, indicative of T cell activation. [Figure 14F] Characterization of further clones from supercluster 1 compared to clones MF8057 and MF8058. Cytokine production in supernatants from cytotoxicity assays. [Figure 14G] Characterization of further clones from supercluster 1 compared to clones MF8057 and MF8058. Cytokine production in supernatants from cytotoxicity assays. [Figure 15A] Characterization of clones from supercluster 4. Binding of selected MF clones to HPB-ALL human cells. [Figure 15B]Characterization of clones from supercluster 4. T cell cytotoxicity assay with BxPC3 cells showing % killing of BXP3 cells. [Figure 15C] Characterization of clones from supercluster 4. Cytokine production in supernatants from cytotoxicity assays. [Figure 15D] Characterization of clones from supercluster 4. Cytokine production in supernatants from cytotoxicity assays. [Figure 15E] Characterization of clones from supercluster 4. Cytokine production in supernatants from cytotoxicity assays. [Figure 16A] Assessment of CD3 functional activity. Affinity on the X-axis for HCT-116 lysis (HPB-ALL) on the Y-axis for additional clones from supercluster 1 (MF8048, MF8101, MF8056), supercluster 3 (MF8562) and supercluster 4 (MF8998). [Figure 16B] Evaluation of CD3 functional activity. Antibodies belonging to supercluster 1 and supercluster 4 show similar activity in cytotoxicity assays and different binding affinities. [Figure 16C] Assessment of CD3 functional activity. Antibodies belonging to supercluster 1 and supercluster 3 display similar binding affinities and differential lytic activity. [Figure 17] Activity of CD3 Fabs MF8998 and MF8058 in the bispecific CD3xEGFR format. [Figure 18] FACS binding data for a large panel of IgGs specific for CD3. For antibodies MF5196, MF6955, and MF6964, binding was determined by BIAcore™ to the CD3δε-Fc antigen, while FACS binding data to HPB-ALL cells is shown for the rest of the clones. [Figure 19-1] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-2]1 is the nucleotide sequence of human CLEC12A. [Figure 19-3] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-4] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-5] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-6] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-7] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-8] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-9] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-10] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-11] 1 is the nucleotide sequence of human CLEC12A. [Figure 19-12] 1 is the nucleotide sequence of human CLEC12A. [Figure 20] Amino acid sequences of human CD3 γ-, δ-, ε-, and ζ-chains.

[0229] The following examples illustrate the invention. [Example]

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

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

[0232] Immunization of Memo® mice with CD3 To generate human antibodies that bind to CD3, mice transgenic for a human common light chain and a human heavy chain (HC) minilocus (containing selected human V gene segments, all human D, and all human J) (see WO 2009 / 157771, incorporated herein by reference) were immunized with TCR / CD3-containing lipoparticles (Intergral Molecular). These mice are referred to as "MeMo®" mice. For specific heavy chain variable regions or trivalent multimers having the sequences disclosed herein, these can be prepared by any means known to those of skill in the art.

[0233] MeMo® mice were immunized with Hek293T-derived human 5D5M TCR / CD3 containing lipoparticles, followed by immunization with human T cells for the generation of anti-TCR / CDR3 immune responses and generation of an anti-TCR / CD3 antibody panel.

[0234] Lipoparticles concentrate structurally intact membrane proteins directly from the cell surface, allowing these complex proteins to be manipulated as soluble, highly abundant 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, 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® mice were used for immunization with TCR / CD3 lipoparticles and primary human T cells.

[0238] The immunization schedule included time points on days 35, 56, 77, and 98, and antigen-specific Ig serum titers were determined by ELISA using 3SDX TCR / CD3 positive and negative lipoproteins from QTG 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 developed relevant anti-TCR / CD3 responses.

[0239] For all immunized mice, lymphoid material for antibody detection was collected and stored as follows: The titer is 1 / 300 in human TCR / CD3 (in an ELISA using lipoparticles), or Titers were less than 1 / 300 and more than 1 / 100 in human TCR / CD3 and did not increase during the final booster immunization.

[0240] Priming immunisation with lipoparticles To prime the humoral immune response in MeMo® mice for TCR / CD3, lipoparticles containing the human 5D5M TCRαβ combination were used for immunization. The lipoparticles were used with Gerbu adjuvant for the first and second injections.

[0241] Booster immunization with 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 adjuvant in PBS, and all subsequent injections consisted of cells in PBS alone. Mice that developed a serum IgG titer of 1 / 300 against human TCR / CD3 (measured by ELISA using lipoparticles) on day 35 received additional injections of cells on days 42, 43, and 44. Mice that failed to meet these criteria received booster immunizations with cells (days 42 and 49). All subsequent immunizations were given as subcutaneous injections of cells in PBS. After the final immunization, mice were sacrificed, and serum, spleens, and left inguinal lymph nodes were collected.

[0242] Screening of sera from immunized mice in ELISA Preliminary serum IgG titers were screened by ELISA using TCR / CD3-containing lipoparticles and "null" lipoparticles. Serum IgG titers were measured using anti-mouse IgG staining, as this staining was shown to be the most sensitive.

[0243] Generation of "immune" phage antibody repertoires by RT-PCR cloning of VH genes Inguinal lymph nodes from successfully immunized mice were used to construct "immune" phage antibody repertoires. RNA was extracted from lymphoid tissue using Trizol LS, and 1 μg of total RNA was used in RT reactions using IgG-CH1-specific primers. The resulting cDNA was then used to amplify a polyclonal pool of VH-encoding cDNAs using proprietary VH-specific primers, essentially as described by Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581-97). To display Fab fragments on phage, the resulting PCR products were then cloned into phagemid vectors as described by de Haard et al. (J Biol Chem. 1999 Jun 25;274(26):18218-30), except that the light chains were the same for all antibodies and encoded by the vector. After ligation, the phagemids were used to transform E. coli TG1 bacteria, and the transformed bacteria were plated on LB-agar plates containing ampicillin and glucose. All phage libraries contained >10e6 transformants and had insert frequencies of >80%. Bacteria were harvested after overnight growth and used to prepare phage according to established protocols (de Haard et al., J Biol Chem. 1999 Jun 25;274(26):18218-30).

[0244] Selection of phage carrying Fab fragments that specifically bind to human CD3. The phage library was rescued according to standardized procedures (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 immune phage antibody repertoire selection. In the first round, recombinant CD3 protein was coated onto the wells of maxisorp™ ELISA plates or NUNC immunotubes, whereas 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 library was also blocked with 4% ELK, and human IgG was used in excess to deplete Fc region binders before adding the phage library to the coated antigen.

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

[0246] The infected bacteria were then plated 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, then rescued and amplified to prepare enriched first-round phage pools 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 As used herein, bispecific antibodies typically differ from each other only in specific amino acid sequences in the heavy chain variable region of one or both of the variable domains. The heavy chain variable region is generated by cloning into an expression vector for the expression of the heavy and light chains. Methods for producing bispecific antibodies are known in the art.

[0248] Briefly, DNA encoding the heavy chain variable region of the CD3-targeting variable domain was cloned into the MV1624 vector (see Figure 10A), which encodes KK residues (L351K, T366K) in the CH3 region for the generation of IgG heavy chain heterodimers (WO 2013 / 157954 and WO 2013 / 157953). The Fc constant region contains mutations in CH2 to silence Fc effector function. The DNA encoding the heavy chain variable region replaces the stuffer region in the construct. The variable region is preceded by an encoded HC signal peptide (not shown). 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 with L351D-L368E mutations in the CH3 region (WO 2013 / 157954 and WO 2013 / 157953). The DNA encoding the heavy chain variable region replaces the stuffer region in the construct. The variable region is preceded by an encoded HC signal peptide (not shown). Both constructs also contain an expression cassette for the expression of the IGKV1-39 / jk1 light chain. Expressing the two heavy chains together with the light chains described above results in the production of a bispecific antibody.

[0249] 293-F cells were used for expression of the designed antibodies in a 24-well plate format. Two days before transfection, 293-F cell stock was split at a 1:1 ratio into 293-F culture medium and incubated overnight at 37°C and 8% CO with an orbital shaking speed of 155 rpm. Cells were diluted to 5x10 cells the day before transfection. 5The cells were diluted to a density of 1000 cells / mL. 4 mL of suspended cells were seeded into a 24-deep-well plate covered with a breathable seal and incubated overnight at 37°C and 8% CO2 with an orbital shaking speed of 285 rpm. On the day of transfection, 4.8 mL of 293-F culture medium was mixed with 240 μg of linear polyethyleneimine (PEI) (MW 25,000). 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 for IgG heterodimers). The mixture was incubated at room temperature for 20 minutes before being gently added to the cells. The day after transfection, 500 μL of penicillin-streptomycin (Pen Strep) diluted in 293F medium was added to each well. Plates were incubated at 37°C and 8% CO with an orbital shaking speed of 285 rpm until harvested 7 days after transfection. Plates were centrifuged for 5 min at 500 g and the supernatant containing IgG was filtered using 10-12 μm melt-blown polypropylene filter plates and stored at -20°C before purification.

[0250] Purification of antibodies from culture supernatant The antibody-containing medium is collected and centrifuged to remove cell debris. Protein A Sepharose beads are then added to the medium. The medium and Protein A Sepharose beads are incubated with the antibody to allow binding.

[0251] After incubation, the beads are isolated from the medium and washed by vacuum filtering. The antibody is eluted from the beads by incubation with an elution buffer.

[0252] Optionally, buffer exchange / desalt 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. PBS or the required buffer is then added to the fraction to replace the buffer with a low-salt buffer. Optionally, this centrifugation step followed by the addition of buffer is repeated to further desalt the antibody storage buffer.

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

[0255] Resting T cells were isolated from whole blood of healthy donors using Ficoll and EasySep human T cell isolation kits according to standard procedures. Flow cytometry analysis was used to confirm T cell purity at >95% with an anti-CD3 antibody, followed by cryopreservation. For cytotoxicity assays, cryopreserved T cells were thawed and used if their viability at thawing was >90%, as measured by standard trypan blue staining. Briefly, thawed resting T cells were co-cultured with BxPC3 or HCT116 target cells at a 5:1 E:T ratio for 48 hours. Antibodies were tested at a range of dilutions. CD3 and EGFR monospecific antibodies, as well as an irrelevant IgG1 isotype control mAb, were included in the assay as controls (e.g., an antibody that binds to CD3 and another antigen, such as tetanus toxoid (TT)). T cell activation was quantified using flow cytometry; CD8 T cells were gated based on CD8 expression and subsequently analyzed for their activation status by measuring CD69 expression on 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 on an Envision microplate reader yielded relative light unit (RLU) values, which were analyzed using GraphPad Prism.

[0256] The target cell lysis for each sample was calculated as follows: % Killing = (100 - (RLU sample / RLU no IgG) x 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 of MF8233. The CD3-targeting binding domain has a VH with the amino acid sequence of one of the MFs designated for CD3. The bispecific antibody contains mutations in CH2 to silence Fc effector function.

[0258] Antibodies MF8233 x MF8397 induced upregulation of CD69 (Figures 4-6) and CD25 (Figures 4-6) on CD4 and CD8 T cells after 48 hours of coculture at an E ± T ratio of 5:1. 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 assessed by flow cytometry (following the FACS procedure previously described in WO 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 standard procedures known in the art. Binding to cell-expressed CD3 can be confirmed using CHO cells transfected with CD3δ / ε or CD3γ / ε. Binding of candidate bispecific IgG1s to EGFR can be determined using BxPC3 and HCT-116, as well as CHO cells transfected with an EGFR expression construct; CD3 and EGFR monospecific antibodies, as well as an irrelevant IgG1 isotype control mAb, are included in the assay as controls (e.g., an antibody that binds to CD3 and another antigen, such as tetanus toxoid (TT)).

[0260] Generation of further clones from superclusters 1, 3 and 4 From immune phage library screening (as described in the section "Selection of phages carrying Fab fragments that specifically bind to human CD3"), additional clones were characterized as carrying Fab fragments that specifically bind to human CD3. Additional clones were identified from supercluster 1, including MF8048, MF8101, and MF8056. Additional clones were identified from supercluster 3 and supercluster 4, including MF8562 in supercluster 3 and MF8998 in supercluster 4.

[0261] Further new clones were identified from supercluster 4 using next-generation sequencing (NGS) analysis. NGS was performed on the VH gene pool present from MeMo® mice used to generate the anti-CD3 panel. For this purpose, sequence datasets obtained from different mice were compared with the MF sequences belonging to supercluster 4. This led to the identification of sequence variant clones MF10401 and MF10428 belonging to supercluster 4. For the different sequences, several different 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 CD3xEGFR bispecific format for further characterization as described in the section "Antibody cloning and production" above.

[0263] Characterization of further clones from superclusters 1 and 4 Additional clones from Supercluster 1 were characterized for their functional activity in a bispecific format. The EGFR-binding domain of the bispecific CD3×EGFR antibody 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 those of previously characterized MF clones from Supercluster 1, as described above in "Antibody Tumor Antigen-Specific T Cell Activation and Lysis of BxPC3 Cells or Lysis of HTC-116 Cells" and "Characterization of CD3 Bispecific Antibodies." Binding affinity to HPB-All cells expressing the human CD3-TCR complex was assayed using flow cytometry (Figures 14A and 18), as well as T cell activation and lysis of tumor antigen-positive target cells (HCT-116) in a cytotoxicity assay (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 with MF8048. Expression levels of 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 with the negative control. Finally, cytokine production of IFN-γ and TNF-α was measured in supernatants from the EGFR×CD3 cytotoxicity assay using HCT-116 cells after 48 hours using the Luminex® assay (eBiosciences™) according to the standard manufacturer's instructions (Figure 14F-G).

[0264] To further characterize clones belonging to supercluster 4, their binding affinity to HPB-ALL cells was determined by FACS (Figure 15). PG1337, a monovalent antibody with two identical MF1337 arms specific for 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 x TAA bispecific antibody with known high activity was included as a positive control. Target cell lysis was quantified using cell viability measurements. Supernatants from the cytotoxicity assays were used to measure cytokine levels of IL-6, IFN-γ, and TNF-α using a Luminex® assay.

[0265] Thus, the three Supercluster 4 clones tested were found to exhibit different binding but similar lytic activity. Although the lytic activity of these clones was similar, reduced cytokine production was observed. [Table 1] [Table 2] [Table 3]

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

[0267] Characterization of the 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 the Fab arm binding EGFR as the tumor cell antigen (Figure 17). In this experiment, lytic activity against HCT-116 cells was measured as described herein above. MF9257xMF8233 was used as a positive control, and MF9257xMF1337 served as a negative control. As can be seen from Figure 17 and Table 5, a high dose-dependent killing rate was observed for multiple tested bispecific antibodies. [Table 5]

[0268] binding affinity As described in the section "Characterization of CD3 Bispecific Antibodies," the binding affinities of additional CD3 clones were analyzed by FACS on HPB-ALL cells expressing human CD3. The affinities of MF6955 and MF6964 for CD3 were measured by surface plasmon resonance (SPR) technology using a BIAcore™ T100. An anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat. Nr. 555784) was coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The CD3×TAA bispecific antibody was then captured on this sensor surface. Subsequently, recombinant purified antigen human CD3δε-Fc was flowed over the sensor surface at a range of concentrations to measure the on- and off-rates. After each cycle, the sensor surface was regenerated with a pulse of HCl, and the CD3×TAA bispecific antibody was recaptured. From the resulting sensorgrams, the on- and off-rates were determined using BIAevaluation software. Figure 18 illustrates the binding affinity range of the generated CD3 panel.

Claims

1. 1. An antigen binding protein that binds to human CD3, 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 amino acid sequence: CDR1: GSGIS, CDR2: GFIPFGSANYAQKFRD, CDR3: RGNWNPX 13 DP (wherein X 13 =L or F) or CDR1:RX 3 WIG, CDR2: IIYPGDSDTRYSPSFQG, CDR3:X 4 IRYFX 5 WSEDYHYYX 6 DV (In the sequence, X 3 =F or Y; X 4 =H or N; X 5 =D or V; X 6 =L or M) or CDR1: SYALS, CDR2:GISGSGRTTWYADSVKG, CDR3: DGGYSYGPYWYFDL or CDR1: SYALS, CDR2: AISGSGRTTWYADSVKG, CDR3: DGGYTYGPYWYFDL an antigen binding protein comprising CDR1, CDR2 and CDR3 comprising:

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 amino acid sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPLDPWGQGTLVTVSS, or EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGGFIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRGNWNPFDPWGQGTLVTVSS, or EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHIRYFDWSEDYHYYLDVWGKGTTVTVSS, or EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNIRYFVWSEDYHYYMDVWGKGTTVTVS, or QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSGISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSYGPYWYFDLWGRGTLVTVSS, or QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS, An antigen-binding protein having 0 to 10, preferably 0 to 5, amino acid mutations, insertions, deletions, substitutions, additions, or a combination thereof, at one or more positions other than the CDRs.

3. The antigen-binding protein of claim 1, wherein the light chain variable region comprises a common light chain variable region.

4. The antigen-binding protein of claim 3, wherein the common light chain variable region comprises an IgVκ1-39 light chain variable region.

5. The antigen-binding protein according to claim 1, wherein the light chain variable region is a germline IgVκ1-39*01 variable region.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein the light chain variable region comprises kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01.

7. An antigen-binding protein according to any one of claims 1 to 6, wherein the light chain variable region comprises germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01.

8. The light chain variable region comprises 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 8. The antigen-binding protein of any one of claims 1 to 7, comprising EIK and having 0 to 5 amino acid mutations, insertions, deletions, substitutions, additions or combinations thereof.

9. An antigen-binding protein according to any one of claims 1 to 8, which is an antibody, preferably a bispecific antibody.

10. The antigen-binding protein according to claim 9, comprising an H / L chain combination according to any one of claims 1 to 8 and an H / L chain combination that binds to a tumor antigen.

11. The antigen-binding protein of claim 10, wherein the H / L chain combination that binds to a 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.

12. The antigen-binding protein of any one of claims 9 to 11, which is a human or humanized antibody.

13. An antigen-binding protein according to any one of claims 9 to 12, wherein the bispecific antibody comprises two different immunoglobulin heavy chains with compatible heterodimerization domains.

14. The antigen-binding protein of claim 13, wherein the compatible heterodimerization domain is a compatible immunoglobulin heavy chain CH3 heterodimerization domain.

15. An antigen-binding protein according to any one of claims 9 to 14, wherein the bispecific antibody is an IgG antibody and has a mutated CH2 and / or lower hinge domain such that interaction of the bispecific IgG antibody with an Fc gamma receptor is reduced.

16. An antigen-binding protein according to claim 15, wherein the mutated CH2 and / or lower hinge domain comprises an amino acid substitution at position 235 and / or 236 (according to EU numbering), preferably an L235G substitution and / or a G236R substitution.

17. The antigen-binding protein of any one of claims 9 to 16, wherein the bispecific antibody comprises a common light chain.

18. An antigen-binding protein according to claim 17 for use in treating a subject in need thereof.

19. The antigen-binding protein of claim 18, wherein the treatment comprises administering the antigen-binding protein of claim 1 or 2 to a subject with an overactive immune system.

20. The antigen-binding protein of claim 19, wherein the subject has an autoimmune disease.

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