Human CD3 binding antibodies

Abstract

It is an object of the present invention to provide variants of antibodies 3056 with essentially the same kind of CD3 binding properties but with improved, not necessarily quantitative, characteristics.SOLUTION: The present invention relates to human CD3 binding antibodies comprising a heavy chain and a light chain, wherein said heavy chain comprises a variable region comprising the amino acid sequence QVQLVQSGGGVVQPGRSLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVAAIWYX1X2RKQDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGTGYNWFDPWGQGTLVTVSS with 0-5 amino acid insertion (s), deletion (s), substitution (s), addition (s) or a combination thereof. human CD3 binding antibodies. The present disclosure also relates to a bispecific antibody having a heavy chain as defined herein above. Methods of production of the antibodies, cells producing the antibodies and (medical) uses of the antibodies are provided.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 treatment. More particularly, the present invention relates to antibodies, preferably bispecific 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 generally used, for example, in transplant rejection, due to their immunosuppressive properties. Bispecific antibodies directed against CD3 on T cells and surface target antigens on cancer cells are capable of binding 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 hold great promise for the treatment of various cancers and neoplastic growths.

[0003] WO2014 / 051433 (herein incorporated by reference) describes CD3 mAbs that are suitable candidates to serve as building blocks in the generation of bispecific antibodies that act as T cell-associated molecules. These CD3 mAbs are designated 3056 and 3896, and the VH and VL sequences of these mAbs are disclosed in Figure 22. Both 3056 CD3 mAb and 3896 CD3 mAb have good properties in terms of functional activity. They bind to CD3 / TCR expressed on the cell surface of human T cell lines with significantly lower affinity (KD) than that of the well-known murine anti-CD3 antibody mOKT3, resulting in a lower affinity for CD3. POS They produce lower mean fluorescence intensities in flow cytometry analysis of cells. Similarly, they induce proliferation of T cells when immobilized on tissue culture plates, but to a lesser extent than mOKT3.

[0004] Without being bound by theory, it is believed that a significantly lower affinity for CD3 than that of mOKT3 is preferred in bispecific T cell engagement formats. Preferably, the bispecific antibody binds to CD3 with an affinity lower than its binding affinity for the tumor antigen. Without being bound by theory, it is believed that this affinity difference allows the bispecific antibody to preferentially opsonize tumor cells, thereby marking them for destruction by nearby immune effector cells, including NK cells and / or T cells.

[0005] The inventors observed that the results obtained with antibody 3056 showed batch-to-batch variation, which surprised them, since no variation occurred with antibody 15C3 (described in WO2005 / 118635), which has the same VH sequence as antibody 3056 but a different light chain. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2014 / 051433 [Patent Document 2] WO2005 / 118635 [Patent Document 3] WO2004 / 009618 [Patent Document 4] WO2009 / 157771 [Patent Document 5] U.S. Patent Application No. 20030078385 [Patent Document 6] US13 / 866,747 (US9,248,181) [Patent Document 7] US14 / 081,848 (US9,358,286) [Patent Document 8] PCT / NL2013 / 050294(WO2013 / 157954) [Non-patent literature]

[0007] [Non-licensed Document 1] Bakker A.ら, Cancer Res. 2004, 64, pages 8843~50 [Non-licensed Document 2] AS Marshall, J Biol Chem, 2004, 279, pp. 14792-802. [Non-licensed Document 3] Y.Hanra, Blood, 2004, 104, pp. 2858-66 [Non-licensed Document 4] CH Chenら, Blood 2006, pages 107, 1459~67 [Non-licensed Document 5] Van Rhenenら, 2007 Blood 110 [Non-licensed Document 6] Moshaverら, 2008 Stem Cells 26: 3059 pages [Non-licensed Document 7] Oncogene. May 23, 2013;32(21):2670-81. doi: 10.1038 / onc.2012.280. Epub July 16, 2012 [Non-licensed Document 8] Spiess, C., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015) http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003 [Non-licensed Document 9] Cui, JBC 2012 (287) pp. 28206~28214 [Non-licensed Document 10] Kontermann, MABS, 2012(4), pp. 182-197 [Non-licensed Document 11] Chames and Baty, MABS 2009(1) pp. 539-547 [Non-licensed Document 12] Moore, Blood, 2011 (117), pp. 4542-4551 [Non-Patent Document 13] Loffler et al., 2000, Blood 95:2098 [Non-Patent Document 14] Zeidler et al., 1999, J. Immunol. 163:1246 [Non-Patent Document 15] Gunasekaran et al., JBC 2010(285) 19637-19646 [Non-Patent Document 16] Sali et al., 1993: J. Mol. Biol. 234, pp. 779-815. [Non-Patent Document 17] Liu et al., Journal of Pharmaceutical Sciences, Vol. 97, pp. 2426-2447 (2008) [Non-Patent Document 18] De Kruif et al., Biotechnol Bioeng. 2010(106)741-50 Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to provide variants of antibody 3056 which have essentially the same CD3 binding properties in kind, but not necessarily in quantity, and which have improved characteristics. [Means for solving the problem]

[0009] The present invention provides an antibody that binds to human CD3, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof at one or more positions other than the positions indicated by X1X2: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] The present invention provides an antibody comprising a variable region comprising:

[0010] The present invention further provides an amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than the positions represented by X1X2: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] Nucleic acid molecules encoding the

[0011] The combination X1 and X2 in the antibodies described herein is preferably such that X1=N and X2=A.

[0012] The invention further provides cells that express the antibodies and / or contain the nucleic acid molecules.

[0013] Unless otherwise specified, the antibodies of the present invention are preferably bispecific antibodies. Bispecific antibodies preferably bind to at least human CD3. In addition, bispecific antibodies preferably bind to at least a surface molecule preferentially expressed on human tumor cells. In preferred embodiments, the bispecific antibody binds to 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, or PSMA. In a more preferred embodiment, the bispecific antibody binds to CLEC12A.

[0014] The present invention further provides a pharmaceutical composition comprising an antibody according to the present invention.

[0015] Further provided is an antibody according to the invention further comprising a label, preferably a label for in vivo imaging.

[0016] The present invention also provides a method for treating a subject having or at risk of having a tumor, comprising the step of administering to the subject a bispecific antibody according to the present invention. A bispecific antibody according to the present invention for use in treating a subject having or at risk of having a tumor is also provided. Furthermore, there is provided use of an antibody of the present invention for preparing a medicament for treating a subject having or at risk of having a tumor. In a preferred embodiment, the tumor is a CLEC12A-positive tumor. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows IEF of PG3055 and PG3056. [Figure 2] FIG. 1 shows CEX-HPLC analysis of PG3055 and PG3056. [Figure 3]Batch-to-batch variability and stability of PG3055 and PG3056 analyzed by CEX-HPLC analysis. Left: Batch aging of 3055; right: Batch aging of 3056. The top sample was stored at -80°C for >3 months, and the bottom sample was stored at 2-8°C for >3 months. [Figure 4] Flow cytometry analysis of the binding of PG3055 and PG3056 to CD3 on purified human T cells after incubation in IMDM + 10% FBS medium at 2-8°C or 37°C for 7 days. PG3055 and PG3056: Incubated in IMDM + 10% FBS medium at 2-8°C for 7 days. PG3055 37°C and PG3056 37°C: PG3055 and PG3056 were incubated in IMDM + 10% FBS medium at 37°C for 7 days. [Figure 5] Left: Homology model of PG3056 Fab with expanded variable domain. VL is light gray, VH is black, and HCDR1-3 loops are light gray. Residues Asn54 and Gly55, which form the deamidation motif in HCDR2, are shown. Right: Superposition of the homology models of PG3055 (light gray) and PG3056 (dark gray) Fabs. [Figure 6] FIG. 1 shows the alignment of VH MF3056 (MF3056_VH) with the VH3-33 germline sequence. [Figure 7] FIG. 1 shows the alignment of VH MF3056 with VH MF3872, MF3873, and MF3905. [Figure 8] Figure 1 shows flow cytometry analysis of the binding of variants PG3872, PG3873 and PG3905 to CD3 on HPB-ALL cells. PG3056 was included as a benchmark control. [Figure 9] FIG. 1 shows the alignment of VH of MF3056 with VH of MF3874, MF3878, MF3883, MF3886, and MF3891. [Figure 10]Figure 1 shows flow cytometry analysis of binding of variants PG3874, PG3878, PG3883, PG3886 and PG3891 to CD3 on HPB-ALL cells. PG3056 was included as a benchmark control. [Figure 11] FIG. 1 shows CEX-HPLC analysis of PG3891. [Figure 12] FIG. 1 shows the alignment of VH of MF3056 with VH of MF5192 to MF5197. [Figure 13] Figure 1 shows flow cytometry analysis of the binding of variants PG5192-5197 to CD3 on HPB-ALL cells. PG3056 was included as a benchmark control. [Figure 14] FIG. 1 shows IEF analysis of PG5196p06. [Figure 15] FIG. 1 shows a CEX-HPLC chromatogram of PG5196. [Figure 16] FIG. 1 shows binding of 5196x4327DM-Fc bsAb to membrane-expressed CD3 (HPB-ALL) and CLEC12A (HL-60). [Figure 17] Figure 1 shows that 5196x4327DM-Fc bsAb maintained CLEC12A-specific activation of CD4 and CD8 T cells, as reflected by upregulation of the early activation marker CD69. bsAb and control IgG were tested at 1000 ng / mL. Flow cytometry data of T cell activation are expressed as the percentage of CD69-positive cells within the CD4+ or CD8+ T cell population. HD1 and HD2 reflect two different experiments using T cells obtained from different healthy donors. [Figure 18] FIG. 5 shows that 5196×4327 induced antigen-mediated target cell lysis. [Figure 19]Sensorgrams showing the binding and dissociation rates of CD3δε-Fc (A) and CLEC12A protein (B) to immobilized 5196x4327 DM-Fc bsAb. The sensorgrams show the response (amount of protein bound to the chip (in artificial units)) as a function of time (seconds). Both columns show sensorgrams obtained using a range of antigen concentrations (colored) and the respective curve fits (black) performed using BIAevaluation software. [Figure 20] Figure 1 shows that primary AML patient samples were phenotyped by flow cytometry for CLEC12A expression and fractions of T cells and AML blasts at the initiation of co-culture (day 0). After 7 days of co-culture with 5196x4327 DM-Fc bsAb or isotype control, fractions and total numbers of T cells and AML blasts were quantified by flow cytometry analysis. [Figure 21] FIG. 1 shows the DSC analysis described in Example 9. [Figure 22]

[0023] Figure 1 shows the heavy chain variable sequence (VH) of antibody 15C3 as described in WO2005 / 118635. That patent publication describes two variants of antibody 15C3. Both variants have the same heavy chain variable domain, but this heavy chain variable domain is combined with two different light chain variable domains. Reference herein to antibody 15C3 refers to the antibody having the heavy chain variable domain shown in this figure in combination with the light chain variable domain L2 shown in this figure. The Fab product of the 15C3 VH plus the L2 light chain is further referred to herein as MF3055. Antibody PG3056 has the VH shown herein in combination with the light chain variable region IGKV1-39 shown in this figure. The Fab product of the 15C3 VH plus the IGKV1-39 light chain is further referred to as MF3056. [Figure 23]Figure 1 shows that antibodies of the invention preferably have a common light chain. A) The common light chain preferably comprises the amino acid sequence shown in this figure as the sequence IGKV1-39. B) In a preferred embodiment, the common light chain comprises the amino acid sequence shown as IGKV1-39 / jk1 or IGKV1-39 / jk5. In a particularly preferred embodiment, the common light chain comprises the amino acid sequence of IGKV1-39 / jk1 shown in this figure. [Figure 24] 23 shows the amino acid sequences of the VH regions of an anti-CLEC12A-binding antibody Fab (MF4327) and an anti-tetanus toxoid (TT)-binding Fab (MF1337). These VH regions, together with the amino acid sequence of the common light chain shown in FIG. 23, form the variable domains of MF4327 and MF1337, which are Fabs that bind to CLEC12A and tetanus toxoid, respectively. [Figure 25] Examples of CD3-binding variants of MF5196 are MF5603, MF5616, MF5626, MF5630, MF5648, MF5661 and MF5694, which all contain rearranged human IGKV1-39 / IGKJ1 VL regions. The amino acid sequences of the VH of the Fabs are shown. [Figure 26] Figure 23B shows the binding of antibodies comprising MF5196 VH; MF5603 VH; MF5616 VH; MF5626 VH; MF5630 VH; MF5648 VH; MF5661 VH and MF5694 VH and the common light chain as shown in Figure 23B to membrane-expressed CD3 on HPB-ALL cells as analyzed by flow cytometry. [Figure 27] FIG. 1 shows a CIEX-HPLC chromatogram of PG5661. [Figure 28] FIG. 1 shows MF5351, MF5354, and MF5356, CD3-binding Fabs composed of rearranged human IGKV1-39 / IGKJ1 VL regions with their respective VH as indicated. [Figure 29] Figure 1 shows the binding of PG formats to membrane-expressed CD3 on HPB-ALL cells. All of these examples bound to CD3. DETAILED DESCRIPTION OF THE INVENTION

[0018] The antibodies of the present invention are preferably bispecific antibodies. Bispecific antibodies preferably bind to at least human CD3. In addition, bispecific antibodies preferably bind to at least a surface molecule expressed on human tumor cells. In preferred embodiments, the bispecific antibody binds to 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, or PSMA. In particularly preferred embodiments, the bispecific antibody binds to CLEC12A.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] CD30 is also known as tumor necrosis factor receptor superfamily, member 8 (TNFRSF8); Ki-1 antigen; CD30; Ki-1; D1S166E; cytokine receptor CD30; leukocyte-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.

[0023] CD33 is also known as CD33 molecule; SIGLEC-3; CD33 antigen (Gp67); myeloid cell 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.

[0024] 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.

[0025] CD44 is also known as the CD44 molecule (Indian Blood Group); IN; MDU2; CD44 antigen (homing function and Indian blood group system); MDU3; CDW44; MIC4; CSPG8; chondroitin sulfate proteoglycan 8; HCELL; hematopoietic cell E- and L-selectin 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.

[0026] 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.

[0027] 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.

[0028] CEA is also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); meconium antigen 100; CD66e; carcinoembryonic antigen; and CD66e antigen. Id: HGNC:1817; Entrez Gene:1048; Ensembl: ENSG00000105388; OMIM:114890; UniProtKB: P06731.

[0029] CLEC12A is a member of the C-type lectin domain family 12, member A; C-type lectin protein CLL-1; MICL; dendritic cell-associated lectin 2; C-type lectin superfamily; myeloid suppressive C-type lectin-like receptor; C-type lectin-like molecule-1; CLL-1; DCAL2; CLL1; C-type lectin-like molecule 1; DCAL-2; killer cell lectin-like receptor subfamily L, member 1 (KLRL1); CD371 (Bakker A. et al., Cancer Res. 2004, 64, 8843-50; GenBank™ Accession No.: AY547296; Zhang W. et al., GenBank™ Accession No.: AF247788; A.S. Marshall et al., J Biol Chem 2004, 279, 14792-802; GenBank™ Accession No.: AY498550; Y. Han et al., Blood 2004, 104, 2858-66; H. Floyd et al., GenBank™ Accession No.: AY426759; C. H. Chen et al., Blood 2006, 107, 1459-67). Id: HGNC: 31713; Entrez Gene: 160364; Ensembl: ENSG00000172322; OMIM: 612088; UniProtKB: Q5QGZ9. CLEC12A is an antigen expressed on leukemic blast cells and leukemic stem cells, including CD34-negative or CD34-low expressing leukemic stem cells (side populations), in acute myeloid leukemia (AML) (AB Bakker et al., Cancer Res 2004, 64, 8443-50; Van Rhenen et al., 2007, Blood 110:2659; Moshaver et al., 2008, Stem Cells 26:3059). CLEC12A expression is otherwise thought to be restricted to the hematopoietic lineage, particularly to myeloid cells in peripheral blood and bone marrow, namely, granulocyte, monocyte, and dendritic cell precursors. More importantly, CLEC12A is not present on hematopoietic stem cells. This expression profile makes CLEC12A a particularly advantageous target in AML.The full-length form of CLEC12A contains 275 amino acid residues, including an additional intracellular stretch of 10 amino acids absent from most other isoforms, and exhibits a strictly myeloid expression profile (surface expression and mRNA level). The term "CLEC12A or its functional equivalents" refers to all of the above-referenced variants (such as splices and mutations) and isoforms thereof that retain the strictly myeloid expression profile (both surface expression and mRNA level) as described in Bakker et al., Cancer Res 2004, 64, 8443-50 and Marshall 2004, J Biol Chem 279(15), 14792-802. The CLEC12A-binding antibodies of the present invention bind to human CLEC12A. When CLEC12A is referred to herein, human CLEC12A is the reference unless otherwise specified.

[0030] The CD3 (cluster of differentiation 3) T cell coreceptor is a protein complex composed of four different chains. In mammals, this complex includes the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains bind to a molecule known as the T cell receptor (TCR) and the ζ chain to generate an activation signal in T lymphocytes. The TCRα, TCRβ, ζ chain, and CD3 molecule together form the TCR complex. CD3 is expressed on T cells. Antibodies that bind to CD3 can bind to the CD3γ chain, the CD3δ chain, the CD3ε chain, or a combination of CD3δ / CD3ε or CD3γ / CD3ε. The CD3-binding antibodies of the present invention bind to the CD3ε chain. CD3ε is known by various aliases, some of which are "CD3e molecule, epsilon (CD3-TCR complex)"; "CD3e antigen, epsilon polypeptide (TiT3 complex)"; T cell surface antigen T3 / Leu-4 epsilon chain; T3E; T cell antigen receptor complex, epsilon subunit of T3; CD3e antigen; CD3-epsilon 3; IMD18; TCRE. The IDs for the CD3E gene are HGNC:1674; Entrez Gene:916; Ensembl: ENSG00000198851; OMIM: 186830 and UniProtKB: P07766. Bispecific CD3-binding antibodies targeting the CD3ε chain have been shown to be effective in recruiting T cells to abnormal cells. Therefore, a (bispecific) antibody according to the invention preferably comprises one heavy / light chain combination that binds to CD3ε. The CD3 binding antibodies of the present invention bind to human CD3. When CD3 is referenced herein, it is a reference to human CD3 unless specifically stated otherwise.

[0031] CS-1 is also known as citrate synthase; EC 2.3.3.1; citrate synthase, mitochondrial; EC 2.3.3. Id: HGNC:2422; Entrez Gene:1431; Ensembl:ENSG00000062485; OMIM:118950; UniProtKB:O75390.

[0032] EGFR is also known 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 growth inhibitory protein 40; cell proliferation-inducing protein 61; HER1; mENA; EC2.7.10.1; EC2.7.10; epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog). Id: HGNC:3236; Entrez Gene:1956; Ensembl:ENSG00000146648; OMIM:131550; UniProtKB:P00533.

[0033] EGFRvIII is a common variant of EGFR (Oncogene. 2013 May 23;32(21):2670-81. doi: 10.1038 / onc.2012.280. Epub 2012 July 16).

[0034] Delta-like 3 (DLL3) is also called Delta-like 3; Drosophila Delta homolog 3; Delta 3; Delta (Drosophila)-like 3; SCDO1. The IDs for DLL3 are HGNC: 2909; Entrez Gene: 10683; Ensembl: ENSG00000090932; OMIM: 602768 and UniProtKB: Q9NYJ7.

[0035] LGR5 is leucine-rich repeat-containing G protein-coupled receptor 5. Alternative names for the 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 invention that bind to LGR5 bind to human LGR5. Due to sequence and tertiary structure similarities between human and other mammalian orthologs, LGR5-binding proteins or antibodies of the invention may, but do not necessarily, also bind to such orthologs. 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).

[0036] MSLN or mesothelin is also called mesothelin; prepro-megakaryocyte potentiating factor; CAK1 antigen; MPF; soluble MPF mesothelin-related protein; megakaryocyte potentiating factor and SMRP. The IDs for MSLN are: HGNC:7371; Entrez Gene: 10232; Ensembl: ENSG00000102854; OMIM: 601051; UniProtKB: Q13421.

[0037] Folate receptor 1 is also referred to as FOLR1; folate receptor 1; 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.

[0038] 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.

[0039] EPCAM is also known as epithelial cell adhesion molecule; EGP40; M4S1; ESA; MIC18; KS1 / 4; tumor-associated calcium signaling factor 1; MK-1; TACSTD1; human epithelial glycoprotein-2; TROP1; membrane-constituent 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; KS1 / 4 antigen; EGP-2; and ACSTD1. Id: HGNC: 11529; Entrez Gene: 4072; Ensembl: ENSG00000119888; OMIM: 185535; UniProtKB: P16422.

[0040] HER2 is a member of the V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2; ERBB2; CD340; NGL; HER-2; HER-2 / neu2; NEU2; TKR1; neuro / glioblastoma-derived oncogene homolog; C-Erb B2 / Neu protein; metastatic lymph node gene 19 protein; herstatin; proto-oncogene C-ErbB-2; neuroblastoma / glioblastoma-derived oncogene homolog; proto-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; MLN19; 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 (neuronal / glioblastoma-derived oncogene homolog). Id: HGNC: 3430; Entrez Gene: 2064; Ensembl: ENSG00000141736; OMIM: 164870; UniProtKB: P04626.

[0041] HM1.24 is also known as BST2; bone marrow stromal cell antigen 2; tetherin; BST-2; bone marrow stromal antigen 2; HM1.24 antigen; tetherin; CD317; CD317 antigen; and NPC-A-7. Id: HGNC:1119; Entrez Gene:684; Ensembl:ENSG00000130303; OMIM:600534; UniProtKB:Q10589.

[0042] MCSP is also called sperm mitochondrial-associated cysteine-rich protein (SMCP); MCSP; MCS; mitochondrial sheath selenoprotein; HSMCSGEN1; sperm mitochondrial-associated cysteine-rich protein. Id: HGNC:6962; Entrez Gene:4184; Ensembl:ENSG00000163206; OMIM:601148; UniProtKB:P49901.

[0043] 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; NAALADase I; PSMA; EC 3.4.17.21; glutamic acid carboxylase II; GCP2; cell growth 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.

[0044] PSMA should not be confused with proteasome (prosome, macropain) subunit alpha type 1, also known as PSMA1.

[0045] The accession number is provided primarily to provide a method of further identification of the target, and the actual sequence of the binding protein may vary due to mutations in the encoding gene, such as those that occur in some cancers, etc. The antigen-binding site binds to the antigen and its various variants, such as those expressed by some antigen-positive immune cells or tumor cells.

[0046] Reference herein to a gene or protein preferably refers to the human form of the gene or protein. Reference herein to a gene or protein refers to the native gene or protein and to variant forms of the gene or protein that are detectable in tumors, cancers, etc., preferably human tumors, cancers, etc.

[0047] Bispecific antibodies of the 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, PSMA protein, or a variant thereof. Needless to say, the antigen-binding heavy / light chain combination preferably binds to the extracellular portion of the antigen. Bispecific antibodies according to the invention preferably bind to human CLEC12A or a variant thereof. A preferred bispecific antibody according to the invention binds to human CD3 and human CLEC12A or a variant thereof.

[0048] HGNC stands for HUGO Gene Nomenclature Committee. The number after the abbreviation is an accession number that allows information about the gene and the protein encoded by it to be retrieved from the HGNC database. Entrez Gene provides an accession number or gene ID that allows information about the gene or the protein encoded by it to be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensemble provides an accession number that allows information about the gene or the protein encoded by it to be obtained from the Ensemble database. Ensembl is a collaborative project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system for creating and maintaining automated annotations of selected eukaryotic genomes.

[0049] The present invention provides an antibody that binds to human CD3, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof at one or more positions other than the positions indicated by X1X2: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] The present invention provides an antibody comprising a variable region comprising:

[0050] In a preferred embodiment, the light chain comprises a light chain variable region comprising the amino acid sequence of the O12 / IgVκ1-39*01 gene segment shown in Figure 23A, with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. The phrase "O12 light chain" is used throughout this specification as an abbreviation for "a light chain comprising a light chain variable region comprising the amino acid sequence of the O12 / IgVκ1-39*01 gene segment as shown in Figure 23A, with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof." IgVκ1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39; O12a, or O12. The external IDs for this gene are HGNC:5740; Entrez Gene:28930; Ensembl:ENSG00000242371. A preferred amino acid sequence for IgVκ1-39 is shown in Figure 23A, which sets forth the sequence of the V region. The V region can be combined with one of five J regions. Figures 23B and 23C set forth two preferred sequences for IgVκ1-39 combined with a J region. The combined sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designation according to the IMGT database world wide web at imgt.org).

[0051] Preferably, the O12 / IgVκ1-39*01 comprising the light chain variable region is a germline sequence. More preferably, the IGJκ1*01 or IGJκ5*01 comprising the light chain variable region is a germline sequence. In a preferred embodiment, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence.

[0052] In a preferred embodiment, the light chain variable region comprises germline O12 / IgVκ1-39*01. In a preferred embodiment, the light chain variable region comprises kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. In a preferred embodiment, it comprises IgVκ1-39*01 / IGJκ1*01. The light chain variable region preferably comprises 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*01.

[0053] Mature B cells producing antibodies with O12 light chains often produce light chains that have undergone one or more mutations relative to the germline sequence, i.e., the normal sequence in nonlymphoid cells of an organism. The process responsible for these mutations is often referred to as somatic (hyper)mutation. The resulting light chain is referred to as an affinity-matured light chain. If such a light chain is derived from the O12 germline sequence, it is an O12-derived light chain. As used herein, the term "O12 light chain" includes O12-derived light chains. Mutations introduced by somatic hypermutation can, of course, also be artificially introduced in the laboratory. Other mutations can also be introduced in the laboratory without necessarily affecting the characteristics of the light chain in type, but not necessarily in quantity. A light chain is at least an O12 light chain if it contains a sequence as shown in Figure 23A, 23B, or 23C, with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising a sequence as shown in Figure 23A, 23B, or 23C with 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising a sequence as shown in Figure 23A, 23B, or 23C with 0 to 5, preferably 0 to 4, and more preferably 0 to 3 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising a sequence as shown in Figure 23A, 23B, or 23C with 0 to 2, more preferably 0 to 1, and most preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the O12 light chain is a light chain comprising a sequence as shown in Figure 23A or 23B with the recited amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the light chain comprises the sequence of Figure 23B.

[0054] The antibody is preferably a bispecific antibody. The 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 / light (H / L) chain 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 be combined with 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 be combined with 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. The light chain preferably comprises a light chain variable region comprising the amino acid sequence of the O12 / IgVκ1-39*01 gene segment as shown in Figure 23A, with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof, as further defined elsewhere herein. Preferably, the consensus 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 O12, as described herein above. A preferred sequence for O12 / IgVκ1-39 is shown in Figure 23A.The sequences of the V regions are set forth in this figure. Figures 23B and 23C set forth two preferred sequences for IgVκ1-39 in combination with a J region. The combined sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01.

[0055] Preferably, the O12 / IgVκ1-39*01 light chain variable region is a germline sequence. Preferably, the O12 / IgVκ1-39*01 comprising the light chain variable region is a germline sequence. More preferably, the IGJκ1*01 or IGJκ5*01 comprising the light chain variable region is a germline sequence. In a preferred embodiment, the IGKV1-39 / jk1 or IGKV1-39 / jk5 light chain variable region is a germline sequence. The O12 light chain of the bispecific antibody is preferably the O12 light chain described herein above.

[0056] Tumor antigens are defined by their expression pattern. Tumor-specific antigens are generally present only on tumor cells and not on any other cells in the postnatal, preferably adult, human body. Tumor-associated antigens are generally present on tumor cells and also on some normal cells in the postnatal, preferably adult, human body. As used herein, tumor antigens are generally tumor-specific antigens or tumor-associated antigens. Tumor antigens may or may not be involved in the carcinogenic process. They may or may not differ from "normal" proteins in healthy individuals. It should be noted that various tumor-specific antigens have subsequently been shown to be expressed on some other non-tumorigenic cells. Preferred tumor antigens are tumor antigens that are expressed on the cell surface and have an extracellular portion. Antibodies generally bind to the extracellular portion of the antigen.

[0057] The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins, containing one or more domains that bind to an epitope on an antigen, where such domains are derived from or share sequence homology with the variable region of an antibody. Antibodies generally consist of basic structural units, each having two heavy chains and two light chains. Preferably, therapeutic antibodies are as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies for human subjects). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. Binding or "specifically recognizing" is defined as binding with an affinity (KD) of at least 1×10e-6 M, 1×10e-7 M, 1×10e-8 M, or at least 1×10e-9 M. Antibodies for therapeutic applications may have an affinity of 1×10e-10 M or even higher. The antibodies of the present invention are generally bispecific antibodies and of the human IgG subclass. Preferably, the antibodies of the present invention are of the human IgG1 subclass. Most preferably, the antibodies of the present invention are full-length IgG molecules. The present invention also provides derivatives and / or analogs of the antibodies of the present invention. Such derivatives and / or analogs preferably have the VH / VL domains of the antibodies of the present invention, including the common light chain variable region as defined elsewhere herein. Suitable derivatives are single chain Fv fragments, monobodies, VHH and Fab fragments. Derivatives may be derived from the C heavy chain of the antibody. H 2 and C H The derivative may be fused to the C3 domain of the heavy chain of an antibody. H 1 domain and C of the antibody light chain LThe derivatives may also be in a multivalent, preferably bispecific, format, in which one of the VH / VL domains of the antibody comprises a heavy chain variable region / light chain variable region (VH / VL) combination that binds CD3 of the invention and at least one other VH / VL domain of the antibody that binds to an antigen other than the CD3 antigen. At least one other VH / VL domain of the antibody preferably binds to a tumor antigen, preferably CLEC12A. Multivalent formats are readily produced, for example, by producing the derivative as a fusion protein, with or without suitable and / or conventional peptide linkers or spacers between the VH / VL domains.

[0058] A "bispecific antibody" is an antibody as described herein above that comprises 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 CD3, preferably 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 comprising one heavy / light chain combination that binds to CD3 and one heavy / light chain combination that binds to another antigen, preferably a tumor antigen.

[0059] Binding of the heavy / light chain combination to an antigen is achieved via an antigen-binding site in the variable regions of the heavy / light chain combination.

[0060] 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 classical antibodies with two H / L combinations have at least one 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, VHH, or Fab fragment that provides a second binding activity.

[0061] The term bispecific antibody may be interchanged with the broader term "bispecific binding protein," which includes an immunoglobulin variable domain that binds CD3 and an antigen-binding (poly)peptide that binds to another antigen, having heavy and light chain variable regions of the invention. In this embodiment, the binding (poly)peptide is preferably a (poly)peptide as specified in Spiess et al. (supra).

[0062] In the bispecific antibodies of the present invention, the light chain in the CD3-binding H / L chain combination is preferably similar to the light chain in the H / L chain combination that can bind to an antigen other than CD3, preferably a tumor antigen. In a more preferred embodiment, the light chains in both H / L chain combinations are identical, i.e., the human light chain is a so-called "common light chain," which is a light chain that can combine with a different heavy chain to form an antibody having 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 O12, preferably the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01, or a fragment or functional equivalent thereof (i.e., the same IgVκ1-39 gene segment but a different IGJκ gene segment) (designation according to the IMGT database World Wide Web at imgt.org).

[0063] The term "common light chain" as used herein refers to the two light chains (or their VL portions) in a bispecific antibody. The two light chains (or their VL portions) may be identical or may have some amino acid sequence differences, while the binding specificity of the full-length antibody is not affected. For example, it is within the scope of the definition of common light chain used herein to prepare or discover a non-identical but still functionally equivalent light chain, for example, by introducing and testing conservative amino acid changes, i.e., amino acid changes in regions that do not contribute or only partially contribute to binding specificity when paired with a heavy chain. The terms "common light chain," "common VL," "single light chain," and "single VL," with or without the addition of the term "rearranged," are all used interchangeably herein.

[0064] Preferably, the common light chain has a germline sequence. A preferred germline sequence is a light chain variable region that is frequently used in the human repertoire. A preferred germline light chain is O12, preferably the rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01, or a fragment or functional equivalent thereof (i.e., the same IgVκ1-39 gene segment but a different IGJκ gene segment) (as designated by the IMGT database World Wide Web at imgt.org). The terms rearranged germline human kappa light chain IgVκ1-39*01 / IGJκ1*01, IGKV1-39 / IGKJ1, huVκ1-39 light chain, or simply huVκ1-39, are used interchangeably throughout this application. Obviously, those skilled in the art will recognize that "common" also refers to functional equivalents of light chains that are not identical in amino acid sequence. There are many variants of the light chain, in which there are mutations (deletions, substitutions, insertions and / or additions) that do not significantly affect the formation of a functional binding region. The light chain of the present invention can also be a light chain as defined herein above, with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof.

[0065] The antibody of the present invention is preferably an IgG antibody, preferably an IgG1 antibody. The term "full-length IgG" according to the present invention is defined as an IgG that essentially includes a complete IgG, but does not necessarily have all the functions of an intact IgG. For the avoidance of doubt, a full-length IgG comprises two heavy chains and two light chains. Each chain comprises a constant (C) region and a variable (V) region, which can be broken down into domains designated CH1, CH2, CH3, VH, and 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, often via the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations that provide desired characteristics may be present. Full-length IgGs should not have substantial deletions of any of the regions. However, IgG molecules in which one or more amino acid residues have been deleted without essentially altering the binding characteristics of the resulting IgG molecule are encompassed within the term "full-length IgG." For example, such IgG molecules may have a deletion of between 1 and 10 amino acid residues, preferably in the non-CDR regions, where the deleted amino acids are not essential for the binding specificity of the IgG.

[0066] Full-length IgG antibodies are used due to their favorable half-life and the desire to remain similar to fully self (human) molecules due to immunogenicity. IgG1 is preferred due to its long circulating half-life in humans. To prevent or avoid immunogenicity in humans, it is preferred that the bispecific full-length IgG antibody according to the present invention is human IgG1. The term "bispecific" means that one heavy and light chain combination (H / L combination) or arm of the antibody binds to a first antigen and the other H / L combination or other arm binds to a second antigen, where the first and second antigens are not identical. An antigen is generally a molecule that serves as a target for an antibody. In the present invention, the antigen is preferably a protein expressed on the membrane of an individual's cells. According to the present invention, the first and second antigens are located on two different molecules, preferably located on two different cell types. The term "one arm [of an antibody]" preferably refers to a heavy / light chain combination comprising one Fab portion of a full-length IgG antibody. Bispecific antibodies, which mediate cytotoxicity by recruiting and activating endogenous immune cells, are an emerging class of next-generation antibody therapeutics. This can be achieved by combining antigen-binding specificities for target cells (i.e., tumor cells) and effector cells (i.e., T cells, NK cells, and macrophages) in a single molecule (Cui et al., JBC 2012 (287) 28206-28214; Kontermann, MABS 2012 (4) 182-197; Chames and Baty, MABS 2009 (1) 539-547; Moore et al., Blood 2011 (117) 4542-4551; Loffler et al., 2000, Blood 95:2098; Zeidler et al., 1999, J. Immunol. 163:1246). According to the present invention, bispecific antibodies are provided in which one heavy / light chain combination binds to the CLEC12A antigen on abnormal (tumor) cells and a second heavy / light chain combination binds to CD3 on immune effector cells.

[0067] The present invention provides bispecific IgG antibodies in which one heavy / light chain combination specifically recognizes CLEC12A or a functional equivalent thereof, including a functional CLEC12A equivalent lacking the additional intracellular stretch of 10 amino acids described above. Bispecific IgG antibodies in which one heavy / light chain combination binds to the full-length form of CLEC12A are preferred. It goes without saying that the tumor antigen-binding heavy / light chain combination binds to the extracellular portion of the tumor antigen.

[0068] The terms "variable region domain", "variable region", "variable domain", "VH / VL pair", "VH / VL", "VH", "VL", "Fab portion", "Fab arm", "Fab" or "arm" are used interchangeably herein.

[0069] Antigen binding by an antibody is generally mediated by the complementary regions and the specific three-dimensional structure of both the antigen and variable domains of the antibody, precisely binding these two structures together (an interaction similar to a lock and key), as opposed to random, nonspecific fixation of the antibody. Because antibodies generally recognize epitopes of antigens, and such epitopes may exist in other proteins as well, antibodies according to the present invention that bind to CD3 or CLEC12A may recognize other proteins if such other proteins contain the same epitope. Thus, the term "binding" does not exclude antibody binding to another protein or proteins containing the same epitope. The heavy / light chain combinations of the antibodies of the present invention that bind to CD3 do not bind to other proteins on the membrane of cells in postnatal, preferably adult, humans. The heavy / light chain combinations of the antibodies of the present invention that bind to CLEC12A do not bind to other proteins on the membrane of cells in postnatal, preferably adult, humans.

[0070] Bispecific antibodies according to the invention that bind to CD3 and a tumor antigen bind to CD3 (preferably CD3 on effector cells) with a binding affinity of at least 1×10e-6 M, as outlined in more detail below. In a preferred embodiment, the CD3 binding affinity is between 1×10e-6 M and 1×10e-10 M, preferably between 1×10e-7 M and 1×10e-9 M.

[0071] Bispecific antibodies according to the present invention that bind to CD3 and a tumor antigen preferably bind to the tumor antigen with a higher binding affinity than that with which they bind to CD3. In preferred embodiments, the binding affinity of the tumor antigen on tumor cells is at least 2-fold, more preferably 4-fold, more preferably 6-fold, or 10-fold higher than the binding affinity to CD3. In preferred embodiments, the tumor antigen binding affinity is 1×10e-6 M to 1×10e-10 M, preferably 1×10e-7 M to 1×10e-10 M, more preferably at least 1×10e-8 M, and preferably at least 1×10e-9 M. This is preferably combined with a CD3 affinity that is at least 2-fold, more preferably 4-fold, more preferably 6-fold, or 10-fold lower than the binding affinity to the indicated tumor antigen. In preferred embodiments, the tumor antigen binding affinity is 1×10e-8 M to 1×10e-10 M.

[0072] As used herein, the term "abnormal cells" includes tumor cells, more specifically tumor cells of blood 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.

[0073] 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 target cells. Immune effector cells include cells of the lymphoid lineage, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells. However, cells of the myeloid lineage, such as monocytes or macrophages, dendritic cells, and neutrophilic granulocytes, can also be considered immune effector cells. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophilic granulocytes. According to the present invention, recruitment of effector cells to abnormal cells means that the immune effector cells are brought into the vicinity of the abnormal target cells, whereby the effector cells can directly kill or indirectly initiate the killing of the recruited abnormal cells. Preferably, the CD3-binding antibody binds to CD3 on the surface of the effector cell.

[0074] The antibody of the present invention that binds to human CD3 has the amino acid sequence: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] The heavy chain variable region comprises:

[0075] The heavy chain variable region may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the indicated amino acid sequence. Of course, amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the indicated sequence are only at positions other than those designated by X1X2. Only the indicated amino acids are allowed at the positions designated by X1X2. 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 insertions, deletions, substitutions, additions, or combinations thereof at positions other than those designated by X1X2 relative to the indicated amino acid sequence. Combinations of insertions, additions, deletions, or substitutions are claimed, provided that the aligned sequences differ at no more than 10, preferably no more than 5, positions. Gaps in one of the aligned sequences are equal in number to the number of amino acids skipped in the other sequence.

[0076] The amino acid insertions, deletions, substitutions, additions, or combinations thereof are preferably not within the CDR3 region of the heavy chain variable region, and preferably not within 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 insertions relative to the displayed sequence. In this embodiment, the heavy chain variable region may have 0 to 10, preferably 0 to 5, amino acid substitutions relative to the displayed amino acid sequence. The amino acid substitutions are preferably conservative amino acid substitutions.

[0077] An amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than the positions indicated by X1X2: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] The antibody of the present invention that binds to human CD3 and comprises a heavy chain variable region comprising the amino acid sequence identified by numbers 5192, 5193, 5196, 5197, 5351, 5354, 5356, 5603, 5616, 5626, 5630, 5648, 5661, or 5694 as shown in Figures 12, 25, and 28. The heavy chain variable region preferably comprises the amino acid sequence identified by numbers 5196, 5197, 5603, 5616, 5626, 5630, 5648, 5661, or 5694 as shown in Figures 12 and 25. The heavy chain variable region of the antibody preferably comprises the amino acid sequence identified by number 5196 as shown in Figure 12.

[0078] The present invention further relates to a bispecific antibody that binds to human CD3 of the present invention, comprising a heavy chain and a light chain, wherein the heavy chain variable region of the heavy chain has the amino acid sequence: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] The present invention provides a bispecific antibody comprising:

[0079] The light chain is preferably a common light chain as defined elsewhere herein. The bispecific antibody further comprises a heavy chain and light chain combination that binds to another antigen, preferably a tumor antigen. The light chain of the heavy chain and light chain combination that binds to another antigen is preferably a common light chain as defined elsewhere herein. An amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof at one or more positions other than the positions indicated by X1X2: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] or 5694 as shown in Figures 12, 25 and 28. The heavy chain variable region of a bispecific antibody preferably comprises the amino acid sequence identified by the numbers 5196, 5197, 5603, 5616, 5626, 5630, 5648, 5661 or 5694 as shown in Figures 12 and 25. The heavy chain variable region of a bispecific antibody preferably comprises the amino acid sequence identified by the number 5196 as shown in Figure 12.

[0080] An amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than the positions indicated by X1X2: QVQLV QSGGG VVQPG RSLRL SCVASG FTFSS YGMHW VRQAP GKGLE WVAAI WYX1X2R KQDYA DSVKG RFTIS RDNSK NTLYL QMNSL RAEDT AVYYC TRGTG YNWFD PWGQG TLVTV SS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] Preferably, the bispecific antibody of the present invention further comprises a heavy / light chain combination that binds to human CLEC12A. In a preferred embodiment, the heavy chain of the heavy / light chain combination that binds to human CLEC12A has the amino acid sequence: QVQLV QSGAE VKKPG ASVKV SCKAS GYTFT SYYMH WVRQA PGQGL EWMGI INPSG GSTSY AQKFQ GRVTM TRDTS TSTVY MELSS LRSED TAVYY CAKGT TGDWF DYWGQ GTLVT VSS; EVQLV QSGAE VKKPG ASVKV SCKAS GYTFT SYYMH WVRQA PGQGL EWMGI INPSG GSTSY AQKFQ GRVTM TRDTS TSTVY MELSS LRSED TAVYY CARGN YGDEF DYWGQ GTLVT VSS; or QVQLV QSGAE VKKPG ASVKV SCKAS GYTFT GYYMH WVRQA PGQGL EWMGW INPNS GGTNY AQKFQ GRVTM TRDTS ISTAY MELSR LRSDD TAVYY CARDG YFADA FDYWG QGTLV TVSS; The variable region comprises:

[0081] The heavy chain variable region of a heavy / light chain combination that binds human CLEC12A can have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof 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 insertions, deletions, substitutions, additions, or a combination thereof relative to the amino acid sequence shown. A combination of insertions, deletions, additions, or substitutions is claimed, provided that the aligned sequences differ at no more than 5 positions. Gaps in one of the aligned sequences are equal in number to the number of amino acids skipped in the other sequence.

[0082] Amino acid insertions, deletions, substitutions, additions, or combinations thereof in the CD3 heavy chain variable region described herein preferably leave H35, A61, Y102, N103, and W104, as well as positions in the CDR3 VH, unchanged. When A50 is modified, it is preferably substituted with S, Y, M, or Q. When D59 is modified, it is preferably substituted with Y or E, although substitution of D59 with L, I, V, F, R, A, N, H, S, or T is also possible. When A61 is substituted, it is preferably substituted with N, I, H, Q, L, R, Y, E, S, T, D, K, or V. When F105 is modified, it is preferably substituted with Y or M. For the CD3 VH of the present invention, residues H35, Y102, N103, and W104 are considered to be involved in CD3 binding. Other specific substitutions at positions A50, D59, A61, and F105 are also relevant but need not affect CD3 binding. The second column of Table 2 lists the introduced amino acid substitutions. Similarly, the recovered substitutions are listed in the third column. Amino acid substitutions that were introduced but not recovered are likely to affect the antibody and are undesirable. For example, the A50I mutation is undesirable. After storage, tolerated amino acid substitutions can be readily found using the method described in Example 5A in conjunction with CIEX-HPLC.

[0083] The amino acid insertions, deletions, substitutions, additions or combinations thereof preferably are not made in the binding interphase of the heavy and light chains.

[0084] When an amino acid is changed at the interface of H / L chain interaction, 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.

[0085] The addition of an amino acid can be considered in principle the same as an insertion. The addition of an amino acid to one of the termini of a polypeptide chain is sometimes considered a strict addition (lengthening) rather than an insertion. For the purposes of the present invention, both addition within the chain or addition to one of the termini are considered insertions.

[0086] The amino acid 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, or insertions with respect to the sequence shown. In this embodiment, the heavy chain variable region may have 0 to 5 amino acid substitutions with respect 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 amino acid sequences GFTFSSYG (according to IMGT) for CDR1, IWYNARKQ for CDR2, and GTGYNWFDP for CDR3, respectively. The CDR1, CDR2, and CDR3 of the CLEC12A-binding VH of the present invention preferably comprise the amino acid sequences GYTFTSYY for CDR1, INPSGGST for CDR2, and GTTGDWFDY for CDR3, respectively.

[0087] The light chain variable region preferably comprises a germline O12 variable region V-segment. In a preferred embodiment, the light chain variable region comprises the kappa light chain V-segment IgVκ1-39*01. In a particularly preferred embodiment, the light chain variable region comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05. In a most preferred embodiment, the light chain variable region comprises the germline kappa light chain IgVκ1-39*01 / IGJκ1*01 sequence.

[0088] In the bispecific antibodies of the present invention, the light chains are preferably the same for both heavy / light chain combinations. Such light chains are also referred to as "common light chains." The term "common light chain" according to the present invention refers to light chains that are identical or have some amino acid sequence differences while retaining the binding specificity of the antibody. For example, it is possible to prepare or discover non-identical but still functionally equivalent light chains within the definition of a common light chain as used herein, for example, by introducing and testing conservative amino acid changes, i.e., amino acid changes in regions that do not contribute or only partially contribute to binding specificity when paired with a heavy chain. The terms "common light chain," "common VL," "single light chain," and "single VL," with or without the term "rearranged," are all used interchangeably herein. The use of a human light chain that can be combined with different heavy chains to form antibodies with functional antigen-binding domains as a common light chain is one aspect of the present invention (WO2004 / 009618, WO2009 / 157771, Merchant et al., 1998, Nissim et al., 1994). 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, have a good ability to pair with many different VH regions, and have good thermodynamic stability, yield, and solubility.

[0089] In a preferred embodiment, the common light chain comprises a light chain variable region comprising germline O12 / 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, it comprises IgVκ1-39*01 / IGJκ1*01. The light chain variable region preferably comprises the germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or the germline kappa light chain IgVκ1-39*01 / IGJκ5*01. In a preferred embodiment, it comprises the germline IgVκ1-39*01 / IGJκ1*01. Obviously, those skilled in the art will recognize that "common" also refers to functional equivalents of light chains that are not identical in amino acid sequence. Many variants of the light chain exist in which there are mutations (deletions, substitutions, additions) that do not significantly affect the formation of a functional binding region.

[0090] In a preferred embodiment, 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 EIK, having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In a preferred embodiment, the light chain variable region contains 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, preferably 0-3, preferably 0-2, preferably 0-1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the amino acid sequence shown. A combination of insertions, deletions, additions, or substitutions is claimed provided that the aligned sequences differ at no more than 5 positions. Gaps in one of the aligned sequences equal the number of amino acids skipped in the other sequence. In a preferred embodiment, 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 EIK. In a preferred embodiment, 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.In another preferred embodiment, 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 PITFG QGTRL EIK.

[0091] The amino acid insertions, deletions, substitutions, additions, or combinations thereof are preferably not in the CDR3 region of the light chain variable region, and preferably not in the CDR1 or CDR2 regions of the light chain variable region. In a preferred embodiment, the light chain variable region does not contain deletions, additions, or insertions with respect to the sequence shown. In this embodiment, the light chain variable region may have 0 to 5 amino acid substitutions with respect to the amino acid sequence shown. 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, respectively, i.e., the CDRs of IGKV1-39 (according to IMGT).

[0092] As described herein above, the antibodies of the present invention are preferably bispecific antibodies. In a preferred embodiment, the bispecific antibody comprises a CD3-binding heavy / light chain combination as described herein and a heavy / light chain combination that binds to a tumor antigen. In a preferred embodiment, the tumor antigen-binding heavy / light chain combination binds to CLEC12A.

[0093] The constant regions of the (bispecific) antibodies of the invention are preferably human constant regions. The constant regions may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the constant regions of natural human antibodies. The various variable domains of the antibodies produced 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 invention are preferably human or humanized antibodies.

[0094] There are various methods in the art for producing antibodies. Antibodies are generally produced by cells expressing nucleic acids encoding the antibody. 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.

[0095] Various institutions and companies have developed cell lines for the large-scale production of antibodies, for example, for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. These cells are also used for other purposes, such as protein production. Cell lines developed for 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.

[0096] In one embodiment, the present invention provides a cell comprising an antibody according to the present invention and / or a nucleic acid according to the present 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 present invention and / or a nucleic acid according to the present invention.

[0097] The present invention further provides a cell comprising an antibody of the present invention. Preferably, the cell (generally 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. Furthermore, a cell culture comprising the cell of the present invention is provided. Various institutions and companies have developed cell lines for large-scale production of antibodies, for example, 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, said nucleic acid molecule encodes the VH identified by number 5196 in Figure 12, the VH identified by number 4327 in Figure 24, or a combination thereof.

[0098] The present invention further provides a method for producing an antibody, comprising culturing the cells of the present invention and recovering 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.

[0099] The cells of the present invention may be, for example, hybridoma cell lines, CHO cells, 293F cells, NS0 cells, or other cell types known to be suitable for antibody production for clinical purposes. In particularly preferred embodiments, 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 particularly preferred embodiments, the cells are CHO cells or variants thereof, preferably variants that utilize the glutamine synthetase (GS) vector system for antibody expression.

[0100] The present invention further provides a method for producing an antibody, comprising culturing the cells of the present invention and recovering 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.

[0101] Bispecific antibodies are generally also 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. For this purpose, 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, the cells generally produce two monoclonal antibodies (homodimers) in addition to the bispecific antibody (heterodimer). If the cells express more than one light chain, the number of possible heavy / light chain combinations in the produced antibodies increases. To reduce the number of different antibody species (combinations of different heavy and light chains) produced, the "common light chain" described above is preferred.

[0102] Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies (i.e., with the same heavy / light chain combination). Several methods have been published that favor the production of bispecific antibodies or, conversely, monospecific antibodies. In the present invention, it is preferred that cells favor the production of bispecific antibodies over the production of individual monospecific antibodies. This is generally achieved by modifying the heavy chain constant region to favor heterodimerization (i.e., dimerization with heavy chains of other heavy / 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 (see, for example, Gunasekaran et al., JBC 2010 (285) pp. 19637-19646). The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. The art describes various ways in which such heterodimerization of heavy chains can be achieved. One method is to generate "knob-into-hole" bispecific antibodies. See U.S. Patent Application No. 20030078385 (Arathoon et al., Genentech).

[0103] US 13 / 866,747 (now published as US 9,248,181), US 14 / 081,848 (now published as US 9,358,286), and PCT / NL2013 / 050294 (published as WO 2013 / 157954), which are incorporated herein by reference, disclose methods and procedures for producing bispecific antibodies using compatible heterodimerization domains. These procedures and procedures can also be advantageously employed in the present invention. Specifically, preferred mutations for producing essentially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (numbering according to Kabat) in the first CH3 domain ("KK variant" heavy chain) and the amino acid substitutions L351D and L368E in the second domain ("DE variant" heavy chain), or vice versa. Previously, our US Patents 9,248,181 and 9,358,286 and PCT Application WO2013 / 157954 demonstrated that DE and KK variants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE variant heavy chains or KK variant heavy chains (DEDE homodimers or KKKK homodimers, respectively) is highly unlikely 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 variant 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 variant of the heavy chain. In a preferred embodiment, the antigen other than CD3 is CLEC12A. In a preferred embodiment, the VH of the variable domain that binds to CLEC12A is MF4327_VH, as shown in Figure 24.

[0104] Some antibodies are modified downstream of the CH2 / 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 having a mutant CH2 and / or downstream hinge domain, thereby reducing interaction of the bispecific IgG antibody with Fc-gamma receptors. Such mutant CH2 and / or downstream hinge domains preferably contain amino acid substitutions at positions 235 and / or 236 (Kabat numbering), preferably L235G and / or G236R substitutions.

[0105] The present invention further provides a method of treating cancer or the risk of cancer in a subject, comprising the step of administering to a subject in need thereof a bispecific antibody that binds to human CD3, the bispecific antibody comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence: QVQLV QSGGG VVQPG RSLRL SCVAS GFTFS SYGMH WVRQA PGKGL EWVAA IWYX1X2RKQDY ADSVK GRFTI SRDNS KNTLY LQMNS LRAED TAVYY CTRGT GYNWF DPWGQ GTLVT VSS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] wherein the antibody comprises a variable region comprising:

[0106] The light chain preferably comprises a consensus light chain variable region. The consensus light chain variable region preferably comprises an O12 / IgVκ1-39 light chain variable region. The light chain variable region is preferably a germline O12 / IgVκ1-39*01 variable region. The light chain variable region preferably comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01. The light chain variable region preferably 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: Preferably, the heavy chain / light chain (H / L) combination that binds to the tumor antigen binds to CLEC12A.

[0107] 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 preferably an IgG antibody having a mutated CH2 and / or hinge downstream domain, thereby reducing the interaction of the bispecific IgG antibody with Fc-gamma receptors. The mutated CH2 and / or hinge downstream domain preferably comprises an amino acid substitution at position 235 and / or 236 (Kabat numbering), preferably an L235G and / or G236R substitution. The antibody preferably comprises a common light chain.

[0108] A bispecific antibody that binds to human CD3, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof at one or more positions other than the positions indicated by X1X2: QVQLV QSGGG VVQPG RSLRL SCVAS GFTFS SYGMH WVRQA PGKGL EWVAA IWYX1X2RKQDY ADSVK GRFTI SRDNS KNTLY LQMNS LRAED TAVYY CTRGT GYNWF DPWGQ GTLVT VSS [In the formula, X1=N and X2=A; X1=N and X2=T; X1=S and X2=G; X1=H and X2=G; X1=D and X2=G; or X1=H and X2=A] Also provided is a bispecific antibody comprising a heavy chain and a light chain that binds to a tumor antigen, the heavy chain and the light chain comprising a variable region comprising:

[0109] The present invention further provides an antibody or derivative thereof, 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 / light chain combination that binds to a tumor antigen. The bispecific antibody is preferably a CD3 / CLEC12A-binding antibody.

[0110] 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 those of epithelial origin; gynecological cancers such as ovarian and endometrial tumors; prostate cancer, brain cancer, or any other solid tumor.

[0111] Also provided are CD3 / tumor antigen bispecific antibodies or derivatives thereof of the present invention, or pharmaceutical compositions comprising such bispecific antibodies or derivatives thereof, for use in treating various leukemias and preleukemias of myeloid origin as well as B-cell lymphomas. Diseases that can be treated by the present invention include myeloid leukemias or preleukemias, such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and chronic myeloid leukemia (CML), as well as Hodgkin's lymphoma and most non-Hodgkin's lymphomas. B-ALL, T-ALL, and mantle cell lymphoma are also preferred targets for treatment with the antibodies of the present invention. Accordingly, the present invention provides bispecific full-length IgG antibodies according to the present invention for use as a pharmaceutical for the treatment of myelodysplastic syndromes (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. Preferably, the tumor antigen is CLEC12A.

[0112] The amount of an antibody according to the present invention to be administered to a patient generally falls within a therapeutic window, meaning that an amount sufficient to achieve a therapeutic effect is used without exceeding a threshold that leads to unacceptable side effects. The smaller the amount of antibody required to achieve the desired therapeutic effect, the larger the therapeutic window generally. Therefore, antibodies according to the present invention that exert a sufficient therapeutic effect at low dosages are preferred.

[0113] 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 a major negative determinant of outcome in AML, with long-term survival (at 5 years) rates of approximately 10% for intensively treated elderly AML patients. In nearly all patients who achieve remission with induction chemotherapy, disease progression is observed within 3 years. Current postremission treatments have shown limited, if any, value in elderly patients with AML. Thus, 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 sustain complete remission, novel types of drugs with completely different mechanisms of action are needed to target these chemotherapy-unresponsive AML tumor cells. Although 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, improving response or survival remains a major research goal. In a recently published network meta-analysis of 65 randomized clinical trials (15,110 patients) in elderly patients with AML, the majority of modified investigational induction regimens had similar or even 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 lower toxicity, especially for elderly AML patients.

[0114] Treatment of chemotherapy-unresponsive AML could be achieved by using bispecific antibodies to redirect T cells from the patient's own immune system to AML tumor cells, followed by tumor-specific activation of the T cells. This process is also known as the "T cell-engaged approach." In this way, the patient's immune system is strengthened and retargeted to attack and eradicate AML tumor cells. The present invention provides a CD3×CLEC12A bispecific IgG antibody that efficiently redirects T cells to AML tumor cells, thereby inducing AML tumor cell lysis. Therefore, the CD3×CLEC12A bispecific antibody is a targeted therapy with fewer side effects that specifically eradicates AML blasts and leukemic stem cells to improve the prognosis of AML patients. Because CLEC12A is expressed on leukemic stem cells (LSCs) but not on normal hematopoietic stem cells, therapy against this antigen is expected to eradicate LSCs while sparing normal stem cells. These full-length IgG bispecific antibodies are being clinically evaluated in patients with relapsed and / or refractory AML. Clinical efficacy has been analyzed using a reduction in AML blasts in the bone marrow as an objective response criterion. Effective bispecific IgG for AML offers a novel therapeutic option for a large patient segment for which no treatment is currently available. In addition to providing a means to achieve long-term remission, this treatment option also has the potential to cure AML when applied during remission. Most likely, it will have the greatest impact in the setting of minimal residual disease (MRD). Relapse rates are expected to decrease due to eradication of MRD. Thus, the impact of this new treatment modality for AML patients will be a less toxic treatment with a lower relapse rate, leading to improved outcomes associated with a better quality of life.

[0115] The antibodies 15C3 and 3056 described in the art share the same heavy chain variable region but different light chain variable regions. Batch-to-batch variation in antibody binding capacity was observed in the 3056 batches. This was not observed with 15C3. Because the light chains of the antibodies are different, the reason for this difference in behavior may be the difference in the light chains. SDS-page analysis revealed that the 3056 antibody was intact even in batches with less active antibodies. 3D modeling of the antibody variable domains indeed revealed some changes in the folding of the VH / VL domains between the two antibodies. Because these changes may explain the different behavior of the antibodies, experiments were designed to make the folding of the VH / VL domains of 3056 more similar to 15C3. Unfortunately, this did not explain the differences between 15C3 and 3056. Isoelectric focusing (IEF) revealed only minor differences between 15C3 (3055) and 3056 relative to the main antibody band at a higher isoelectric point. Subsequent analysis using CIEX-HPLC, a chromatographic technique that allows for the separation of charge variants, revealed a very complex retention spectrum with a broad elution profile for antibody 3056 depending on the batch, which contrasted with the profile observed for 15C3 (3055). Furthermore, it was found that the pattern of 3056 changed significantly over time, suggesting that the 3056 antibody is inherently unstable.

[0116] Only when variants of the 3056_VH chain were designed that were altered at one or two specific positions did the behavior change, ranging from erratic and completely non-binding to binding without significant batch-to-batch variation.

[0117] The 3056 heavy chain contains a NG deamidation motif, WY, which may be responsible for the observed charge heterogeneity of the 3056 antibody. NGThe 15C3 and 3056 Fabs contain RKQ in their CDR2 regions. In silico modeling of the 15C3 and 3056 Fabs revealed no significant differences in the folding of the NG deamidation motif in the HCDR2 region, demonstrating that this motif is not involved in the observed instability of the 3056 antibody. Surprisingly, however, the claimed changes significantly reduced the observed batch-to-batch variability of the antibodies of the present invention.

[0118] The present invention further provides antibodies comprising a CD3-binding variable domain and a CLEC12A-binding variable domain, wherein the CLEC12A-binding variable domain has a VH comprising an amino acid sequence identified by number 4327 in Figure 24 having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof, and the CD3-binding variable domain has a VH comprising an amino acid sequence identified by number 5192, 5193; 5196; 5197; 5351; 5354; 5356; 5603; 5616; 5626; 5630; 5648; 5661; or 5694 as shown in Figures 12, 25 and 28 having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than positions 54 and 55. The light chain preferably comprises a variable domain having the amino acids as shown in Figure 23, preferably Figure 23B.

[0119] The present invention further provides an antibody comprising a CD3-binding variable domain and a CLEC12A-binding variable domain, wherein the CLEC12A-binding variable domain has a VH comprising the amino acid sequence identified by number 4327 in Figure 24 which has 0 to 10, and preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof; and the CD3-binding variable domain has a VH comprising the amino acid sequence identified by number 5196 in Figure 12 which has 0 to 10, and preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof at one or more positions other than positions 54 and 55.

[0120] The heavy chain variable domain that binds to CD3 has an amino acid sequence identified by numbers 5196; 5197; 5603; 5616; 5626; 5630; 5648; 5661; or 5694 as shown in Figure 12 and Figure 25, preferably with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than positions 54 and 55. The heavy chain variable domain that binds to CD3 has an amino acid sequence identified by number 5196 as shown in Figure 12, preferably with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than positions 54 and 55.

[0121] In preferred embodiments, the heavy chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, preferably 0-3, preferably 0-2, preferably 0-1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the indicated amino acid sequence. For the CD3 heavy chain, the indicated amino acid insertions, deletions, substitutions, or additions are at positions other than 54 and 55. Combinations of insertions, additions, deletions, or substitutions are claimed provided that the aligned sequences differ at no more than 10 positions, preferably no more than 5 positions. Gaps in one of the aligned sequences are equal in number to the number of amino acids skipped in the other sequence.

[0122] The present invention further provides an antibody comprising a CD3-binding variable domain and a CLEC12A-binding variable domain, wherein the CLEC12A-binding variable domain has a VH comprising the amino acid sequence identified by number 4327 in Figure 24, and the CD3-binding variable domain has a VH comprising the amino acid sequence identified by number 5192; 5193; 5196; 5197; 5351; 5354; 5356; 5603; 5616; 5626; 5630; 5648; 5661; or 5694 as shown in Figures 12, 25 and 28. The heavy chain of the CD3-binding variable domain preferably has the amino acid sequence identified by number 5196; 5197; 5603; 5616; 5626; 5630; 5648; 5661; or 5694 as shown in Figures 12 and 25. The variable domain that binds to CD3 preferably has the amino acid sequence identified by number 5196 as shown in FIG. [Example]

[0123] As used herein, "MFXXXX" [where X is independently a number from 0 to 9] refers to a Fab in which the VH comprises a variable domain having an amino acid sequence identified by a four-digit number. Unless otherwise indicated, the light chain variable region of the variable domain generally has the sequence of Figure 23, generally 23B. "MFXXXX VH" refers to the amino acid sequence of the VH, identified by a four-digit number. MF further comprises a light chain constant region and a heavy chain constant region that normally interacts with the light chain constant region. PG refers to a monospecific antibody comprising identical heavy and light chains. PB refers to a bispecific antibody having two different heavy chains. The heavy chain variable regions differ, and generally the CH3 regions also differ, where one heavy chain has a KK mutation in its CH3 domain and the other has a complementary DE mutation in its CH3 domain (see PCT / NL2013 / 050294 (published as WO2013 / 157954) for reference).

[0124] Example 1 Charge heterogeneity of 3055 and 3056 mAbs Isoelectric focusing (IEF) was used to determine the pI and charge heterogeneity of PG3056, a full-length IgG1 monoclonal antibody containing the MF3056 VH paired with the IGVK1-39 / JK1 common light chain, and PG3055, a full-length IgG1 monoclonal antibody containing the same VH paired with the 15C3 VL2-IGKV1-13 light chain. For this purpose, Focusgel (Webscientific, catalog no. 1006-03) with a pI range of 6-11 was run using a GE Healthcare Multiphor II electrophoresis instrument equipped with a cooling plate cooled to 10°C. Ten micrograms of untreated sample was loaded into the sample slot next to a high pI range marker (GE Healthcare, catalog no. 17047301V). The electrophoresis program consisted of three phases: an initial focusing phase of 10 min at 500 V, followed by a final focusing phase of 90 min at 1,500 V, and 10 min at 2,000 V. The gel was subsequently fixed and stained with colloidal Coomassie dye (Pierce, Cat. No. 24590).

[0125] IEF analysis of PG3056 and PG3055 resulted in the gel shown in Figure 1. For PG3055, a major band was seen at a high pI (approximately 9) with satellite bands at somewhat higher pI values. PG3056 showed a similar IEF pattern, but with a slightly higher pI value and a slightly more diffuse major band.

[0126] Both IgGs were analyzed using cation exchange chromatography HPLC (CEX-HPLC) to analyze their charge heterogeneity using an orthogonal method. CEX-HPLC was performed at room temperature using a Dionex HPLC system equipped with an SP STAT 7 μm column and a UV / Vis detector. Ten μg of sample was injected, and a gradient of 25 mM phosphate buffer (pH 6.0) with increasing NaCl concentration was used to separate the antibody charge variants. Data were analyzed using Chromeleon software.

[0127] CEX-HPLC chromatograms of PG3055 and PG3056 are shown in Figure 2. PG3055 exhibits a major peak at 18.8 minutes, flanked by minor peaks corresponding to the acidic and basic IgG isoforms. Surprisingly, the chromatogram of PG3056 contains multiple peaks over a large time interval, with two broad major peaks visible at 18.4 and 20.4 minutes. The chromatogram demonstrates a significant increase in charge heterogeneity in the IGVK1-39 / JK1 common light chain anti-CD3 antibody (PG3056) compared to the 15C3 VL2-IGKV1-13 light chain IgG (PG3055).

[0128] CEX-HPLC was also used to determine the batch-to-batch variability and stability of the anti-CD3 antibodies. Both antibodies were analyzed after >3 months of storage at 2-8°C and after prolonged storage at -80°C. The overlay of the PG3055 sample (Figure 3, left) shows that minor differences were observed between the samples, with the relative peak areas of the minor peaks at 17.1 and 19.8 minutes slightly altered upon prolonged storage at 2-8°C. The differences observed between the PG3056 sample (Figure 3, right) were much greater. Upon storage at 2-8°C, the peak at 20.4 minutes significantly decreased, while the peak at 18.4 minutes increased to the point where it became the main peak of the sample. Other early-eluting peaks, corresponding to other acidic isoforms of IgG, such as the peak at 16.4 minutes, also showed a relative increase in peak area. The changes observed in the CEX-HPLC chromatograms after prolonged storage at 2-8°C indicate that the anti-CD3 antibody containing the IGVK1-39 / JK1 common light chain is not stable during storage under these conditions, whereas the PG3055 antibody containing the 15C3 VL2-IGKV1-13 light chain is much more stable.

[0129] Example 2 Effect of batch-to-batch variation of PG3056 antibody on antigen binding To evaluate the stability of anti-CD3 antibodies in the presence of serum, PG3055 and PG3056 antibodies were diluted to 10 μg / mL in IMDM (Invitrogen, Catalog No. 21980-065) supplemented with 10% FBS (PAA, Catalog No. A15-101) and subsequently incubated at 37°C for 7 days. An isotype control IgG (PG1207) was included in the analysis. After 7 days, IgG binding to CD3 was assessed by flow cytometry on MACS-purified T cells from healthy donors. For comparison, PG3055 and PG3056 stored in the same medium at 2-8°C for 7 days were included in the flow cytometry analysis. CD3 binding of all antibodies was tested at concentrations of 10, 1, 0.1, and 0.01 μg / mL diluted in IMDM + 10% FBS. Bound antibodies were visualized using a goat anti-human Fc antibody (Southern Biotech, 2043-02) according to the FACS procedure previously described in WO2014 / 051433, except that staining was performed in IMDM + 10% FBS. The data (Figure 4) show that PG3055 and PG3056 exhibited similar binding to T cells when incubated at 2-8°C in the presence of serum. PG3055 binding was unaffected by incubation in IMDM + 10% FBS at 37°C for 7 days (PG3055 vs. PG3055 37°C), whereas PG3056 binding to CD3 on T cells was significantly reduced (PG3056 vs. PG3056 37°C).

[0130] In conclusion, PG3056, an anti-CD3 antibody containing the IGVK1-39 / JK1 common light chain, showed severely reduced binding to CD3 after 7 days of incubation at 37°C in serum. In contrast, the PG3055 antibody retained full CD3 binding under the same conditions.

[0131] Example 3 What is the difference between PG3055 and PG3056 antibodies? Antibodies PG3055 and PG3056 have identical sequences except for the light chain variable region. Apparently, the common light chain does not work well in the context of other amino acid sequences of antibodies, especially antibodies with heavy chain variable regions that the common light chain contacts closely. The most obvious way to try and correct the defects of the 3056 antibody would be to see if we could somehow change the common light chain to make it more similar to the light chain in antibody 3055. In addition, the light chain of antibody PG3056 was not selected as part of the antigen-binding site that binds the CD3 molecule; the heavy chain variable region was selected for that purpose. This is another reason to see if we could make the common light chain more similar to the light chain in the parent antibody PG3055.

[0132] To determine where the PG3055 and PG3056 variable regions differed as a result of their different light chains, their Fab regions were modeled. MODELLER (Sali et al., 1993: J. Mol. Biol. 234, 779-815) was used to construct homology models of the PG3056 and PG3055 Fab regions. From the resulting structure set, the model structure with the best energy score was selected. Side-chain optimization and energy minimization algorithms were used to replace template side chains where they differed from the target sequence. The homology models of 3055 Fab and 3056 Fab were visually verified using Yasara (http: / / www.yasara.org). The overlay of the models for PG3055 and PG3056 (Figure 5, right panel) shows that sequence differences in the light chains alone cause minor structural changes, and that the orientation of some portions of the light chain relative to the heavy chain varies to some extent between the two IgGs. The altered moieties may be inherently more unstable and may be the underlying cause of the observed stability differences. Alternatively, the altered moieties may be more or less susceptible to different heterogeneity-inducing processes. Various processes, known or unknown, may be responsible for the characteristics observed for the PG3056 antibody. Enzymatic and non-enzymatic modifications, including disulfide bond formation, glycosylation, N-terminal glutamine cyclization, C-terminal lysine processing, deamidation, oxidation, glycation, and peptide bond cleavage, are among the processes that can cause heterogeneity (Liu et al., Journal of Pharmaceutical Sciences, 97:2426-2447 (2008)). Depending on the type of modification, heterogeneity of monoclonal antibodies may be introduced by intracellular or extracellular processes, for example, those carried out in serum, ascites, and / or cell culture medium. Heterogeneity may also be introduced by the purification process under different stress conditions, such as exposure to high temperature or strong light, or by incubation with buffers during storage.

[0133] Modeling did not pinpoint specific amino acids or regions where the effects of mutating the variable regions of PG3056 should be focused. The differences observed between the homology models of PG3055 and PG3056 were minor.

[0134] In the present invention, it has been found that it is not necessary to change the amino acid sequence of the light chain of the PG3056 antibody. By adapting the heavy chain, it is possible to produce antibodies with good binding properties, good stability, and good homogeneity, even when the heavy chain is present in the variable region with a common light chain. As exemplified in more detail herein below, it has surprisingly been found that certain changes in the CDR2 region of the heavy chain are tolerated with respect to binding and provide antibodies with the desired stability and homogeneity.

[0135] This change results in a change in the NG deamidation motif in the CDR2 region of the heavy chain. The HCDR2 regions of both PG3055 and PG3056 contain residues Asn54 and Gly55. As shown in Figure 5, these residues are surface-exposed in both antibodies. The structural location and surface exposure are very similar in both molecules, with only slight differences in orientation relative to the asparagine side chain. Considering that the surface exposure of this motif in both antibodies makes it readily accessible to the surrounding environment and that there are no significant differences in the folding of the HCDR2 NG motif between PG3055 and PG3056, enhanced deamidation of Asn54 in PG3056 is unlikely to be the underlying cause of the binding variability observed between different PG3056 batches.

[0136] Example 4 Generation and characterization of PG3056 variants Analytical characterization of PG3056 by CIEX-HPLC indicated that the IgG was highly heterogeneous. Because this heterogeneity could hinder CIEX-HPLC-based purification of the CD3xCLEC12A bispecific IgG, we sought to improve the CIEX-HPLC profile of the MF3056 Fab. The heavy chain variable region of MF3056 contains several residues and / or motifs that may contribute to the heterogeneity of PG3056. These are the C-terminal lysine residue, the NG asparagine deamidation motif in HCDR2, and the acid-labile DP motif in HCDR3 (see Figure 6 for an alignment of MF3056_VH versus VH3-33 germline sequences).

[0137] Although in silico modeling did not identify any HCDR2 NG motifs as a likely source of the observed heterogeneity, we sought to identify MF3056 VH variants that lack this post-translational modification motif and might simultaneously exhibit improved stability along with improved CIEX-HPLC retention profiles. To this end, the following MF3056 VH variants were generated and tested: MF3872, MF3873, and MF3905 (Figure 7).

[0138] These Fabs containing these VH variants and the common light chain (MF3872, MF3873, and MF3905) were expressed as full-length monoclonal IgGs (PG3872, PG3873, and PG3905) and tested for binding to membrane-expressed CD3 on HPB-ALL cells by flow cytometry (using the FACS procedure previously described in WO2014 / 051433). The following results were obtained (Figure 8).

[0139] These results show that variants PG3873 and PG3905 completely lost CD3 binding, whereas variant PG3872 retained very low binding to CD3.

[0140] As an alternative approach to obtain improved variants with reduced heterogeneity and reduced immunogenicity, the VH of MF3056 was germlined toward the VH3-33 sequence by making individual or compound amino acid substitutions at several residues, generating the following variants of MF3056_VH: MF3874_VH, MF3878_VH, MF3883_VH, MF3886_VH, and MF3891_VH (Figure 9).

[0141] Furthermore, Fabs containing these VH variants and the common light chain (MF3874_VH, MF3878_VH, MF3883_VH, MF3886_VH, and MF3891_VH) were expressed as full-length monoclonal IgGs (PG3874, PG3878, PG3883, PG3886, and PG3891) and tested for CD3 binding as described above. The following results were obtained (Figure 10).

[0142] As shown in Figure 10, all individual (PG3874(Q6E), PG3878(V23A), PG3883(A50V), PG3886(T97A)) and combined (PG3891(Q6E / V23A / T97A)) germlined variants retained full CD3 binding capacity. PG3891 was subsequently analyzed by CIEX-HPLC (as described in Example 1) to assess whether germlining resulted in reduced heterogeneity.

[0143] The CIEX-HPLC profile of PG3891 still showed significant charge heterogeneity, as shown in Figure 11. Similar CIEX-HPLC profiles were obtained for the individual germlined variants (data not shown).

[0144] In a next attempt to reduce the charge heterogeneity of PG3056, the MF3056_VH variant, which lacks the HCDR2 NG motif, was generated (Figure 12).

[0145] Fabs containing these VH variants and the common light chain (MF5192-5197_VH) were generated, expressed as full-length monoclonal IgG (PG numbers), and tested for binding to membrane-expressed CD3 on HPB-ALL cells by flow cytometry as described above. From this analysis (Figure 13), PG5196 (N 54 G 55 N 54 A 55 It was shown that MF3056 variants (PG5192, PG5193, or PG5197) retained CD3 binding comparable to PG3056, whereas all other tested MF3056 variants showed significantly reduced CD3 binding (PG5192, PG5193, or PG5197) or no binding of CD3 (PG5194 and PG5195).

[0146] To assess the charge heterogeneity of the PG5196 mAb, isoelectric focusing was performed as described in Example 1. After staining the gel, a thin band at high pI and minor satellite bands at somewhat higher pI values ​​are observed for PG5196 (see Figure 14).

[0147] To further evaluate the charge heterogeneity of PG5196, CIEX-HPLC was performed according to the procedure described in Example 1. The resulting chromatogram is shown in Figure 15. The chromatogram shows a major peak at a retention time of 19 min preceded by a minor peak at 16.9 min. Surprisingly, these data demonstrate that PG5196 exhibits a significantly improved charge heterogeneity profile compared to PG3056.

[0148] Example 5 Generation of additional variants of PG5196 using phage display selection A phage display library was designed based on the MF5196 VH to obtain additional CD3-binding Fabs with similarly reduced charge heterogeneity. A phage display library was generated containing a panel of VH regions based on the rearranged human IGKV1-39 / IGKJ1 VL region (De Kruif et al., Biotechnol Bioeng. 2010 (106) 741-50) and MF5196 incorporating amino acid substitutions that could potentially improve the VH / VL interface. Specific substitutions and allowed alternative amino acids are listed for each position in Table 1. For each mutated position, all indicated substitutions and the original amino acid were introduced in equal ratios.

[0149] [Table 1]

[0150] Bacteriophages from these phage display libraries were selected in one or two rounds using HBP-ALL cells and / or recombinant human CD3δε-Fc protein using procedures known to those skilled in the art. Binding phages were chemically eluted and used to reinfect bacteria. After picking several surviving bacterial colonies, the phages were rescued and screened by flow cytometry for binding to the CD3 / TCR complex expressed on the cell surface. All phages that showed CD3 binding were subjected to colony PCR, and the VH regions were amplified and sequenced.

[0151] Analysis of the VH gene revealed which substitution variants retained CD3 binding. The selected MF5196_VH variants had the following substitutions: A50 to QSYL, D59 to LIVFRANEHST, A61 to NIHQLRYESTDKV, and / or F105 to MY (Table 2). In contrast, substitutions at H35, Y102, N103, and W104 listed in Table 1 were not tolerated, as all selected CD3-binding variants retained the original amino acids at positions 35, 102, 103, and 104. This indicates that residues H35, Y102, N103, and W104 are critical for CD3 binding.

[0152] Examples of CD3-binding variants of MF5196_VH are MF5603_VH, MF5616_VH, MF5626_VH, MF5630_VH, MF5648_VH, MF5661_VH, and MF5694_VH, all combined with rearranged human IGKV1-39 / IGKJ1 VL regions. The VH sequences of these MFs are listed in Figure 25. Testing these MF variants in a monospecific IgG format for binding to membrane-expressed CD3 on HPB-ALL cells by flow cytometry as described above showed that all examples bound to CD3 (Figure 26). As an example of their inherent stability, PG5661 was analyzed by CIEX-HPLC using the procedure described in Example 1. The resulting chromatogram is shown in Figure 27. The chromatogram shows a major peak with a retention time of approximately 20 minutes, similar to the profile shown for PG5196.

[0153] [Table 2]

[0154] Example 5B To obtain additional stable CD3-binding Fabs based on the MF5196 VH, a phage display library was designed containing a set of VH3-33 variants fused to the rearranged human IGKV1-39 / IGKJ1 VL region (De Kruif et al., Biotechnol Bioeng. 2010 (106) 741-50) and the heavy chain CDR3 region of MF5196.

[0155] Bacteriophages from these phage display libraries were selected in one or two rounds using HBP-ALL cells and / or recombinant human CD3δε-Fc protein using procedures known to those skilled in the art. Binding phages were chemically eluted and used to reinfect bacteria. After picking several surviving bacterial colonies, the phages were rescued and screened by flow cytometry for binding to the CD3 / TCR complex expressed on the cell surface. All phages that showed CD3 binding were subjected to colony PCR, and the VH regions were amplified and sequenced.

[0156] The resulting examples of additional CD3-binding variants of MF5196_VH are MF5351_VH, MF5354_VH and MF5356_VH, all combined with rearranged human IGKV1-39 / IGKJ1 VL regions (listed in Figure 28).

[0157] Testing these MF variants for binding to membrane-expressed CD3 on HPB-ALL cells in a monospecific IgG format by flow cytometry as described above showed that all examples bound to CD3 (Figure 29).

[0158] Example 6 Functional characterization of MF5196 Fab in a CD3 x CLEC12A bispecific IgG format To investigate the functional activity of MF5196 Fab, this CD3 Fab and MF3056 CD3 Fab were expressed as a fixed arm together with CLEC12A Fab MF4327. The CD3 x CLEC12A bispecific IgG was expressed in the full-length bispecific IgG format described in WO2014 / 051433, including a hinge downstream / CH2 engineering at positions 235-236 (referred to as DM-Fc, a CH2 double mutation in the Fc region). Like MF3056 and MF5196, MF4327 Fab uses the human IGKV1-39 / IGKJ1 light chain. The MF4327 Fab sequence is shown in patent application WO2014 / 051433. First, binding of 5196x4327 DM-Fc bsAb to CD3 and CLEC12A was demonstrated by flow cytometry using CD3+ HPB-ALL cells and CLEC12A+ HL-60 cells (per a procedure previously described in WO2014 / 051433). 3056x4327 DM-Fc bsAb was included as a reference, and an irrelevant IgG1 isotype control, PG1337, was included as a control (Figure 16). Both CD3xCLEC12A DM-Fc bsAbs bound to membrane-expressed CD3 and HPB-ALL, with slightly improved binding relative to 5196x4327 DM-Fc bsAb.

[0159] Next, the functional activity of the 5196x4327 CD3xCLEC12A DM-Fc bsAb was examined. Initially, its T cell stimulatory ability was examined using resting T cells from healthy donors. Purified resting T cells were obtained using the procedure described in patent WO2014 / 051433. Subsequently, as described in WO2014 / 051433, purified resting T cells were incubated with cells from the leukemia-derived HL-60 cell line in 10% human serum (HS) at an effector:target cell ratio of 5:1 for 2 days. The 3056x4327 DM-Fc bsAb was included as a reference. As negative control benchmark IgGs, an isotype control IgG (control IgG) and the 5196x1337 DM-Fc bsAb were included. The 5196x1337 DM-Fc bsAb binds CD3 in one arm (MF5196) and tetanus toxoid (TT) in the second arm (MF1337), and was therefore included to investigate potential off-target induction activity by MF5196. The bsAb and control IgG were tested at 1,000 ng / mL. T cell activation data are expressed as the percentage of CD69-positive cells within the CD4+ or CD8+ T cell population (Figure 17). The 5196x4327 DM-Fc bsAb induced activation of both CD4 and CD8 T cells, as reflected by upregulation of the CD69 early activation marker. Similarly, the 3056x4327 DM-Fc bsAb induced upregulation of CD69 on CD4 and CD8 T cells, but to a lesser extent compared to the 5196x4327 DM-Fc bsAb. The observed upregulation of CD69 on both T cell subsets was specific for the CLEC12A antigen, as the 5196x1337 DM-Fc bsAb did not induce upregulation of CD69.

[0160] This analysis showed that the 5196x4327 DM-Fc bsAb was slightly better than the 3056x4327 DM-Fc bsAb at inducing antigen-specific activation of CD4 and CD8 T cells (Figure 17). The T cell activation induced by the 5196x4327 DM-Fc bsAb was specific for CLEC12A, as the control 5196x1337 DM-Fc bsAb did not induce T cell activation. Collectively, this indicates that the N-to-A amino acid substitution in HCDR2, which generates the CD3-binding MF5196 Fab, fully retained CLEC12A-specific T cell activation.

[0161] To examine whether the degree of T cell activation by the 5196x4327 vs. 3056x4327 CD3xCLEC12A DM-Fc bsAb was sufficient to induce target cell lysis, HL-60 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cocultured with T cells from healthy donors at a 5:1 effector:target cell ratio in the presence of 10% HS. The 5196x1337 CD3xTT DM-Fc bsAb was included to investigate potential off-target lysis. Bispecific IgGs were tested over a 4-fold dilution range starting at 1,000 ng / ml. After 2 days, viable CFSE-positive HL-60 cells were quantified by flow cytometry. Results were expressed as percent specific lysis relative to the PBS control condition (Figure 18).

[0162] This analysis showed that the 5196x4327 DM-Fc bsAb fully retained its ability to induce CLEC12A antigen-specific target cell lysis. Surprisingly, the potency of 5196x4327 DM-Fc was significantly better than that of 3056x4327 DM-Fc bsAb.

[0163] In conclusion, this example demonstrates that the MF5196 Fab is functional, as the 5196x4327 CD3xCLEC12A DM-Fc bsAb efficiently induced CLEC12A antigen-specific T cell activation and lysis of CLEC12A+HL-60 cells. Furthermore, this example demonstrates that the 5196x4327 DM-Fc bsAb has improved potency compared to the 3056x4327 DM-Fc bsAb.

[0164] Example 7 Affinity of the anti-CD3 and anti-CLEC12A arms of 5196×4327 DM-Fc bsAb The affinity of MF5196 CD3 Fab and MF4327 CLEC12A Fab for their targets was measured by surface plasmon resonance (SPR) using a BIAcore T100. Anti-human IgG mouse monoclonal antibody (Becton and Dickinson, catalog no. 555784) was conjugated to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). 5196x4327 DM-Fc bsAb was then captured onto the sensor surface. Subsequently, recombinant purified antigen human CLEC12A (Sino Biological Co., catalog no. 11896-H07H) and human CD3δε-Fc protein were flowed over the sensor surface at a concentration range to measure binding and dissociation rates. After each cycle, the sensor surface was regenerated with a pulse of HCl, and 5196x4327 DM-Fc bsAb was recaptured. From the resulting sensorgrams, the association and dissociation rates were determined using BIAevaluation software.

[0165] These data (Figure 19) showed that 5196x4327 DM-Fc bsAb had an affinity of 3 nM for human CLEC12A and 177 nM for CD3, indicating that the affinity of the CLEC12A arm of 5196x4327 DM-Fc bsAb was approximately 60-fold higher than the affinity of the human CD3-binding arm.

[0166] [Table 3]

[0167] Example 8 Efficacy of 5196x4327 DM-Fc bsAb to induce lysis of AML blasts in primary AML patient samples In Example 6, the 5196x4327 DM-Fc bsAb was shown to potently induce lysis of HL-60 target cells by T cells derived from healthy donors. In patent WO2014 / 051433, the inventors demonstrated that the CD3xCLEC12A DM-Fc bsAb was capable of inducing lysis of AML blasts by autologous T cells derived from AML patients at an effector-to-target ratio of 5:1. In this Example, we investigated the efficacy of the CD3xCLEC12A DM-Fc bsAb, more specifically, the 5196x4327 CD3xCLEC12A DM-Fc bsAb, in inducing lysis of AML blasts in primary AML samples at low effector-to-target ratios, i.e., T cell-to-AML blast ratios.

[0168] AML patient samples collected at diagnosis (AML FAB classification M1, M2, M4, M4 / M5, Table 4) were thawed and characterized for T cell and AML blast fractions by flow cytometry analysis for CD4, CD8, CD14, CD33, CD34, CD45, and 7AAD. The analyzed AML samples had effector-to-target ratios ranging from 1:7 to 1:40.

[0169] Primary AML patient bone marrow samples were then cultured in IMDM medium supplemented with 10% normal HS, 20 ng / mL IL-15 (Miltenyi, no. 130-095-766), 2.5 ng / mL GM-CSF (Immunotools, no. 11343125), 12.5 ng / mL G-CSF (described in Norde et al., 2009), 6.25 ng / mL IL-3 (Immunotools, no. 11340035), 3.0 ng / mL SCF (Immunotools, no. 11343325), and 2.5 ng / mL Flt3L (Immunotools, no. 311340035). Test conditions included PBS, isotype control Ab WT-Fc, 5196x4327 DM-Fc bsAb, 5196x1337 DM-Fc bsAb, and positive control CD3 WT-Fc Ab (all antibodies at 1,000 ng / mL). After 7 days of culture, T cell expansion and AML blast killing were determined by flow cytometry analysis using the same markers used on day 0. Results were expressed as fold T cell expansion or frequency of AML blast lysis relative to PBS conditions.

[0170] These data (Table 4 and Figure 20) demonstrated that the 5196x4327 DM-Fc bsAb efficiently induced T cell expansion after 7 days (5-30 fold T cell expansion). More importantly, these data showed that the 5196x4327 DM-Fc bsAb efficiently induced lysis of patient AML tumor cells (26-88%) in 5 of 5 primary AML patient samples tested, even in AML samples with very low effector-to-target ratios.

[0171] [Table 4]

[0172] The efficacy of 5196x4327 DM-Fc bsAb to induce T cell proliferation and lysis of AML blasts was analyzed in additional AML patient samples taken at the time of diagnosis.

[0173] AML patient samples collected at diagnosis (AML FAB classifications M1, M2, M4, and M4 / M5, Table 5) were thawed and characterized for T cell and AML blast fractions by flow cytometry analysis for CD4, CD8, CD14, CD33, CD34, CD45, and 7AAD. The analyzed AML samples had effector-to-target ratios ranging from 1:3 to 1:97. Primary AML patient samples were then cultured as described in Example 8 above. Test conditions included PBS, isotype control Ab WT-Fc, 5196x4327 DM-Fc bsAb, 5196x1337 DM-Fc bsAb, and positive control CD3 WT-Fc Ab (all antibodies at 200 ng / mL). After 7 and 10 days of culture, T cell expansion and AML blast killing were determined by flow cytometry analysis using the same markers used on day 0. Results were expressed as fold T cell expansion or AML blast lysis frequency relative to PBS conditions.

[0174] These data (Table 5) demonstrated that the 5196x4327 DM-Fc bsAb efficiently induced T cell expansion (7-226 fold T cell expansion) after 10 days. More importantly, these data showed that the 5196x4327 DM-Fc bsAb efficiently induced lysis of patient AML tumor cells (38-99%) in 6 of 8 primary AML patient samples tested, even in AML samples with very low effector-to-target ratios of 1:45-1:97.

[0175] [Table 5]

[0176] Example 9 Differential scanning calorimetry (DSC) was used to measure the thermal stability of the IgG domains described herein. DSC experiments were performed on a MicroCal VP-DSC using Origin v7.0 (VPViewer and VPAnalyzer) software. The antibody was first dialyzed against 10 mM phosphate, 150 mM NaCl buffer (pH 6.5). IgG samples were analyzed at a protein concentration of 0.25 mg / mL, as determined by UV absorption, and the dialysis buffer was used as the reference sample. Scans were performed from 50°C to 95°C at a scan rate of 1°C / min and analyzed using GraphPad Prism 5 and Microsoft Excel 2010 software.

[0177] DSC analysis of wild-type (WT) IgG1 resulted in two peaks, as shown in Figure 21 (labeled WT|WT for CH2|CH3). Tm1 at 70.9 °C corresponds to the melting of the CH2 domain, while the peak at 85.0 °C (Tm2) corresponds to the melting of the Fab and CH3 domains. The DSC graph of IgG1 (DM|WT) with the same Fab containing two mutations in the CH2 domain (L235G, G236R) shows a very similar Tm2 peak at 85.0 °C. However, the Tm1 peak shifted to 73.5 °C, indicating that these mutations significantly increase the stability of the CH2 domain. Since the CH2 domain is the most vulnerable domain not only of WT IgG1 but also of CH3-engineered bispecific IgG1, it can be concluded that the L235G, G236R-engineered CH2 domain also confers additional stability to the CD3xCLEC12A bispecific IgG antibody with these CH2 mutations.

Claims

1. An antibody that binds to human CD3, comprising a heavy chain and a light chain, wherein the heavy chain is 1 X 2 an amino acid sequence having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than the positions indicated by: QVQLV QSGGG VVQPG RSLRL SCVAS GFTFS SYGMH WVRQA PGKGL EWVAA IWYX 1 X 2 RKQDY ADSVK GRFTI SRDNS KNTLY LQMNS LRAED TAVYY CTRGT GYNWF DPWGQ GTLVT VSS [In the formula, X 1 =N and X 2 =A; X 1 =N and X 2 =T; X 1 = S and X 2 =G; X 1 =H and X 2 =G; X 1 =D and X 2 =G; or X 1 =H and X 2 =A] An antibody comprising a variable region comprising:

2. The antibody of claim 1, wherein the light chain comprises a common light chain variable region.

3. The antibody of claim 1 or 2, wherein the common light chain variable region comprises an O12 / IgVκ1-39 light chain variable region.

4. The antibody of claim 3, wherein the light chain variable region is a germline O12 / IgVκ1-39*01 variable region.

5. The antibody of claim 4, wherein the light chain variable region comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01.

6. 6. The antibody of claim 5, wherein the light chain variable region comprises the germline kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01.

7. the light chain variable region having 0 to 5 amino acid insertions, deletions, substitutions, additions, or a combination thereof, the amino acid sequence: The antibody of any one of claims 1 to 3, comprising 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.

8. 8. The antibody of claim 1, which is a bispecific antibody.

9. 9. The bispecific antibody according to claim 8, comprising an H / L chain combination as defined in any one of claims 1 to 7 and an H / L chain combination that binds to a tumor antigen.

10. 10. The bispecific antibody of claim 9, wherein the H / L chain combination that binds to a tumor antigen binds to CLEC12A.

11. 11. The antibody or bispecific antibody of claim 1, which is a human antibody or a humanized antibody.

12. 12. The bispecific antibody of any one of claims 8 to 11, comprising two different immunoglobulin heavy chains with compatible heterodimerization domains.

13. 13. The bispecific antibody of claim 12, wherein the compatible heterodimerization domain is a compatible immunoglobulin heavy chain CH3 heterodimerization domain.

14. 14. The bispecific antibody of any one of claims 8 to 13, which is an IgG antibody having a variant CH2 and / or downstream hinge domain, thereby reducing interaction of the bispecific IgG antibody with Fc-gamma receptors.

15. 15. The bispecific antibody of claim 14, wherein the variant CH2 and / or hinge downstream domain comprises an amino substitution at position 235 and / or 236 (Kabat numbering), preferably an L235G and / or a G236R substitution.

16. 16. The bispecific antibody of any one of claims 8 to 15, comprising a common light chain.

17. 17. The antibody of any one of claims 1 to 16 for use in treating a subject in need thereof.

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