Bispecific antibodies against CD277 and tumor antigens

Abstract

The present invention relates to a bispecific antibody that binds to CD277 and a human tumor antigen. The present invention also relates to a polynucleotide encoding said bispecific antibody, and to a vector and a host cell comprising said polynucleotide. Furthermore, the present invention relates to a method for producing said antibody, a method for using said antibody in the treatment of disease and its therapeutic use.

Description

[Technical field]

[0001] The contents of the sequence listing submitted electronically with this patent application (name: Evo-PCT sequence list.xml) are incorporated herein by reference in their entirety.

[0002] The present invention relates to a bispecific antibody that binds to the butyrophilin 3 family member CD277 (BTN3A) and a human tumor antigen. The present invention also relates to polynucleotides encoding said bispecific antibody, and vectors and host cells comprising said polynucleotides. Furthermore, the present invention relates to methods of producing said antibody, methods of using said antibody in the treatment of disease and therapeutic uses thereof. [Background technology]

[0003] Vγ9Vδ2 T cells are the major subset of γδ T cells in peripheral blood, accounting for approximately 60%–95%. Bioinformatics analysis of large metagenomic datasets determined the relative abundance of Vγ9Vδ2 T cells in tumors and correlated this with patient outcome. Tumor-infiltrating γδ T lymphocytes (γδ TILs), although in low numbers, were found in all tumor tissues. Importantly, a correlation was shown between the relative amount of γδ TILs and better responsiveness to immune checkpoint therapy in various cancers. (Gentles, AJ et al.; Nat.Med.2015, 1-12; Tosolini, M.; et al.; Oncoimmunology 2017, 6, 1-10). Cancer therapies based on in vivo stimulation or adoptive T cell transfer of Vγ9Vδ2 T cells have been tested over the past decades but failed to provide consistent clinical efficacy. Further concepts such as γδ chimeric antigen receptor (CAR)-T cells and γδ T cell engagers are currently under preclinical evaluation (Kuenkele KP., et al.; Cells 2020, 9, 829).

[0004] The butyrophilin 3 family member BTN3A (CD277; UniProtKB - O00481 (BT3A1_HUMAN)) is a transmembrane receptor with two extracellular immunoglobulin (Ig)-like domains and an intracellular B30.2 domain. CD277 plays a role in T cell activation and adaptive immune responses, controlling the proliferation of activated T cells, regulating the release of cytokines and IFNγ by activated T cells, and mediating T cell responses to infected and transformed cells characterized by high levels of phosphorylated metabolites such as isopentenyl pyrophosphate (Afrache, H., et al., Immunogenetics 64, 781-794 (2012)).

[0005] (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate) HMBPP is an essential intermediate in the prokaryotic non-mevalonate / 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP) pathway for isoprenoid synthesis. BTN3A is exquisitely tuned to recognize this pathogen-derived molecule in a manner similar to how TLRs recognize conserved pathogen structures such as LPS and DNA (O'Neill, LAJ; et al.; Nat.Rev. Immunol.2013, 13, 453-460; Gu, S.et al.; Front.Immunol.2014, 5, 688; Vavassori, S. et al.; Nat.Immunol.2013, 14, 908-916). The intracellular domain B30.2 of BTN3A1 directly interacts with the bacterial metabolite HMBPP (Rhodes, DAet al.; J. Immunol.2015, 194, 2390-2398; Harly, C.; et al. Blood 2012, 120, 2269-2279; Sandstrom, A.; et al.; Immunity 2014, 40, 490-500). The interaction of BTN3A1 with HMBPP allows BTN3A1 to bind to components of the immunological synapse, including γδTCR, and subsequently activates Vδ2 T cells. Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vγ9Vδ2 T cells (Wang H. et al. J Immunol 2013; 191:1029-1042; Sandstrom A. et al.; Immunity Volume 40, Issue 4, 17 April 2014, Pages 490-500, Janssen O. et al., J Immunol 1991; 146; 35-39).

[0006] CD277 is an essential compound for all tumors (Liang, F. et al., Febs Open Bio 2021 11, 2586-2599; Ghigo, C. et al., J Immunother Cancer 2020 8, A3-A3). Payne KK. et al.; Science 369, 942-949 (2020) describes that BTN3A1 governs antitumor responses by regulating αβT cells and γδT cells. De Bruin et al. (De Bruin RCG. et al.; Oncoimmunology 2018, VOL.7, NO.1, e1375641) describe a bispecific nanobody approach targeting both Vγ9Vδ2 T cells and EGFR, which induces Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in vivo in a mouse xenograft model, demonstrating the cytolytic potential of Vγ9Vδ2 T cells.

[0007] Palakodeti et al. (Palakodeti A. et al.; JBC vol. 287, no. 39, pp. 32780-32790, 2012) describe the regulation of human Vγ9Vδ2 T cell responses by CD277-specific antibodies. WO2012080769 and WO2020025703 relate to anti-BTN3A1 antibodies and their uses. BTN3A1 agonists are also described in WO2012080769; WO2010106051 (US20150353643); WO2011014438; WO2017144668; WO2019211370, WO2011 / 014438, and WO2012080351.

[0008] WO2012080351 and WO2012080769 refer to anti-C277 antibodies (7.2 and 20.1). ScFv is mentioned as a possible antibody format. Agonistic anti-C277 antibodies in the art activate the cytolytic function, cytokine production and proliferation of Vγ9Vδ2 T cells. According to De Gassart A. et al., in Science Translational Medicine 13, (2021), (https: / / doi.org / 10.1126 / scitranslmed.abj0835), activation of Vγ9Vδ2 T cells in peripheral blood causes a transient drop in circulating Vγ9Vδ2 T cells as a result of trafficking and migration, not exhaustion. The relevance of exhaustion was first recognized by the inventors.

[0009] Imbert C. and Olive D. (A. Birbrair (ed.), Tumor Microenvironment, Advances in Experimental Medicine and Biology 1273 (https: / / doi.org / 10.1007 / 978-3-030-49270-0_5) and Imbert C. et al. (Advances in Experimental Medicine and Biology, (2020), Springer, Vol. 1273, 91-104) suggest bispecific antibodies targeting both CD277 and tumor antigens for activation of Vγ9Vδ2 T cells. Multispecific antibodies such as bispecific antibodies containing one arm containing Fab or scFv containing VH and VL of anti-CD277 antibody as the format of bispecific molecules mAb x mAb, mAb x Fab, Fab x F(ab´)2 or ligand x Fab fusion proteins are also proposed in WO2020025703.

[0010] Bispecific antibodies are known in many different formats (see e.g. review by Brinkmann U. and Kontermann E.; MAbs. 2017 Feb-Mar; 9(2):182-212; Figure 2 in Brinkmann and Kontermann). In 2019, more than 20 different commercialized technology platforms are available for the generation and development of bsAbs (review: Lanrijn AF et al; Nature Reviews; https: / / doi.org / 10.1038 / s41573-019-0028-1). Bispecific antibody formats are described in Coloma MJ and Morrison SL, Nat.Biotechnol.15:159-163 (1997); see also Ulrich Brinkmann & Roland E. Kontermann (2017), The making of bispecific antibodies, mAbs, 9:2, 182-212, DOI:10.1080 / 19420862.2016.1268307. These bispecific molecules consist of an IgG antibody, called the master or parent module, and an scFv of different specificity attached to the C-terminus of the heavy chain (IgG-HC-scFv, "Morrison bispecific"; see Figure 1).

[0011] WO2010112193 (US009382323; EP2414391B1) relates to a multispecific antibody comprising a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains; and one or more single-chain Fv fragments that bind to one or more further antigens, said single-chain Fv fragments being fused to said full-length antibody via a peptide connector at the C-terminus or N-terminus of the heavy or light chain of said full-length antibody.

[0012] Presti et al. (Presti, EL et al., Frontiers in immunology 2017, 8, 975-11) state that γδ T cells can be redirected to cancer cells using antibodies. This can be achieved, for example, by using bispecific antibodies in which one binding site recognizes a tumor-specific cell surface molecule (e.g., EpCAM or HER2 / neu) and the other binding site targets CD3 or the Vγ9 chain of the Vγ9Vδ2 TCR. Such bispecific antibodies have demonstrated efficacy in preclinical models (Hoh A, et al. Liver Int (2013) 33:127-36. doi:10.1111 / liv.12011; Oberg HH, et al.; Cell Immunol (2015) 296:41-9).

[0013] WO2018041827 describes adenoviruses armed with bispecific T cell engagers (BiTEs), in which one of the binding domains is specific for a non-TCR activating protein such as BTN3A1 and one of the binding domains is specific for a tumor antigen, e.g., CEA, MUC-1, EpCAM, HER receptors HER1, HER2, HER3, HER4, PEM, A33, G250, carbohydrate antigens Ley, Lex, Leb, PSMA, TAG-72, STEAP1, CD166, CD24, CD44, E-cadherin, SPARC, ErbB2, ErbB3, etc. WO2012080769 relates to anti-CD277 antibodies (e.g., mAb 7.2, mAb 20.1). Common examples include antibody fragments such as Fv, Fab, F(ab')2, Fab', dsFv, scFv, Sc(Fv)2, and diabodies.

[0014] WO2020060406 describes an antibody comprising a first binding moiety capable of binding to human CD1d and a second binding moiety capable of binding to the Vγ9 chain of a T cell receptor on γδ T cells for use in the treatment of chronic lymphocytic leukemia, multiple myeloma or acute myeloid leukemia.

[0015] Tumor antigens are known from a variety of studies, for example studies comparing mRNA or protein expression levels in tumor and normal tissues or cell lines, respectively, and studies comparing antigen density on the surface of tumor and normal cells. (Woell, S. et al.,Int. J. Cancer 134, 731-739 (2014); Herlyn, M. et al., PNAS 76, 1438-1442 (1979); Rusnak, DW et al., Cell Prolif 580-594 (2007); Karhemo, P.-.R. et al., Frontiers in pharmacology 3, 192 (2012); Imai, K. et al., Clin Cancer Res 14, 6487-6495 (2008); Coto-Llerena, M. et al, Frontiers Oncol 10, 979 (2020); Moreaux J., Biochem Biophys Res Commun 14, 148-155 (2012); Owen, DH et al. al., J Hematol Oncol 12, 61 (2019); Wu, M. et al, Cancer Epidemiology Biomarkers Prev Publ Am Assoc Cancer Res Cosponsored Am Soc Prev Oncol 8, 775-82 (1999); Tarn, C. et al., Proc National Acad Sci 105, 8387-8392 (2008)). Claudin18 (CLD18) molecule (UniProtKB - P56856 (CLD18_HUMAN)) is a transmembrane protein with a molecular weight of approximately 27,9 / 27,72 kD. Claudins are membrane proteins present in epithelial and endothelial tight junctions. Tight junctions organize a network of interconnected chains of intramembrane particles between adjacent cells. In tight junctions, Occludin and Claudins are the most prominent transmembrane protein components.Due to its strong cell-cell adhesion properties, the cell membrane creates a first barrier preventing and controlling the outward transport of solutes and restricting the lateral diffusion of membrane lipids and proteins to maintain cell polarity. Tight junction forming proteins are crucially involved in the formation of epithelial tissue structure. Such proteins are hardly accessible to antibodies in well-structured epithelia, but are assumed to be exposed on tumor cells. Antibodies against Claudin18 and its splice variant Claudin18.2 are described, for example, in WO2007059997, WO2008145338, US20150374789, WO2013174403, and US9770487 (US10314890; EP2958945; IMAB362). WO2021024020 describes a combination therapy of anti-Claudin18.2 antibodies and immune checkpoint inhibitors for cancer treatment.

[0016] STEAP-1 (six-transmembrane epithelial antigen of prostate-1) is a 339 amino acid cell surface protein that is expressed primarily on prostate cells in normal tissues. STEAP-1 protein expression is maintained at high levels in various states of prostate cancer, and STEAP-1 is also highly expressed in other human cancers, such as lung and colon cancer. The expression profile of STEAP-1 in normal and cancer tissues suggested its potential as a target for immunotherapy. WO2008 / 052187 reports an anti-STEAP-1 antibody and its immunoconjugate. STEAP-1xCD3 bispecific antibodies are described in WO2014165818 and WO2017055388.

[0017] FOLR1 is expressed in epithelial tumor cells of various origins, including ovarian, lung, breast, renal, colon, and endometrial cancers. 10.1517 / 17425247.2012.694863.Epub 2012.WO2012119077 describes antibodies against FOLR1. Bispecific antibodies targeting FOLR1 and CD3 are described in WO2016 / 079076 and WO2021255143.

[0018] DLL3 is selectively expressed in high-grade pulmonary neuroendocrine tumors, including SCLC and LCNEC. Increased expression of DLL3 was observed in xenograft tumors from SCLC and LCNEC patients and was also confirmed in primary tumors. See Saunders et al ., Sci Translational Medicine 7(302):302ral36 (2015). Increased expression of DLL3 has also been observed in extrapulmonary neuroendocrine carcinomas, including prostate neuroendocrine carcinoma (Puca et al., Sci TranslMed 11(484): pii: eaav0891 (2019)). DLL3 is expressed on the surface of such tumor cells, but not in normal tissues. WO2021007371 is directed to anti-DLL3 antibodies, proposing humanized, chimeric and bispecific antibodies. WO2019195409 refers to a multispecific protein that binds to the NKG2D receptor, CD16 and tumor antigens. Summary of the Invention

[0019] Agonistic anti-C277 antibodies in the art activate the cytolytic function, cytokine production and proliferation of Vy9V52 T cells. Agonistic anti-C277 antibodies in the art induce a transient decrease in circulating Vy9V52 T cells, which has been explained as a result of trafficking from the circulation to tissues, including cancer tissues, rather than depletion of Vy9V52 T cells.

[0020] However, the inventors have recognized that such activation of Vy9V52 T cells by agonistic anti-C277 antibodies in the art in the absence of tumor cells induces self-elimination of Vy9V52 T cells. The inventors have found that a bispecific antibody that specifically and agonistically binds to CD277 (hereinafter also referred to as a "bispecific anti-CD277 antibody"), which specifically binds to a human tumor antigen (hereinafter also referred to as a "tumor antigen") and has the properties described below, exhibits excellent killing effect against human tumor cells bearing the tumor antigen, is highly safe with respect to lysis of non-tumor cells, and does not induce self-elimination of Vy9V52 T cells.

[0021] In one embodiment, the invention comprises a bispecific antibody comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, wherein the first binding moiety is a full-length bivalent antibody and the second binding moiety consists of two identical single chain Fv antibodies that specifically bind to the tumor antigen, each of the single chain Fv antibodies being linked to each C-terminus of the first binding moiety by a peptide linker.

[0022] In one embodiment, each of said single chain Fv antibodies is linked by a peptide linker between the N-terminus of the variable light chain and each C-terminus of the first binding moiety.

[0023] In one embodiment the bispecific antibody according to the invention is characterized in that it comprises in the first binding moiety the heavy chain CDR sequences CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and the light chain CDR sequences CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8.

[0024] In one embodiment, the antibody according to the invention is characterized in that it comprises the following substitutions in CDRH2 (SEQ ID NO: 44): N5S and K10N (also called N53S, K58N (Kabat), or N185S-K190N).

[0025] In one embodiment, the antibody according to the invention is characterized in that it comprises, in addition to said CDRH2 substitutions, a substitution of L8V (also called L31V) in CDRL1 (SEQ ID NO: 75).In one embodiment, the antibody according to the invention is characterized in that it comprises the additional substitutions L8V and H1R in CDRL1 (SEQ ID NO: 140).

[0026] In one embodiment, a bispecific antibody according to the invention is characterized in that it comprises a first binding moiety which specifically and agonistically binds to human CD277 and a second binding moiety which specifically binds to a tumor antigen, said first binding moiety being a full-length bivalent antibody comprising in the first binding moiety the heavy chain CDR sequences CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44 and CDRH3 of SEQ ID NO: 45 and the light chain CDR sequences CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8, and said second binding moiety consists of two identical single chain Fv antibodies which specifically bind to said tumor antigen, each of said single chain Fv antibodies being linked by a peptide linker to the respective C-terminus of said first binding moiety.

[0027] In one embodiment the bispecific antibody according to the invention is characterized in that CDRH2 is SEQ ID NO: 68, SEQ ID NO: 72 or SEQ ID NO: 110.

[0028] In one embodiment the bispecific antibody according to the invention is characterized in that CDRL1 is SEQ ID NO:75, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:140 or SEQ ID NO:141.

[0029] In one embodiment, the antibody according to the invention is characterized in that it comprises the N5S and K10N substitutions in CDRH2 (SEQ ID NO: 44).

[0030] In one embodiment, the antibody according to the invention is characterized in that it comprises, in addition to said CDRH2 substitutions, a substitution of L8V in CDRL1 (SEQ ID NO: 75).In one embodiment, the antibody according to the invention is characterized in that it comprises the additional substitutions L8V and H1R in CDRL1 (SEQ ID NO: 140).

[0031] In one embodiment, the first binding portion of an antibody according to the invention is a human antibody, a humanized antibody or a CDR-grafted antibody.

[0032] In one embodiment, the invention features a bispecific antibody comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, the bispecific antibody comprising heavy chain CDR sequences CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1) and b) as light chain CDR sequences, a set of CDRs selected from the group consisting of: b1) CDRL1 of SEQ ID NO: 75, CDRL2 of SEQ ID NO: 76, and CDRL3 of SEQ ID NO: 77; b2) CDRL1 of SEQ ID NO: 79, CDRL2 of SEQ ID NO: 80, and CDRL3 of SEQ ID NO: 81; b3) CDRL1 of SEQ ID NO: 83, CDRL2 of SEQ ID NO: 84, and CDRL3 of SEQ ID NO: 85; b4) CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89; b5) CDRL1 of SEQ ID NO: 117, CDRL2 of SEQ ID NO: 118, and CDRL3 of SEQ ID NO: 119; b6) CDRL1 of SEQ ID NO: 121, CDRL2 of SEQ ID NO: 122, and CDRL3 of SEQ ID NO: 123; b7) CDRL1 of SEQ ID NO: 125, CDRL2 of SEQ ID NO: 126, and CDRL3 of SEQ ID NO: 127; b8) CDRL1 of SEQ ID NO: 129, CDRL2 of SEQ ID NO: 130, and CDRL3 of SEQ ID NO: 131; b9) CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135; b10) CDRL1 of SEQ ID NO: 137, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139; b11) CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139; b12) CDRL1 of SEQ ID NO: 140, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135; b13) CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135; b14) CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 135; b15) CDRL1 of SEQ ID NO: 151, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8; b16) CDRL1 of SEQ ID NO: 152, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8; b17) CDRL1 of SEQ ID NO: 153, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8; b18) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 156; b19) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 157; b20) CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 158; b21) CDRL1 of SEQ ID NO: 154, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8; b22) CDRL1 of SEQ ID NO: 155, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 The present invention is characterized in that it comprises c) the second binding moiety consists of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each linked to the respective C-terminus of the first binding moiety.

[0033] In one embodiment the bispecific antibody according to the invention is characterized in that for the first binding moiety the variable heavy chain is SEQ ID NO: 42 and the variable light chain is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 65, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86.

[0034] In one embodiment the bispecific antibody according to the invention is characterized in that it comprises humanized versions of said variable chains.

[0035] In one embodiment, the bispecific antibody according to the invention is characterized in that the tumor antigen is selected from the group consisting of CLDN18.2 (UniProtKB-P56856-2, CLD18_HUMAN), FOLR1 (UniProtKB-P15328, FOLR1_HUMAN), STEAP1 (UniProtKB-Q9UHE8, STEA1_HUMAN), or DLL3 (UniProtKB-Q9NYJ7, DLL3_HUMAN). Further useful tumor antigens are described, for example, in Middleburg et al., Cancers (2021) 13, 287, pp 4-6.

[0036] In one embodiment, the antibody according to the invention is characterized in that the first binding moiety comprises a combination of heavy and light chain CDRs selected from the group consisting of the CDR combinations shown for compounds EvB#21-136 in Table 3, or the variable light chain and variable heavy chain combinations shown for compounds EvB#21-136 in Table 3, and the second binding moiety consists of two identical single chain Fv antibodies that specifically bind to a tumor antigen. In one embodiment, the bispecific antibody according to the invention is humanized.

[0037] In one embodiment, the antibody according to the present invention is characterized in that the second binding portion, for FOLR1 as a tumor antigen, comprises light chain CDRs CDRL1 of SEQ ID NO: 11, CDRL2 of SEQ ID NO: 12, and CDRL3 of SEQ ID NO: 13, and heavy chain CDRs CDRH1 of SEQ ID NO: 15, CDRH2 of SEQ ID NO: 16, and CDRH3 of SEQ ID NO: 17 (FOLR1 CDR set).

[0038] In one embodiment, the antibody according to the present invention, directed against STEAP1 as a tumor antigen, comprises in the second binding portion the CDRs CDRL1 of SEQ ID NO: 19, CDRL2 of SEQ ID NO: 20, and CDRL3 of SEQ ID NO: 21, as well as CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25 (STEAP1 CDR set).

[0039] In one embodiment, the antibody according to the present invention, directed against DLL3 as a tumor antigen, comprises in the second binding portion the CDRs CDRL1 of SEQ ID NO: 27, CDRL2 of SEQ ID NO: 28, and CDRL3 of SEQ ID NO: 29, as well as CDRH1 of SEQ ID NO: 31, CDRH2 of SEQ ID NO: 32, and CDRH3 of SEQ ID NO: 33 (DLL3 CDR set).

[0040] In one embodiment, the antibody according to the present invention has a second binding portion for CLDN18.2 as a tumor antigen, which comprises, as CDRs, CDRL1 of SEQ ID NO: 35, CDRL2 of SEQ ID NO: 36, and CDRL3 of SEQ ID NO: 37, as well as CDRH1 of SEQ ID NO: 39, CDRH2 of SEQ ID NO: 40, and CDRH3 of SEQ ID NO: 41 (CLDN18.2 CDR set).

[0041] In one embodiment, the antibody according to the invention is characterized in that the second binding portion comprises a combination of heavy and light chain variable regions of SEQ ID NO: 10 and SEQ ID NO: 14 against FOLR-1 as tumor antigen.

[0042] In one embodiment, the antibody according to the invention is characterized in that the second binding portion comprises a combination of heavy and light chain variable regions of SEQ ID NO: 18 and SEQ ID NO: 22 against STEAP1 as tumor antigen.

[0043] In one embodiment, the antibody according to the invention is characterized in that the second binding portion comprises a combination of heavy and light chain variable regions of SEQ ID NO: 26 and SEQ ID NO: 30 against DLL3-4 as tumor antigen.

[0044] In one embodiment, the antibody according to the invention is characterized in that the second binding portion comprises a combination of heavy and light chain variable regions of SEQ ID NO: 34 and SEQ ID NO: 38 against CLDN 18.2 as tumor antigen.

[0045] In one embodiment, the antibody according to the invention has the following characteristics: a) said bispecific antibody exhibits an EC50 ratio of 0.001 to 0.2 for lysis of a cell line bearing a first tumor antigen compared to lysis by a reference antibody comprising as its heavy chain the heavy chain of SEQ ID NO: 94 and as its light chain the light chain of SEQ ID NO: 93; b) the bispecific antibody exhibits an EC50 ratio of 5 to 1000 for lysis of a second cell line that does not bear the tumor antigen compared to lysis by the reference antibody; All were measured in the same assay under the same conditions in the presence of activated Vγ9Vδ2 T lymphocytes, at an E / T ratio of 5:1 and in the presence of 12.5 IU / mL interleukin-2.

[0046] In one embodiment, the bispecific antibody is in the Mab-scFv format. In one embodiment, the invention is characterized in that it comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; b) the second binding moiety specifically binds to the tumor antigen and comprises, as heavy and light chain CDRs, a set of CDRs selected from the group consisting of: b1) CDRL1 of SEQ ID NO: 11, CDRL2 of SEQ ID NO: 12, and CDRL3 of SEQ ID NO: 13, and CDRH1 of SEQ ID NO: 15, CDRH2 of SEQ ID NO: 16, and CDRH3 of SEQ ID NO: 17 against FOLR1 as a tumor antigen (FOLR1 CDR set); b2) CDRL1 of SEQ ID NO: 19, CDRL2 of SEQ ID NO: 20, and CDRL3 of SEQ ID NO: 21, and CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25 against STEAP1 as a tumor antigen (STEAP1 CDR set); b3) CDRL1 of SEQ ID NO: 27, CDRL2 of SEQ ID NO: 28, and CDRL3 of SEQ ID NO: 29, and CDRH1 of SEQ ID NO: 31, CDRH2 of SEQ ID NO: 32, and CDRH3 of SEQ ID NO: 33 against DLL3 as a tumor antigen (DLL3 CDR set); b4) CDRL1 of SEQ ID NO: 35, CDRL2 of SEQ ID NO: 36, and CDRL3 of SEQ ID NO: 37, and CDRH1 of SEQ ID NO: 39, CDRH2 of SEQ ID NO: 40, and CDRH3 of SEQ ID NO: 41 for CLDN18.2 as a tumor antigen (CLDN18.2 CDR set); c) said bispecific antibody exhibits an EC50 ratio of 0.001 to 0.2 for lysis of a cell line bearing a first tumor antigen compared to lysis by a reference antibody comprising as its heavy chain the heavy chain of SEQ ID NO: 94 and as its light chain the light chain of SEQ ID NO: 93; d) the bispecific antibody exhibits an EC50 ratio of 5 to 1000 for lysis of a second cell line that does not bear the tumor antigen compared to lysis by the reference antibody; All were measured in the same assay under the same conditions, in the presence of activated Vγ9Vδ2 T lymphocytes, an E / T ratio of 5:1, and 12.5 IU / mL interleukin-2.

[0047] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:8 (CDRL set 1); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 47, CDRH2 of SEQ ID NO: 48, and CDRH3 of SEQ ID NO: 49 (CDRH set 2); b3) CDRH1 of SEQ ID NO: 51, CDRH2 of SEQ ID NO: 52, and CDRH3 of SEQ ID NO: 53 (CDRH set 3); b4) CDRH1 of SEQ ID NO: 55, CDRH2 of SEQ ID NO: 56, and CDRH3 of SEQ ID NO: 57 (CDRH set 4); b5) CDRH1 of SEQ ID NO: 59, CDRH2 of SEQ ID NO: 60, and CDRH3 of SEQ ID NO: 61 (CDRH Set 5); b6) CDRH1 of SEQ ID NO: 63, CDRH2 of SEQ ID NO: 64, and CDRH3 of SEQ ID NO: 65 (CDRH Set 6); b7) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 68, and CDRH3 of SEQ ID NO: 69 (CDRH Set 7); b8) CDRH1 of SEQ ID NO: 71, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 73 (CDRH set 8); b10) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 106, and CDRH3 of SEQ ID NO: 107 (CDRH set 10); b11) CDRH1 of SEQ ID NO: 109, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 111 (CDRH set 11); b12) CDRH1 of SEQ ID NO: 113, CDRH2 of SEQ ID NO: 114, and CDRH3 of SEQ ID NO: 115 (CDRH set 12); b13) CDRH1 of SEQ ID NO:59, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:4 (CDRH set 14); b14) CDRH1 of SEQ ID NO:59, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:4 (CDRH set 15); b15) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 4 (CDRH set 20); b16) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH set 18); b17) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH set 19) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0048] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 121, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL set 2); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b3) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22); b4) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 4 (CDRH set 20); b5) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 68, and CDRH3 of SEQ ID NO: 4 (CDRH Set 7); b6) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH set 18); b7) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0049] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 83, CDRL2 of SEQ ID NO: 84, and CDRL3 of SEQ ID NO: 85 (CDRL set 3); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH set 1, b2) CDRH set 2, b3) CDRH set 3, b4) CDRH set 4, b5) CDRH set 5, b6) CDRH set 6, b7) CDRH set 7, b8) CDRH set 8, and b9) CDRH1 of SEQ ID NO:2, CDRH2 of SEQ ID NO:3, and CDRH3 of SEQ ID NO:4 (CDRH set 9), b10) CDRH set 10, b11) CDRH set 11, and b12) CDRH set 12; c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0050] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL set 4); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b3) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22); b4) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 4 (CDRH set 1); b5) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH set 21); b6) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 68, and CDRH3 of SEQ ID NO: 45 (CDRH set 7); b7) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 23); b8) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 106, and CDRH3 of SEQ ID NO: 45 (CDRH set 24); b9) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH set 25); b10) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 114, and CDRH3 of SEQ ID NO: 115 (CDRH set 26); b11) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 4 (CDRH set 27); b12) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19) b13) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH set 18) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0051] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 75, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL set 5); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b3) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22); b4) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 20); b5) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 68, and CDRH3 of SEQ ID NO: 45 (CDRH set 7); b2) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH set 18); b6) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0052] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 140, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL set 6); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b3) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22); b1) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 20); b2) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 23); b3) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 18); b4) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0053] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 8 (CDRL set 7); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b3) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22); b4) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 18); b5) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0054] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL set 8); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b2) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b3) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22); b4) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 20); b5) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 23); b6) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 18); b7) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19) The present invention is characterized in that it comprises c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0055] In one embodiment, the invention includes a bispecific antibody comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139 (CDR set 12); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 20); b2) CDRH1 of SEQ ID NO: 67, CDRH2 of SEQ ID NO: 68, and CDRH3 of SEQ ID NO: 45 (CDRH set 23); b3) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 18); b4) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 19), and b5) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1); b6) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH set 21); b7) CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH set 22) Including, c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0056] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89 (CDRL set 9); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH Set 1, b2) CDRH Set 2, b3) CDRH Set 3, b4) CDRH Set 4, b5) CDRH Set 5, b6) CDRH Set 6, b7) CDRH Set 7, b8) CDRH Set 8, and b9) CDRH Set 9, b10) CDRH Set 10, b11) CDRH Set 11, and b12) CDRH Set 12; c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0057] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 79, CDRL2 of SEQ ID NO: 80, and CDRL3 of SEQ ID NO: 81 (CDRL set 10); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH Set 1, b2) CDRH Set 2, b3) CDRH Set 3, b4) CDRH Set 4, b5) CDRH Set 5, b6) CDRH Set 6, b7) CDRH Set 7, b8) CDRH Set 8, and b9) CDRH Set 9, b10) CDRH Set 10, b11) CDRH Set 11, and b12) CDRH Set 12; c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0058] In one embodiment, the invention includes a bispecific antibody comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; Light chain CDR sequences: CDRL1 of SEQ ID NO: 75, CDRL2 of SEQ ID NO: 76, and CDRL3 of SEQ ID NO: 77 (CDRL set 11); b) as heavy chain CDR sequences, CDR sequences selected from the group consisting of: b1) CDRH Set 1, b2) CDRH Set 2, b3) CDRH Set 3, b4) CDRH Set 4, b5) CDRH Set 5, b6) CDRH Set 6, b7) CDRH Set 7, b8) CDRH Set 8, and b9) CDRH Set 9, b10) CDRH Set 10, b11) CDRH Set 11, and b12) CDRH Set 12; c) said second binding moiety consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0059] In one embodiment the invention comprises a bispecific antibody according to the invention in Mab-scFv format, wherein the first binding moiety comprises a) as light chain CDR sequences CDRL1 set 1 and b) as heavy chain CDR sequences b1) CDRH set 1, b2) CDRH set 2, b3) CDRH set 3, b4) CDRH set 4, b5) CDRH set 5, b6) CDRH set 6, b7) CDRH set 7, and b8) CDRH set 8, b10) CDRH set 10, b11) CDRH set 11, b12) CDRH set 12; Furthermore, the second binding moiety comprises, as heavy and light chain CDRs, a set of CDRs selected from the group consisting of a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set, and a CLDN 18.2 CDR set.

[0060] In one embodiment the invention comprises a bispecific antibody according to the invention in Mab-scFv format, wherein the first binding moiety comprises as light chain CDR sequences CDRL1 set 2 and as heavy chain CDR sequences: b1) CDRH set 1, b2) CDRH set 2, b3) CDRH set 3, b4) CDRH set 4, b5) CDRH set 5, b6) CDRH set 6, b7) CDRH set 7, b8) CDRH set 8, and b9) CDRH set 9, b10) CDRH set 10, b11) CDRH set 11, b12) CDRH set 12; Furthermore, the second binding moiety comprises, as heavy and light chain CDRs, a set of CDRs selected from the group consisting of a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set, and a CLDN 18.2 CDR set.

[0061] In one embodiment the invention comprises a bispecific antibody according to the invention in Mab-scFv format, wherein the first binding moiety comprises as light chain CDR sequences CDRL1 set 3 and as heavy chain CDR sequences: b1) CDRH set 1, b2) CDRH set 2, b3) CDRH set 3, b4) CDRH set 4, b5) CDRH set 5, b6) CDRH set 6, b7) CDRH set 7, and b8) CDRH set 8, and b9) CDRH set 9, b10) CDRH set 10, b11) CDRH set 11, and b12) CDRH set 12; Furthermore, the second binding moiety comprises, as heavy and light chain CDRs, a set of CDRs selected from the group consisting of a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set, and a CLDN 18.2 CDR set.

[0062] In one embodiment the invention comprises a bispecific antibody according to the invention in Mab-scFv format, wherein the first binding moiety comprises as light chain CDR sequences CDRL1 set 4 and as heavy chain CDR sequences: b1) CDRH set 1, b2) CDRH set 2, b3) CDRH set 3, b4) CDRH set 4, b5) CDRH set 5, b6) CDRH set 6, b7) CDRH set 7, b8) CDRH set 8, and b9) CDRH set 9, b10) CDRH set 10, b11) CDRH set 11, b12) CDRH set 12; Furthermore, the second binding moiety comprises, as heavy and light chain CDRs, a set of CDRs selected from the group consisting of a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set, and a CLDN 18.2 CDR set.

[0063] In one embodiment the invention comprises a bispecific antibody according to the invention in Mab-scFv format, characterized in that the first binding moiety comprises as light chain CDR sequences CDRL1 set 5 and as heavy chain CDR sequences a CDR set selected from the group consisting of: b1) CDRH set 1, b2) CDRH set 2, b3) CDRH set 3, b4) CDRH set 4, b5) CDRH set 5, b6) CDRH set 6, b7) CDRH set 7, b8) CDRH set 8 and b9) CDRH set 9, b10) CDRH set 10, b11) CDRH set 11, b12) CDRH set 12, and the second binding moiety comprises, as heavy and light chain CDRs, a set of CDRs selected from the group consisting of a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set, and a CLDN 18.2 CDR set.

[0064] One embodiment of the invention is a bispecific antibody according to the invention in Mab-scFv format, characterized in that the first binding moiety is a humanized antibody.

[0065] In one embodiment, the invention comprises a bispecific antibody in a Mab-scFv format, wherein the first binding moiety is As a variable heavy chain, a variable heavy chain selected from the group consisting of SEQ ID NOs: 42, 46, 50, 54, 58, 62, 66, and 70, or a humanized version thereof having at least 95% sequence identity thereto; and As a light chain sequence, a sequence selected from the group consisting of: a) SEQ ID NO:5; b) SEQ ID NO: 74; c) SEQ ID NO: 78; d) SEQ ID NO: 82; e) SEQ ID NO: 86; or a humanized version thereof having at least 95% sequence identity to said sequence, and a second binding moiety consisting of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of the single-chain Fv antibodies being linked by a peptide linker to each C-terminus of the first binding moiety.

[0066] In one embodiment, the second binding moiety comprises a set of heavy and light chain variable regions selected from the group consisting of: e) SEQ ID NO: 10 and SEQ ID NO: 14 for FOLR-1 as tumor antigen; f) SEQ ID NO: 18 and SEQ ID NO: 22 for STEAP1 as tumor antigen; g) SEQ ID NO: 26 and SEQ ID NO: 30 for DLL3-4 as tumor antigen, and h) SEQ ID NO: 34 and SEQ ID NO: 38 for CLDN18.2 as tumor antigen.

[0067] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format, wherein the first binding moiety comprises as variable heavy chain the variable heavy chain of SEQ ID NO: 1 or a humanized version thereof having at least 95% sequence identity to said sequence, and as light chain sequence a) SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, or a humanized version thereof having at least 95% sequence identity to said sequence, and a second binding moiety consisting of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of the single-chain Fv antibodies being linked by a peptide linker to each C-terminus of the first binding moiety.

[0068] In one embodiment, the second binding moiety comprises a set of heavy and light chain variable regions selected from the group consisting of: e) SEQ ID NO: 10 and SEQ ID NO: 14 for FOLR-1 as tumor antigen; f) SEQ ID NO: 18 and SEQ ID NO: 22 for STEAP1 as tumor antigen; g) SEQ ID NO: 26 and SEQ ID NO: 30 for DLL3-4 as tumor antigen, and h) SEQ ID NO: 34 and SEQ ID NO: 38 for CLDN18.2 as tumor antigen.

[0069] In one embodiment, the invention comprises a bispecific antibody according to the invention in Mab-scFv format and characterized in that: The first binding moiety comprises a variable light chain and a variable heavy chain set selected from the group set forth in Table 3, and the second binding moiety comprises a variable light chain and a variable heavy chain set selected from the group consisting of: a) SEQ ID NO: 10 and SEQ ID NO: 14 for FOLR1 as tumor antigen; b) SEQ ID NO: 18 and SEQ ID NO: 22 for STEAP1 as tumor antigen; c) SEQ ID NO: 26 and SEQ ID NO: 30 for DLL3 as tumor antigen, and d) SEQ ID NO: 34 and SEQ ID NO: 38 for CLDN18.2 as tumor antigen.

[0070] In one embodiment, the invention is characterized in that it comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that: a) the first binding moiety is a full-length bivalent antibody; b) The second binding moiety is a single chain Fv antibody (scFv) in the Mab-scFv format, which specifically binds to the tumor antigen and comprises as heavy and light chain variable regions a set selected from the group consisting of: b1) SEQ ID NO: 10 and SEQ ID NO: 14 for FOLR1 as tumor antigen; b2) SEQ ID NO: 18 and SEQ ID NO: 22 for STEAP1 as tumor antigen; b3) SEQ ID NO: 26 and SEQ ID NO: 30 for DLL3 as a tumor antigen, and b4) SEQ ID NO: 34 and SEQ ID NO: 38 for CLDN18.2 as tumor antigen.

[0071] a) said bispecific antibody exhibits an EC50 ratio of 0.001 to 0.2 for lysis of a cell line bearing a first tumor antigen compared to lysis by a reference antibody comprising as its heavy chain the heavy chain of SEQ ID NO: 94 and as its light chain the light chain of SEQ ID NO: 93; b) the bispecific antibody exhibits an EC50 ratio of 5 to 1000 for lysis of a second cell line that does not bear the tumor antigen compared to lysis by the reference antibody; All were measured in the same assay under the same conditions, in the presence of activated Vγ9Vδ2 T lymphocytes, an E / T ratio of 5:1, and 12.5 IU / mL interleukin-2.

[0072] In one embodiment, the antibody according to the invention is characterized in that said first binding moiety is a CDR-grafted or humanized antibody. In one embodiment, the human VH framework (FRH) is IGHV1-46*01 (X92343) or IGHV4-34*01 (AB019439). In one embodiment, the human VL framework (FRL) is that of IGKV3-11*01 V-KAPPA (X01668) or IGKV1-12*01 V-KAPPA (V01577); see IMGT repertoire. In one embodiment the human VH / VL framework combinations are IGHV1-46*01 and IGKV3-11*01, IGHV1-46*01 and IGKV1-12*01, IGHV4-34*01 and IGKV3-11*01, IGHV4-34*01 and IGKV1-12*01. According to the invention, the framework sequence consists of four parts (FRH1-4 and FRL1-4).

[0073] In one embodiment, the invention comprises a bispecific antibody in Mab-scFv format comprising a first binding moiety that specifically and agonistically binds human CD277 and a second binding moiety that specifically binds to a tumor antigen, a) said first binding moiety is a full length bivalent antibody and comprises a variable light chain in the format of FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, where FRL1 is SEQ ID NO: 142, FRL2 is SEQ ID NO: 143, FRL3 is SEQ ID NO: 144 or 145, and FRL4 is SEQ ID NO: 146, and a variable heavy chain in the format of FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, where FRH1 is SEQ ID NO: 147, FRH2 is SEQ ID NO: 148, FRH3 is SEQ ID NO: 149, and FRH4 is SEQ ID NO: 150, in combination with a CDRH / CDRL set selected from the sets of Table 6 or 7; and c) said second binding moiety is characterized in that it consists of two single chain Fv antibodies (scFv) which specifically bind to said tumor antigen.

[0074] In one embodiment the antibody according to the invention is a humanized antibody and is characterized in that it comprises a variable light chain consisting of the sequence FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 and a variable heavy chain consisting of the sequence FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 or it comprises a variable light chain consisting of the sequence FRL1-CDRL1-FRL2-CDRL2-FRL3a-CDRL3-FRL4 and a variable heavy chain consisting of the sequence FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and a CDRH / CDRL set selected from the sets of Table 6 or 7.

[0075] In one embodiment, the present invention is characterized in that the second binding moiety consists of two identical single chain Fv antibodies that specifically bind to the tumor antigen. In one embodiment, the second binding moiety consists of two identical single chain Fv antibodies (scFv) that specifically bind to the tumor antigen, each linked by its N-terminus to each C-terminus of the first binding moiety. Thus, only one scFv is linked to each C-terminus of the Fc moiety of the first binding moiety (which is a full-length monospecific anti-CD277 antibody). The bispecific antibody format consisting of a full-length bivalent antibody as the first binding moiety and the two scFvs as the second binding moiety is herein named "Mab-scFv format". An exemplary Mab-scFv format is shown in Figure 1a.

[0076] In one embodiment, each of the scFvs is chemically linked to each of the C-terminuses of the first binding moiety by a first peptide linker (Linker 1).

[0077] In one embodiment the invention comprises a bispecific antibody according to the invention, characterized in that said scFv is linked to said C-terminus in the following orientation: peptide linker1-VL-peptide linker2-VH.

[0078] In one embodiment, the peptide linker is selected from the group consisting of the peptides of SEQ ID NOs: 97, 98, 99, 100, and 101.

[0079] In one embodiment, the present invention includes a bispecific antibody according to the present invention, characterized in that the first peptide linker consists of 5 to 25 amino acids, and in one embodiment, 10 to 25 amino acids.

[0080] In one embodiment, the present invention includes a bispecific antibody according to the present invention, wherein the second peptide linker consists of 10 to 25 amino acids.

[0081] In one embodiment, said bispecific antibody does not induce significant lysis in said second cell line, greater than 10-fold, in one embodiment greater than 5-fold, in one embodiment greater than 2-fold over background lysis.

[0082] In one embodiment of the invention, the bispecific antibody exhibits an EC50 ratio for lysis of the first cell line compared to lysis by the reference antibody, which in one embodiment is 0.2 or less, in one embodiment, 0.001 to 0.2, in one embodiment, 0.005 to 0.2, and in one embodiment, 0.01 to 0.2.

[0083] In one embodiment, the bispecific antibody exhibits an EC50 ratio for lysis of the second cell line compared to lysis by the reference antibody of 5 or more, 10 or more, 5 to 1000, in one embodiment, 5 to 2000, in one embodiment, 5 to 5000, in one embodiment, 10 to 1000, in one embodiment, 10 to 2000, and in one embodiment, 10 to 5000.

[0084] By EC50 ratio according to the present invention is meant the ratio of EC50 values ​​measured for cell lysis. An exemplary method is described in Example 6.

[0085] In one embodiment, said second tumor antigen negative cell line is said first cell line in which the tumor antigen has been inactivated (a knockout cell line).

[0086] In one embodiment, the invention comprises a bispecific antibody according to the invention, characterized in that said antibody induces an Emax of 0.5 or more, 0.8 or more, or 0.9 or more compared to a reference antibody. In one embodiment, said bispecific antibody exhibits an Emax ratio for lysis of said first tumor antigen-positive cell line compared to the Emax of said reference antibody of 0.5 to 1.5, in one embodiment 0.8 to 1.5, in one embodiment 0.9 to 1.5.

[0087] The reference antibody is a full-length bivalent monospecific agonistic anti-CD277 antibody, comprising a heavy chain of SEQ ID NO: 94 and a light chain of SEQ ID NO: 93. The reference antibody comprises a variable heavy chain of SEQ ID NO: 1, a variable light chain of SEQ ID NO: 5 and CDRs of SEQ ID NOs: 2, 3, 4, 6, 7, 8.

[0088] In one embodiment, the invention comprises a bispecific antibody according to the invention, characterized in that said tumor antigen is a tumor antigen that does not internalize the bispecific antibody of the invention.

[0089] A further embodiment of the present invention is a recombinant nucleic acid sequence encoding a bispecific antibody according to the present invention.

[0090] A further embodiment of the present invention is a vector comprising a recombinant nucleic acid sequence encoding the bispecific antibody according to the present invention.

[0091] A further embodiment of the present invention is a host cell comprising a vector comprising a recombinant nucleic acid sequence encoding a bispecific antibody according to the present invention.

[0092] In one embodiment the invention comprises a bispecific antibody according to the invention for use in the treatment of a tumor disease.

[0093] In one embodiment the invention comprises a bispecific antibody according to the invention for use in the treatment of a tumor disease.

[0094] In one embodiment, the neoplastic disease is selected from the group consisting of colon cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.

[0095] A further embodiment of the invention is a pharmaceutical composition comprising said bispecific antibody according to the invention.

[0096] In one embodiment, the invention comprises a method of treating cancer comprising administering to a subject in need thereof an effective amount of a bispecific antibody according to the invention or a pharmaceutical composition comprising said bispecific antibody.

[0097] The CD277 Mabs according to the invention and their properties are further described in Tables 3 and 5. In one embodiment, the CD277 Mab comprises an Fc domain consisting of a first and a second subunit. In one embodiment, the CD277 Mab comprises a second antigen binding domain that binds to a second antigen. In one embodiment, the second binding moiety is an scFv molecule that specifically binds to a tumor antigen and the antibody is in Mab-scFv format. [Brief description of the drawings]

[0098] [Figure 1a] One embodiment of the structure of a bispecific antibody according to the invention. [Figure 1b] A bispecific antibody against a tumor antigen and a BTN3A agonist (HC and LC of SEQ ID NO: 94 and 93, "second bispecific antibody") shows enhanced potency against tumor antigen-bearing cells compared to a monospecific BTN3A antibody of the same sequence, but binds to tumor antigen-negative cells with the same potency as a monospecific BTN3A antibody of the same BTN3A antibody sequence (see figure below). Such a "second" bispecific antibody still non-specifically activates Vγ9Vδ2 cells in circulation and in normal tissues. On the contrary, the bispecific antibody of the present invention still shows strong potency against tumor antigen-positive cells, but shows less potency against tumor antigen-negative cells, compared to a "second" bispecific antibody against a BTN3A agonist. Thus, unexpectedly, the bispecific antibody of the present invention has less side effects in treatment. [Diagram 2]Activity of EvB#5 against FOLR1+ and FOLR1- tumor cells. 10.000 Ovcar-3 (FOLR1+) or NCI-H1693 (FOLR1-) cells were cultured in complete medium (RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 100 μg / mL streptomycin, 100 U / mL penicillin, 10% fetal bovine serum). After allowing tumor cells to adhere overnight, cells were further cultured in complete medium and supplemented with the indicated concentrations of antibodies and short-term activated Vγ9Vδ2 T cells in 10 IU / mL rIL-2 at an E / T ratio of 5:1. As a control for spontaneous lysis of the tumor cells themselves, tumor cells in additional wells were cultured in medium supplemented with 12.5 IU / mL rIL-2 without the addition of Vγ9Vδ2 T cells or antibodies ("SL", spontaneous lysis control). As a control for maximum lysis, short-term activated Vγ9Vδ2 T cells were cultured with tumor cells in additional wells at an E / T ratio of 5:1 in medium supplemented with 12.5 IU / mL rIL-2, but with the addition of Triton-X detergent to achieve maximum lysis ("Triton X 100" control). As a control for background lysis of tumor cells by Vγ9Vδ2 T cells, tumor cells in additional wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in medium supplemented with 12.5 IU / mL rIL-2, but without the addition of antibody ("medium control"). Cell index (CI) was then measured every 3 min for 90 h. Tumor cell lysis at time tx was calculated by the following formula: Tumor cell lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100. Curve fitting was performed using Graphpad Prism 9 with a sigmoidal dose-response function to obtain the best fit value (Top value) of the maximum tumor cell lysis (tx) achieved with the reference antibody. The tumor cell lysis % relative to the maximum tumor cell lysis ("Top") achieved with the reference antibody was calculated by the following formula: "Tumor cell lysis % (tx) = tumor cell lysis (tx) / Top * 100". Figure 2a shows the tumor cell lysis rate of FOLR1+ Ovcar-3 tumor cells, and Figure 2b shows the tumor cell lysis rate of NCI-H1693 WT (FOLR1-) cells ± SD at 24 hours. EC50 values ​​of the different constructs are shown.The bispecific antibody according to the invention killed 50% of Ovcar-3 cells at a concentration of 0.12 nM. Background lysis ± SD at 24 hours is shown for the medium control. Figure 2c shows a comparison of the % tumor cell lysis of Ovcar-3 cells for the bispecific antibody according to the invention in scFv format and in the reverse format. (EvB#1: full-length bivalent VH / VL binding antibody of SEQ ID NO: 1 and SEQ ID NO: 5 linking two scFvs of an anti-tumor antigen antibody, EvB#8: full-length VH / VL binding antibody of the same anti-tumor antigen antibody linking two scFvs of SEQ ID NO: 1 and SEQ ID NO: 5 linking as VH / VL. Both formats are shown in Figure 2d). [Diagram 3] Statistical analysis of lysis efficiency. Statistical analysis showed that at a concentration of 0.1 nM, the bispecific antibody EvB#5 induces 48% lysis of the cell line Ovcar-3 with FOLR1 at an E / T ratio of 5:1 in the presence of activated Vγ9Vδ2 T lymphocytes (FIG. 3a). On the other hand, the bispecific antibody does not induce lysis of NCI-H1693 without FOLR1 significantly above background lysis ("medium control") in the same assay and under the same conditions (FIG. 3b). The significance of the differences was determined by unpaired t-test using Graphpad Prism 9 software, and the degree of significance was indicated: ns P>0.05, * P≦0.05, ** P≦0.01, *** P≦0.001. Thus, the bispecific antibody according to the invention enhances the cytotoxicity of Vy9V52 T cells against FOLR1+ Ovcar-3 cells and not against FOLR1- NCI-H1693 cells, whereas the reference antibody ("Ref.Ab") does not enhance the cytotoxicity of Vy9V52 γδ T cells against FOLR1+ Ovcar-3 cells. [Figure 4]Activity of EvB#2 against NCI-H1693sgNT (WT control) and NCI-H1693sgNT with antigen 1 knockout (clone 27). 10.000 NCI-H1693sgNT (WT control) or clone 27 cells were cultured in complete medium. After allowing tumor cells to adhere overnight, cells were cultured in additional complete medium and the indicated concentrations of antibodies and short-term activated Vγ9Vδ2 T cells were added at an E / T ratio of 5:1 in 12.5 IU / mL rIL-2. As a control for spontaneous lysis of the tumor cells themselves, tumor cells in additional wells were cultured in medium supplemented with 12.5 IU / mL rIL-2 without the addition of Vγ9Vδ2 T cells or antibodies ("SL", spontaneous lysis control). As a control for maximum lysis, short-term activated Vγ9Vδ2 T cells were cultured with tumor cells in additional wells at an E / T ratio of 5:1 in medium supplemented with 12.5 IU / mL rIL-2, but with the addition of Triton-X detergent to achieve maximum lysis ("Triton X 100" control). As a control for background lysis of tumor cells by Vγ9Vδ2 T cells, tumor cells in additional wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in medium supplemented with 12.5 IU / mL rIL-2, but without the addition of antibody ("medium control"). The cell index (CI) was then measured every 3 min for 90 h. Tumor cell lysis at time tx was calculated by the following formula: Tumor cell lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100 Curve fitting was performed using Graphpad Prism 9 with a sigmoidal dose-response function to obtain the best fit value (Top value) of the maximum tumor cell lysis (tx) achieved with the reference antibody. The tumor cell lysis % relative to the maximum tumor cell lysis ("Top") achieved with the reference antibody was calculated by the following formula: Tumor cell lysis (tx) = tumor cell lysis (tx) / Top * 100 Figure 4a shows the tumor cell lysis rate of antigen 1 + NCI-H1693 sgNT tumor cells, and Figure 4b shows the tumor cell lysis rate of antigen 1 - clone 27 cells ± SD at 24 hours. EC50 values ​​of the different constructs are shown. Bispecific antibodies according to the invention killed 50% of NCI-H1693 sgNT cells at a concentration of 0.012 nM. Background lysis ±SD at 24 h is shown for the medium control. [Diagram 5] Statistical analysis of lysis efficiency. Statistical analysis showed that at a concentration of 0.01 nM, the bispecific antibody EvB#2 lysed 61% of the antigen 1 bearing cell line NCI-H1693 sgNT in the presence of activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1 (FIG. 5a). On the other hand, the bispecific antibody does not induce lysis significantly above background lysis in NCI-H1693 ko cells (clone 27) not bearing the tumor antigen in the same assay and under the same conditions (FIG. 5b). The significance of the differences was determined by unpaired t-test using Graphpad Prism 9 software and the degree of significance was indicated: ns P>0.05, * P≦0.05, ** P≦0.01, *** P≦0.001. Thus, the bispecific antibody according to the invention enhances Vδ2+ γδ T cell cytotoxicity against NCI-H1693sgNT but not against ko cells. On the other hand, the reference antibody ("Reference Antibody") does not enhance V52+ γδ T cell cytotoxicity against tumor antigen 1-bearing NCI-H1693sgNT cells. [Figure 6]Activity of EvB#3 against STEAP1+ and STEAP1- tumor cells. 10.000 UMUC-3 (STEAP1+) or Ovcar-3 (STEAP1-) cells were cultured in complete medium. After allowing tumor cells to adhere overnight, cells were further cultured in complete medium and supplemented with the indicated concentrations of antibodies and short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in 10 IU / mL rIL-2. As a control for spontaneous lysis of the tumor cells themselves, tumor cells in additional wells were cultured in medium supplemented with 12.5 IU / mL rIL-2 without the addition of Vγ9Vδ2 T cells or antibodies ("SL", spontaneous lysis control). As a control for maximum lysis, short-term activated Vγ9Vδ2 T cells were cultured with tumor cells in additional wells at an E / T ratio of 5:1 in medium supplemented with 12.5 IU / mL rIL-2, but with the addition of Triton-X detergent to achieve maximum lysis ("Triton X 100" control). As a control for background lysis of tumor cells by Vγ9Vδ2 T cells, tumor cells in additional wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in medium supplemented with 12.5 IU / mL rIL-2, but without the addition of antibody ("medium control"). Cell index (CI) was then measured every 3 min for 90 h. The tumor cell lysis at time tx was calculated by the following formula: Tumor cell lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100 Curve fitting was performed using Graphpad Prism 9 with a sigmoidal dose-response function to obtain the best fit value (Top value) of the maximum tumor cell lysis achieved with the reference antibody (tx). The tumor cell lysis % relative to the maximum tumor cell lysis achieved with the reference antibody ("Top") was calculated by the following formula: Tumor cell lysis rate (tx) = tumor cell lysis rate (tx) / Top * 100 Figure 6a shows the tumor cell lysis rate of STEAP1+ UMUC-3 tumor cells, and Figure 6b shows the tumor cell lysis rate of STEAP1- Ovcar-3 tumor cells at 24 hours (tx) ± SD. EC50 values ​​of the different constructs are shown. The bispecific antibody according to the invention killed 50% of UMUC-3 cells at a concentration of 0.17 nM. Background lysis ±SD at 24 hours is shown for the medium control. [Figure 7]Molecular cloning and tumor anchor cassette exchange. [Figure 8] Cytolysis rates of FOLR1+Ovcar-3 and FOL1R- tumor cells are shown (see legend to Fig. 2). BTN3A agonist antibody: reference antibody, EvB#5: bispecific antibody without CDR mutations, EvB#47 and EvB#52: bispecific antibodies with mutations, see Table 2 for example). [Figure 9a] Vy9V52 T cell degranulation assay. Degranulation of Vy9V52 T cells in the absence of tumor antigen positive cells was monitored by FACS analysis of CD107a levels on the cell surface. Antibodies were applied at 10x the effective concentration to reflect higher drug concentrations in the primary distribution compartment after iv administration. The top panel shows significant degranulation of Vy9V52 T cells of four different donors activated with reference antibody 20.1 compared to CD107a surface levels in the presence of medium without antibody. The bottom panel shows no significant degranulation of Vy9V52 T cells from four different donors in the presence of antibodies of the invention when compared to CD107a surface levels in the presence of medium without antibody. [Figure 9b] Vy9V52 T cell autoelimination assay. Autoelimination of V52 T cells in the absence of tumor antigen positive cells was monitored by FACS analysis after staining of dead cells with SytoxGreen. Antibodies were applied at 10x effective concentrations to reflect higher drug concentrations in the primary distribution compartment after iv administration. The top panel shows that activation with reference antibody 20.1 significantly killed V52 T cells from four different donors compared to the percentage killed in the presence of antibody-free medium. The bottom panel shows that the presence of the antibodies of the invention does not significantly kill V52 T cells from four different donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] The present inventors have investigated the cytolysis of tumor antigen-positive and -negative cells using an agonistic mouse anti-CD277 antibody (parent antibody, 20.1) described in Imbert C. et al., WO2012080351 and WO2012080769, a bispecific antibody consisting of the anti-CD277 antibody, and an antibody against an exemplary tumor antigen in the bispecific antibody. As shown in Figure 9, the bispecific antibody in the Mab-scFv format shows superior tumor cell lysis activity to each monospecific anti-CD277 antibody.

[0100] Surprisingly, the inventors further found that a bispecific antibody in the Mab-scFv format comprising said parent anti-CD277 antibody with two point mutations (N53S, K58N, or also called N5S and K10N when counted in CDRH2) in the CDR heavy chain CDRH2 (SEQ ID NO: 44) provides high lysis of tumor antigen positive cells, but reduced lysis of tumor antigen negative cells, compared to a bispecific antibody consisting of a parent antibody without these mutations and an anti-tumor antigen antibody. This surprising effect is further improved by additional point mutations (L31V, L8V when counted in CDRL1) in the light chain CDRL1 (e.g. SEQ ID NO: 75, 140, 141) of the anti-CD277 antibody part. Thus, the present invention provides such a bispecific antibody and a humanized version thereof.

[0101] In one embodiment, the antibody according to the invention is characterized in that it comprises, in addition to said CDRH2 substitution, a substitution of L8V in CDRL1.In one embodiment, the antibody according to the invention is characterized in that it comprises additional substitutions of L8V and H1R in CDRL1.

[0102] As used herein, the term "activated Vy9V52 T cells" according to the invention means that Vy9V52 T cells are activated by stimulation with the aminobisphosphonate (n-BP) zoledronic acid and addition of recombinant IL2 (rIL2); see Example 3.

[0103] The term "first binding moiety" refers to a full-length antibody. As used herein, the term "full-length antibody" refers to a heterotetrameric glycoprotein consisting of two identical light chains (L chains) and two identical heavy chains (H chains). Full-length antibodies are monospecific, bivalent antibodies that contain a variable domain, a constant domain, and an Fc portion. Usually, each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain has disulfide bridges at regular intervals within the chain. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. A full-length antibody is composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3) from the N-terminus to the C-terminus, and is abbreviated as VH-CH1-HR-CH2-CH3. A "full-length antibody light chain" is composed of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) from the N-terminus to the C-terminus, and is abbreviated as VL-CL. The antibody light chain constant domain (CL) is kappa or lambda. It is believed that certain amino acid residues form an interface between the light chain and the heavy chain variable domains [Chothia et al., J. Mol.Biol., 186:651-663 (1985); Novotny and Haber, Proc.Natl. Acad.Sci. USA, 82:4592-4596(1985). ]The light chains of antibodies from any vertebrate species can be classified into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Depending on the amino acid sequence of the constant domain of their heavy chains, full-length antibodies can be assigned to different "classes". There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2.The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0104] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0105] The term "humanized antibody or humanized version thereof" refers to an antibody in which the framework or "complementarity determining regions" (CDRs) have been modified to constitute the CDRs of an immunoglobulin with different specificity compared to the parent immunoglobulin. In one embodiment, mouse CDRs are grafted into the framework regions of a human antibody to prepare a "humanized antibody or version". See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. In one embodiment, the human framework is IGHV1-46*01 (X92343) or IGHV4-34*01 (AB019439), IGKV3-11*01 V-KAPPA (X01668) or IGKV1-12*01 V-KAPPA (V01577). In one embodiment encompassed by the present invention, the constant region is additionally modified or altered from that of the original antibody to generate the properties according to the invention, particularly with regard to C1q binding and / or Fc receptor (FcR) binding.

[0106] The term "variable domain" as used herein refers to an antibody region that contains three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). Native heavy and light chain variable domains each contain four FR regions, which are largely in a β-sheet structure and are connected by the three CDRs, forming loops that connect the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD (1987)). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as being involved in antibody-dependent cellular cytotoxicity.

[0107] The term "Fc region" as used herein refers to the C-terminal region of an immunoglobulin heavy chain. The Fc region may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is usually defined to extend from an amino acid residue at position Cys226, or from an amino acid residue at position Pro230, to the carboxyl-terminus of the Fc region (the numbering system according to Kabat et al., supra, is used herein). The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain IgE.

[0108] As used herein, "Fc region chain" refers to one of the two polypeptide chains of an Fc region.

[0109] The "CH2 domain" (also referred to as the "Cγ2" domain) of the human IgG Fc region typically extends from approximately amino acid residue position 231 to approximately amino acid residue position 340. The CH2 domain is unique in that it is not tightly paired with other domains. Rather, two N-linked branched glycans are interposed between the two CH2 domains in intact native IgG molecules. It has been speculated that the glycans may act as a proxy for domain-domain pairing and help stabilize the CH2 domains. Burton, Mol.Immunol. 22:161-206 (1985). The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.

[0110] A "CH3 domain" comprises the stretch of residues at the C-terminus of the CH2 domain of the Fc region (from an amino acid residue at about position 341 to an amino acid residue at about position 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced "bulge" in one chain and a corresponding "cavity" in the other chain; see U.S. Pat. No. 5,821,333).

[0111] The "hinge region" is generally defined as the region extending from about Glu216, or about Cys226, to about Pro230 of human IgG1 (Burton, Mol.Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S-S bonds in the same positions. The hinge region herein may be a native sequence hinge region or a variant hinge region. The two polypeptide chains of the variant hinge region generally retain at least one cysteine ​​residue per polypeptide chain, such that the two polypeptide chains of the variant hinge region can form disulfide bonds between the two chains. A preferred hinge region herein is a native sequence human hinge region, such as a native sequence human IgG1 hinge region.

[0112] A "functional Fc region" possesses at least one "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of a cell surface receptor (e.g., B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays known in the art for assessing such antibody effector functions.

[0113] A "native sequence Fc region" comprises an amino acid sequence identical to that of a naturally occurring Fc region. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide, compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith, and more preferably at least about 95% sequence identity therewith.

[0114] Antibodies of the IgG4 subclass have reduced binding to the Fc receptor (FcyRIIIa), whereas antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297, Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues that, when altered, reduce Fe receptor binding (Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0 307 434). In one embodiment, an antibody according to the invention has reduced FcγR binding compared to an IgG1 antibody, and the full-length antibody is of IgG4 subclass, or IgG1 or IgG2 subclass with mutations at S228, L234, L235 and / or D265, and / or comprises a PVA236 mutation. In one embodiment, the mutations in the full-length antibody are S228P, L234A, L235A, L235E and / or PVA236. In another embodiment, the mutations in the full-length antibody are IgG4 S228P, IgG1 L234A and L235A.

[0115] In a further embodiment, the antibody according to the invention is characterized in that said full length antibody is of human IgG1 subclass, or of human IgG1 subclass with the mutations L234A and L235A. In a further embodiment, the antibody according to the invention is characterized in that said full length antibody is of human IgG4 subclass, or of human IgG4 subclass with the additional mutation S228P. One embodiment comprises the mutations S228P (Ser228Pro), L235E (Leu235Glu) and P329G (Pro329Gly), or S228P (Ser228Pro) and P329G (Pro329Gly) in the constant heavy chain region of the IgG4 subclass.

[0116] The term "second binding moiety" refers to a single chain Fv molecule. Each identical single chain Fv molecule is attached to the C-terminus of the Fc portion of the first binding moiety. Thus, the second binding moiety contains two single chain Fv molecules.

[0117] As used herein, the term "single-chain Fv molecule (scFv)" refers to a molecule in which the variable domain of the light chain (VL) is linked by a polypeptide chain from its C-terminus to the N-terminus of the variable domain of the heavy chain (VH). Alternatively, an scFv comprises a polypeptide chain in which the C-terminus of the VH is linked by a polypeptide chain to the N-terminus of the VL.

[0118] The term "peptide linker" or "linker" as used in the present invention denotes a peptide having an amino acid sequence, preferably of synthetic origin. The peptide linker according to the present invention is used to fuse a single chain Fab or scFv fragment to the C-terminus of a full-length antibody. Preferably, said peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5-30 amino acids in length, more preferably 10-20 amino acids in length. In one embodiment, said peptide connector is (GxS)n or (GxS)nGm, where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3), preferably x=4, n=2 or 3, more preferably x=4, n=3. In one embodiment, said peptide connector is (G4S)3. Useful peptide linkers are also described in SEQ ID NOs: 97-101.

[0119] The variable regions may be linked directly or, typically, via a linker peptide that allows for the formation of a functional antigen-binding moiety. Exemplary peptide linkers include, but are not limited to, those described herein or known in the art.

[0120] The scFv molecule is further stabilized by disulfide bridges between the heavy and light chain variable domains, for example as described in Reiter et al. (Nat. Biotechnol. 14, 1239-1245 (1996)). Thus, in one embodiment, the T cell activating bispecific antigen binding molecule of the present invention comprises an scFv molecule in which an amino acid in the heavy chain variable domain and an amino acid in the light chain variable domain are substituted with cysteine, such that a disulfide bridge can be formed between the heavy chain variable domain and the light chain variable domain. In a specific embodiment, the amino acid at position 44 of the light chain variable domain and the amino acid at position 100 of the heavy chain variable domain are substituted with cysteine ​​(Kabat numbering).

[0121] As is known in the art, scFvs can also be stabilized by mutation of the CDR sequences, as described in (Miller et al, Protein Eng Des Sel. 2010 Jul;23(7):549-57; Igawa et al, MAbs. 2011 May-Jun;3(3):243-5; Perchiacca & Tessier, Annu Rev Chem Biomol Eng. 2012;3 :263- 86).

[0122] In one embodiment, the scFv can be replaced with a single chain Fab fragment to improve production yield. A "Fab fragment" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker being in any of the following orders from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragments a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 and d) VL-CH1-linker-VH-CL are stabilized via a natural disulfide bond between the CL and CH1 domains. The term "N-terminus" refers to the last amino acid at the N-terminus and the term "C-terminus" refers to the last amino acid at the C-terminus.

[0123] The antigen-binding constructs described herein are bispecific and in a general embodiment comprise at least two antigen-binding polypeptide constructs each capable of specifically binding to two different antigens. The first binding moiety is a full-length bivalent antibody and the second binding moiety consists of two monovalent antibody fragments that do not contain an Fc portion. In a preferred embodiment, the two monovalent antibody fragments are in scFv format (i.e. an antigen-binding domain consisting of a heavy chain variable domain and a light chain variable domain). In one embodiment, the scFv molecule is human. In another embodiment, the first and second binding moieties are humanized.

[0124] Exemplary heavy chains illustrating the preferred Mab-scFv format (see also FIG. 1a) are shown in SEQ ID NO: 102 (DLL3) and SEQ ID NO: 103 (CLDN18.2).

[0125] "Specific binding" or "selective binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured by surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument). In one embodiment, the extent of binding of the antigen-binding moiety to an unrelated protein is less than about 10%, preferably less than 5%, of the binding of the antigen-binding moiety to the antigen as measured by SPR.

[0126] The term "EC50 ratio" in the context of the present invention means the ratio between the value of the bispecific antibody according to the invention as the numerator (top) and the value of the reference antibody as the denominator (bottom).

[0127] In one embodiment, said second tumor antigen negative cell line is said first cell line in which the tumor antigen has been inactivated (knockout cell line; ko cell line).

[0128] In one embodiment, the bispecific antibody exhibits an Emax ratio of 0.5 to 1.5 for lysis of the first tumor antigen positive cell line compared to the Emax of the reference antibody. Hemolysis is measured by monitoring the impedance of tumor cells (see Example 6).

[0129] As used herein, the term "does not induce lysis of human cells not bearing said tumor antigen" refers to lysis of tumor cells by an antibody of the invention, measured in the presence of 12.5 IU / mL interleukin-2 and in the presence of activated Vy9V52 T lymphocytes at an E / T ratio of 5:1, that is not significantly different (p-value>0.05) from background lysis, measured in the same assay and under the same conditions, but without the addition of antibody ("medium control").

[0130] As used herein, "CD277 binding" means binding to BTN3A1, BTN3A2, and / or BTN3A3.

[0131] "Affinity" refers to the strength of interaction between a single binding site of a molecule (e.g., CD277) and a binding partner (e.g., an anti-CD277 antibody), expressed as a dissociation constant (kD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may be composed of different rate constants, as long as the ratio of the rate constants is the same. Affinity can be measured by established methods well known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0132] As used herein, an "affinity matured antibody" refers to an antibody that has one or more modifications in one or more of its CDRs that result in a reduced affinity of the anti-CD277 antibody compared to a parent antibody that does not have those modifications. Preferred mature antibodies with reduced affinity have an affinity for CD277 in the nanomolar to micromolar range. Affinity matured antibodies can be produced by alanine scanning (Tiller KE et al; Front.Immunol., 04 September 2017 https: / / doi.org / 10.3389 / fimmu.2017.00986) or other procedures known in the art (see, for example, Tabasinezhada M. et al; Immunology Letters Volume 212, August 2019, Pages 106-113; 1.Georgiev, IS et al. J Immunol 192, 1100-1106 (2014)).

[0133] As used herein, the terms "agonist" and "agonistic" refer to or describe a molecule capable of substantially inducing, promoting or enhancing, directly or indirectly, the biological activity or activation of Vy9V52 T cells (by promoting the formation of an immunological synapse for the γδTCR). Optionally, an "agonist CD277 antibody" is an antibody that has the activity of achieving said activation of Vy9V52 T cells by binding and activating CD277. Preferably, the agonist is a molecule capable of activating human and cynomolgus Vy9V52 T cells. More preferably, the agonist is an antibody against CD277, which has 5-fold less agonistic activity than antibody 20.1. The agonistic activity of such antibodies can be determined in the assay described in Example 6.

[0134] "Specific binding to a tumor antigen" means that the binding is selective for the tumor antigen and can be distinguished from unwanted or non-specific interactions. The ability of a bispecific antibody (or second binding moiety) according to the invention to bind to a specific tumor antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al, Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding to unrelated proteins is less than about 10% of the binding of a bispecific antibody (or second binding moiety) according to the invention to a tumor antigen, as measured, for example, by SPR.

[0135] As used herein, the term "agonistic antibody that specifically binds to CD277" according to the invention means that such an antibody activates the cytolytic function, cytokine production and proliferation of Vy9 / V52 T cells. In one embodiment, the extent of binding to unrelated proteins is less than about 10% of the binding of the bispecific antibody according to the invention (or the second binding moiety) to a tumor antigen as measured, for example, by SPR. In one embodiment, the bispecific antibody according to the invention does not activate the cytolytic function, cytokine production and proliferation of Vy9 / V52 T cells in the absence of tumor cells bearing said respective tumor antigen in a cytolytic assay as described in Example 6 at a concentration of 5 nM or less, in one embodiment 20 nM or less.

[0136] As used herein, "tumor antigen knockout cell line or knockout cell line" refers to a tumor cell line carrying the respective tumor antigen in a wild-type version and in which the respective tumor antigen gene is inactivated. According to the present invention, CRISPR / Cas9 technology can be used to introduce genetic mutants of said genes to inactivate said antigen expression.

[0137] The term "tumor antigen" refers to an antigen presented on the surface of a tumor cell, including tumor-specific antigens (TSA) and tumor-associated antigens (TAA). In a preferred embodiment, the tumor antigen is Claudin18.2, FOLR1, STEAP, or DLL3. Further useful tumor antigens are described, for example, in Middleburg et al., Cancers (2021) 13, 287, pp 4-6. Some tumor antigens, such as FOLR1, are internalized after binding to natural ligands such as folate (Cheung et al., Oncotarget, 7 (32), 2016, pp 52553-32574) or therapeutic antibodies (Paulos et al., Molecular Pharmacology, 66 (6), 2004, pp 1406-1414).

[0138] Thus, the availability for recruitment of Vy9V52 T cells may be reduced and the bispecific antibodies according to the invention may be co-internalized and vice versa, resulting in depletion of CD277 receptors on the cell surface. In such a case, the receptors will not be able to contact the Vy9V52 T cell receptors of the immune cells to form an immunological synapse. Thus, according to the invention, it is preferred that the bispecific antibodies according to the invention either bind tumor antigens that are not internalized after binding of the respective antibodies or only bind to such an extent that the tumor antigen and CD277 levels remaining after (co)internalization at the cell surface are sufficient to trigger activation of Vg9Vd2 T cells.

[0139] Preferably, the tumor antigen according to the present invention is selected from both antigen-bearing tumor cells and antigen-negative cells treated with each bispecific antibody or reference antibody for 8 hours. Vy9V52 T cells are then added and the tumor cell lysis rate by surface-exposed and activated CD277 is measured as described in Example 6. Emax values ​​are determined by curve fitting and the Emax ratio of bispecific antibody to reference antibody is calculated for each cell line respectively. The Emax ratio on antigen-bearing tumor cells should not be less than half of the Emax ratio on cells without tumor antigen, indicating that the presence of tumor antigen does not lead to a loss of more than 50% of activity due to co-internalization of CD277 by bispecific antibody on antigen-bearing tumor cells.

[0140] As used herein, the term "Emax" refers to the maximum response induced by a given concentration of an antibody or antigen-binding portion thereof in an in vitro or in vivo assay.

[0141] As used herein, the term "EC50" refers to the concentration of an antibody or antigen-binding portion thereof that induces a response in an in vitro assay that is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.

[0142] As used herein, "KD or K DThe term "antibody-antigen binding constant" refers to the equilibrium dissociation constant of the binding reaction between an antibody and an antigen.

[0143] The antibodies according to the invention are produced recombinantly. Thus, one embodiment of the invention is a nucleic acid encoding the antibody according to the invention, and a further embodiment is a cell comprising said nucleic acid encoding the antibody according to the invention. Methods of recombinant production are widely known in the art and include protein expression in prokaryotic and eukaryotic cells followed by isolation of the antibody and purification, usually to a pharma-ceutically acceptable degree of purity. To express the antibodies of the invention in a host cell, the nucleic acids encoding the respective modified light and heavy chains are inserted into an expression vector in standard manner. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the antibody is recovered from the cells (supernatant after lysis or cells). General methods for the recombinant production of antibodies are well known in the art and are described, for example, in the reviews of Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol. 16 (2000) 151-160; Werner, RG, Drug Res. 48 (1998) 870-880.

[0144] The bispecific antibodies according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, etc. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced by conventional procedures.

[0145] The term "host cell" as used in this application refers to any type of cell line that can be engineered to produce an antibody according to the invention. In one embodiment, HEK293 cells and CHO cells are used as host cells.

[0146] One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention. Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the invention provides a composition, e.g. a pharmaceutical composition, comprising an antibody according to the invention, formulated together with a pharmaceutical carrier.

[0147] One embodiment of the present invention is a bispecific antibody according to the invention for use in the treatment of cancer (tumor disease).

[0148] Another aspect of the invention is a pharmaceutical composition for use in the treatment of cancer.

[0149] Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

[0150] Another aspect of the invention is a method for treating cancer in an individual, comprising administering to the individual an effective amount of a bispecific antibody according to the invention.

[0151] Another aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention.

[0152] As used herein, a "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0153] The compositions of the present invention can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. To administer the compounds of the present invention by a certain route of administration, it may be necessary to coat the compounds with a material to prevent inactivation or to co-administer the compounds. For example, the compounds can be administered to a subject in a suitable carrier, such as liposomes, diluents, and the like. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such vehicles for pharma-ceutically active substances is known in the art.

[0154] The term cancer as used herein refers to proliferative diseases such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the digestive system, colon cancer, breast cancer, uterine cancer, Hodgkin's disease, and the like.

[0155] In one embodiment, the cancer (tumor disease) is selected from the group consisting of colon cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14]

[0156] The sequence listing lists each VH or VL followed by its respective CDR. Some VH and VL contain one or more identical CDRs: Sequence number 2 is the same as 43, 59, 63, 71, and 109. Sequence number 4 is the same as sequence numbers 45, 49, 53, 57, 61, 65, 69, 73, 107, 111, and 115. Sequence number 7 is the same as sequence numbers 80, 84, 88, 118, 122, 126, 130, and 134. Sequence number 8 is the same as sequence numbers 119, 89, 123, 127, 131, 135, and 139. SEQ ID NO:44 is the same as SEQ ID NO:64. SEQ ID NO: 47 is a single sequence. SEQ ID NO:51 is the same as SEQ ID NO:54. SEQ ID NO:67 is a single sequence. Sequence number 75 is the same as sequence numbers 79 and 87. SEQ ID NO:105 is the same as SEQ ID NO:113. SEQ ID NO: 138 is a single sequence.

[0157] Materials and Methods Cell culture, transfection, antibody production, purification: CHO-S cells (FreeStyle TM The cells were cultured in CD CHO growth medium (Gibco® / Thermo Fisher Scientific) supplemented with 1% (v / v) GlutaMAX 100x and 1% (v / v) HT Supplement 100x (Thermo Fisher Scientific). Every other day, cells were cultured at 0.3x10 6 The cells were then adjusted to a density of 2x10 cells / ml to maintain exponential growth. One day before transfection, the cells were cultured at 2x10 6 Cells were seeded at a density of 4x10 cells / ml on the day of transfection. 6 Transfection was performed using MaxCyte STX TM The transfection was performed using a transfection unit (MaxCyte, Inc., Gaithersburg, MD, USA) according to the manufacturer's instructions. The MaxCyte Processing Assembly OC-400 and transfection protocol optimized for protein production in CHO-S cells were used. 8 × 10 8 Harvest a total amount of cells: 8 x 10 7 The cells were divided into 10 equal parts. After washing twice with 4 ml of electroporation (EP) buffer, the cells were resuspended in EP buffer and diluted to 8 × 10 7 The cells were incubated at a density of 100 μl / 400 μl. Plasmid DNA was added at a total of 300 μg / ml with a 1:1 ratio of heavy:light chains or a single vector for surface antigen expression. After electroporation, CHO-S cells were seeded directly into culture flasks without the addition of extra buffer or medium and incubated at 37°C, 5% CO2 for 30 min. Culture conditions after transfection were different for protein production and transient expression of surface markers:

[0158] Surface receptor expression: Cells were cultured in CD CHO growth medium for 48 hours and then used for FACS analysis.

[0159] Antibody production: 150 ml of production medium was added (CD OptiCHO TM + 1 % (v / v) GlutaMAX 100x + 1 % (v / v) HT Supplement 100x + 1 % (v / v) Pluronic TM F-68 100x, all Gibco® / Thermo Fisher Scientific). One day after transfection, 1 mM Sodium Butyrate (Thermo Fisher Scientific) was added and cells were fed with 3.5% (v / v) MaxCyte Feed Stock (28 ml Yeastolate Stock Solution 0.5 % + 140 ml CHO CD Efficient Feed A Stock Solution + 7 ml GlutaMAX 100x + 24.8 ml Glucose (450 g / l) Stock Solution, Gibco® / Thermo Fisher Scientific). The culture temperature was reduced to 32°C for the remainder of the production cycle (14 days or until cell viability was below 50%). During the production period, cell density and viability were measured every other day and cells were fed daily with MaxCyte Feed Stock (see above) until production was stopped. The supernatant was collected by centrifugation and filtered to remove cell debris (final volume approximately 200 ml). TMAffinity chromatography was performed using CH1-XL (Hu) Affinity Matrix (Thermo Fisher Scientific). Briefly, 1 ml of beads was added and gently mixed overnight at 4°C. The beads were washed in a gravity flow column three times with 10 ml of PBS. The protein was eluted with 5 ml of 0.1 M glycine, pH 3.0 and immediately neutralized by adding 1 ml of Tris / HCL pH 8.0. The eluted fraction was dialyzed against 2 l of PBS at 4°C for a total of three times. Monomeric antibodies were then isolated by size exclusion chromatography (AKTA Pure 25, GE Healthcare Life Science) using a HiLoad 26 / 600 Superdex 200 pg column (GE Healthcare Life Science) at a flow rate of 1 ml / min (PBS buffer). Antibody preparations were analyzed by SDS-PAGE using standard procedures. The gel was stained with Coomassie blue. The protein concentration of the purified protein was analyzed by BCA assay (Pierce) according to the manufacturer's conditions.

[0160] Flow cytometry: 0.5x10 for individual staining reactions 6 cells were used. The cells are washed once with 1 ml PBA (PBS, 1% BSA, 0.05% NaN3). The cell pellet is resuspended in 50 μl of purified recombinant protein at a concentration of 50 μg / ml diluted in PBA. The cells are incubated on ice for 30 min. The cells are washed twice with 1 ml PBA. The cell pellet is then suspended in 25 μl of 1:20 diluted anti-human-IgGFITC (Jackson Immuno Research, cat no.:109-096-098) and incubated on ice in the dark for 30 min. The cells are then washed twice with 1 ml PBA. The cells are finally resuspended in 500 μl PBA and immediately analyzed on a Navios flow cytometer (Beckman Coulter). EXAMPLES

[0161] Example 1. Cloning of bispecific antibodies (Figure 5) A bispecific CD277 antibody is generated according to the following procedure: Expression vectors for the production of IgG-scFv molecules were designed using standard procedures (Kellner, CS.et al.; Methods Mol Biol, 2018.1827: p. 381-397). pSEC-Tag2-Hygro-C was used as the backbone for mammalian expression vector construction. IgG-scFv antibody derivatives were designed in an improved format based on the prototype IgG-scFv first reported by Coloma, MJ et al.; Nat Biotechnol, 1997. 15(2): p. 159-63.

[0162] Light chain: The design of the light chain was realized as described by Kellner, CS. et al. (supra). For the light chain expression cassette, a secretory leader sequence (L1; Haryadi, RS. et al; PLoS One, 2015.10(2): p. e0116878) was added to the 5´ end of the VL-region. The human C-kappa region was fused at the 3´ end to form the complete kappa light chain coding sequence. A minimal Kozak sequence was added upstream of the start codon to allow optimal initiation of translation. NheI and PmeI restriction sites were introduced at the 5´ and 3´ ends, respectively. Cloning into the vector backbone was performed according to standard procedures.

[0163] Heavy chain derivative: The heavy chain derivative encodes an IgG1 heavy chain with amino acid exchanges of L234A and L235A in the lower hinge region to prevent Fc receptor interaction (Lund, JG. et al.; J Immunol, 1991.147(8): p. 2657-62). A heavy chain secretory leader was added to the 5´ end of the VH region (H7; Haryadi, RS. et al; PLoS One, 2015.10(2): p. e0116878). A minimal Kozak sequence was added upstream of the start codon to allow optimal translation initiation. The stop codon of the IgG heavy chain was removed and a sequence encoding a 15 amino acid flexible linker (G4S)3 (SEQ ID NO:97) was introduced. The last two codons of the flexible linker (GS) at the DNA level have a BamHI restriction site followed by a PmeI restriction site. Each scFv fragment was designed as a BamHI-PmeI cloning cassette in the VL-(G4S)4-VH format. Cloning of the final expression constructs was performed according to standard procedures. In the heavy chain derivatives, additional restriction sites were introduced that do not affect the amino acid composition, allowing modular design and the exchange of specific parts of the molecule: NheI-PpuMI:VH region exchange. PpuMI-BsrGI: exchange of silencing mutations in the CH2 domain. BsrGI-BamHI: exchange of linker sequences. BamHI-PmeI:scFv fragment exchange. [Table 2-1]

[0164] [Table 2-2]

[0165] [Table 2-3]

[0166] Example 2: Generation of humanized bispecific antibodies Antibody affinity maturation proceeds stepwise in immune response. By accumulating additional mutations in the CDR regions compared to germline and undergoing a rigorous selection process, antibody affinity can be increased stepwise as described by Rajewsky and coworkers in 1988 (Allen, D., et al.; EMBO J, 1988. 7(7): p. 1995-2001., Kocks, C. and K. Rajewsky, Proc Natl Acad Sci USA, 1988. 85(21): p. 8206-10.). Thus, the humanized antibody of the present invention can be generated by gradually restoring the germline configuration in the light and heavy chain variable regions.

[0167] Methods of humanization Humanization of mouse monoclonal antibodies was performed using standard CDR grafting techniques. The principle of this method is to recreate a human antibody that contains only the complementarity determining regions (CDRs) of a mouse monoclonal antibody, thereby reducing the immunogenicity of the antibody when used as a therapeutic in humans. Humanization by CDR grafting requires that the antigen-binding residues of the mouse antibody be retained in the humanized antibody, so it is clear that the identification of these residues plays a key role in the protocol. The 4F9L X-ray structure of the mouse monoclonal antibody scFv against BTN3A1 was used to guide the humanization process and determine whether to preserve the parental mouse residues or replace them with human germline residues.

[0168] The CDR grafting protocol used is a modern adaptation of the approach pioneered by Greg Winter and colleagues at the Medical Research Council, Cambridge, UK. CDR definitions are based on the Kabat nomenclature. Selection of the human framework acceptor regions onto which the mouse monoclonal antibody mouse CDR regions were grafted was achieved by searching the IMGT mouse and human V gene databases with the mouse monoclonal antibody mouse variable region sequences as input using IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ), developed at NCBI to facilitate the analysis of immunoglobulin V region sequences. The strategy applied is to use natural human germline sequences that do not contain the specific somatic mutations found in individual human antibody sequences.

[0169] Light chain backmutation: The light chain variable region from the first binding portion of the parent antibody was compared to the mouse germline repertoire (IMGT database) to identify the germline gene showing the closest homology. This led to the identification of the IGKV15-103*01 and IGKJ2*01[F] genes, which were aligned to the parent VL region. Six amino acid residues that differed from the germline were identified. By illustrating the residues identified in the crystal structure of the parent antibody, amino acid residues that are surface exposed and likely to directly contribute to antigen interaction were identified. Individual amino acids or clusters of amino acids were converted to their germline configuration and used to generate expression constructs.

[0170] Backmutation of the heavy chain: A similar strategy was applied to identify potential amino acid positions in the heavy chain variable region. The IGHV1S81*02[F]v-gene was identified as the closest matching gene. The D and J segments were not identified because the CDR3 region appeared to be highly mutated, making it difficult to identify the corresponding gene segments. Similar to the strategy applied to reset the VL region to germline, mutations in the CDR1 and CDR2 regions of the heavy chain were reverted to the germline configuration in a stepwise manner (single mutations or clusters). To identify residues in CDR3, a different strategy was applied, since no germline gene segments could be identified. Here, surface-exposed residues in the CDR3 region were identified by analysis of the co-crystal structure of 20.1 with BNT3A residues (Payne, KK et al.; Science, 2020.369(6506): p. 942-949). Three of these residues have been described as potential contact residues (Payne, KK et al.; supra) and were therefore converted to alanine. Alanine exchange was chosen because alanine scanning has been reported to identify residues important for epitope binding by disrupting antibody / antigen interactions (Parhami-Seren, BM. et al; J Immunol, 2001.167(9): p. 5129-35). Individual amino acids or clusters of amino acids were converted to germline configuration and used to generate expression constructs. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0171] The heavy and light variable chain set of the bispecific antibody according to the invention is defined as two chains in one row of the table. "R162S" means that the amino acid R at position 162 is substituted with the amino acid S, and "N185S" means that the asparagine at position 185 is substituted with a serine. N185S and K190N are in bold and underlined in SEQ ID NO: 44. L31V is in bold and underlined in SEQ ID NO: 75. The counting of other amino acids in the variable chains can start from N185 and L31.

[0172] Example 3: Generation and characterization of activated Vy9V52 T cell lines. To generate expanded γδ T cell lines, 10 IgG1-positive cells were cultured from leukocyte concentrates (LRS) of healthy adult blood donors. 6 Cells / mL were cultured in 6-well plates in complete medium supplemented with 50 IU / mL rIL-2 (Novartis, Basel, Switzerland) and stimulated with 2.5 μM aminobisphosphonate (n-BP) zoledronic acid (Novartis), which induces selective expansion of Vγ9Vδ2-expressing γδ T cells. At rest, initially stimulated γδ T cells produce only small amounts of IL-2, so 50 IU / mL (15 pg / mL) rIL2 was added every other day (Oberg et al., Cancer Res. 2014). After 2 weeks, selective expansion of γδ T cells expressing the Vδ2 chain with a purity of more than 94% was observed. Activation of Vδ2 T cells was indicated by a slight increase in expression of CD25 (Pechhold et al. J Immunol Baltim Md 1950 152, 4984-92 (1994)) and a strong increase in expression of the activation marker CD69. Furthermore, the expanded Vδ2 T cell population displayed a central memory (CM, CD27+ CD45RA-) or effector memory (EM, CD27- CD45RA-) phenotype, indicating activation of these expanded γδ T cells.

[0173] Example 4: Selection of tumor cell lines To test the various antibody constructs, a panel of different tumor cell lines expressing each tumor antigen was selected based on published information on antigen expression levels or FACS analysis. Briefly, for surface staining, 3-5x10 cells were cultured at 10x10 RT. 5 Cells were washed twice with washing buffer (PBS containing 1% BSA, 0.1% NaN3). They were then stained for 25 min with fluorochrome-conjugated or unconjugated antibodies, isotype controls, and washed twice before resuspending in 1% PFA (paraformaldehyde) in PBS buffer or staining with the second antibody, according to the procedure outlined by the manufacturer. After incubation with the second antibody, cells were washed twice and resuspended in 1% PFA buffer. All samples were analyzed using an LSR-Fortessa flow cytometer (BD Biosciences) with Diva 9 and FlowJo software. Literature and FACS analysis results are summarized in Table 4. [Table 4]

[0174] Example 5: Generation of knockout tumor cell lines Generation of tumor antigen KO cells by RNP transfection: Guide RNA (gRNA) was prepared by combining the respective crRNA and tracrRNA at equimolar concentrations (100 μM), annealing at 95°C for 5 min, and reannealing at room temperature. RNP was then prepared by combining gRNA and recombinant SpCas9 protein in PBS and incubating at room temperature for 15 min. RNP was electroporated into parental cells using SF Cell Line 4D-Nucleofector X Kit S (Lonza; # V4XC-2032) and 4D-Nucleofector X unit (Lonza) according to the manufacturer's instructions and program FE-132. Monoclonal cells were then generated and expanded by FACS sorting (BD Aria), and verified by flow cytometry staining and amplicon sequencing (NGS).

[0175] Generation of tumor antigen KO cells by lentiviral transfection: Parental cells were first transfected with Cas9-p2A-Blasticidin-lentivirus and selected with Blastidin to achieve stable expression of Cas9. Then, the respective guide RNA was cloned into CROP-seq-Guide-Puro plasmid (Addgene # 86708) to generate lentivirus. Then, Blastidin-selected Cas-9 expressing cells were transfected with the lentivirus, and the cells were selected with puromycin, expanded, and verified by flow cytometry staining and amplicon sequencing (NGS). As a control, the same transfection protocol was applied using non-targeting guide RNA ("sgNT") to generate the respective sgNT cell lines. The guide RNA sequence suitable for generating FOLR1 KO cells is SEQ ID NO: 96.

[0176] Example 6: Cytolysis assay Cytotoxicity against tumor cell lines such as OVCAR-3 (ovarian cancer), NCI-H1693 (NSCLC), or UM-UC-3 (bladder cancer) was measured using a Real-Time Cell Analyzer (RTCA, X-Celligence, ACEA Biosciences, San Diego, CA, USA) in triplicates as described elsewhere (Oberg et al., 2014 and 2020). Briefly, 7.5–10 × 10 cells were cultured in complete medium RPMI 1640 (supplemented with 25 mM HEPES, 2 mM L-glutamine, 100 μg / mL streptomycin, 100 U / mL penicillin, and 10% fetal bovine serum). 3Adherent tumor cells / well were added to a 96-well microE-plate, and tumor cell impedance was monitored by an electronic sensor every 5 min for up to 24–40 h. The measured tumor cell impedance is expressed in an arbitrary unit called cell index ("CI"), which reflects changes in cellular parameters such as morphological changes (attachment, spreading, etc.), cell proliferation, and cell lysis. Because the initial adhesion of tumor cells in different wells may differ slightly, the CI can be normalized to 1 after the tumor cancer cells reach a linear growth phase. Once a linear growth rate was reached after 24–40 h, medium containing activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1, and 12.5 IU / mL of rIL-2, and the indicated concentrations of various antibody constructs, or various controls, was added ("start of experiment", t=0 h). As controls, tumor cells were treated in some wells with 1% final concentration of Triton X-100 to serve as a positive control for complete lysis, and in other wells with activated Vγ9Vδ2 T lymphocytes (same conditions as above) to serve as a control for background lysis. Lysis of adherent tumor cells was monitored by measuring the normalized CI for at least 3 min at different time points.

[0177] Raw data files were exported to Microsoft Excel or Graph Pad Prism using RTCA software (ACEA Biosciences Inc.) for further evaluation. Mean CIs for Triton-X-100 samples and Vγ9Vδ2 T lymphocytes without added antibody were calculated at the indicated time points after the start of the experiment and defined as complete lysis ("Triton X 100") and background lysis ("medium control"), respectively. Tumor cell lysis induced by antibody constructs was calculated for each sample at the same time points ("tx") as the tumor cells: Lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx))*100 Curve fitting was performed using Graphpad Prism 9 with a sigmoidal dose-response function to obtain the best-fit value (Top value) of maximum tumor cell lysis (tx) achieved with the reference antibody. The % tumor cell lysis relative to the maximum tumor cell lysis achieved with the reference antibody ("Top") was calculated using the following formula: Tumor cell lysis rate (tx)=Tumor cell lysis rate (tx) / Top*100 Oberg, HH; et al.; Front.Immunol.2014, 5, 643 and Oberg, HH; et al.; Methods Enzymol.2020, 631, 429-441. The results are shown in Figures 4, 6, and 8.

[0178] Example 7: SPR Assay The SPR assay was performed according to the latest technology. The results for the reference antibodies are shown in Table 5. Briefly, recombinant CD277 was immobilized on the surface of a Biacore CM5 optical sensor chip by covalent EDC / NHS coupling according to the Biacore amine coupling kit protocol. Antibody samples were applied as analytes in serial dilutions to allow standardization and comparison of all antibodies binding to the same target molecule surface. Kinetic analysis data are based on a 1:1 Langmuir curve fitting model and average Langmuir on-rates, off-rates and KD values: see Table 5. [Table 5]

[0179] Example 8: Degranulation and cell death assays Principle: Cytotoxic T cells, such as γδ T cells, store cytotoxic mediators, such as granzymes, perforin, and granulysin, in secretory lysosomes. Lysosome-associated membrane glycoproteins (LAMPs), such as LAMP-1 (CD107a) and LAMP-2 (CD107b), are embedded in the lipid bilayer of secretory lysosomes. After T cell activation, secretory lysosomes can migrate toward and fuse with the plasma membrane. After fusion, LAMPs are transiently expressed on the cell surface of T cells, and secretory lysosomes degranulate the granule contents.

[0180] Methods: Short-term activated γδ T cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 25 mM Hepes, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% fetal calf serum under standard conditions (5% CO2, humidified, 37°C). γδ T cells supplemented with 12.5 U / mL IL-2 were incubated for 6 h in 96-well microtiter plates (Nunc, Wiesbaden) with medium, 300 nM pyrophosphate bromohydrin, different concentrations of constructs, or the control construct AV#75. For CD107 assays, 0.5 μg / mL PE-labeled anti-CD107a mAb clone H4A3 (Biolegend) and 0.5 μg / mL PE-labeled anti-CD107b mAb clone H4B4 (Biolegend) or appropriate isotype controls were added directly to 96-well microtiter plates, while 3 μM of the secretion inhibitor monensin was added after 3 h of cell culture. After a further 3 h, γδ T cells were washed and PerCP-conjugated anti-CD45 mAb (clone 2D1, BD Biosciences), AlexaF700-conjugated anti-CD3 mAb (clone SK7, Biolegend), BV510-conjugated anti-CD8 mAb (clone SK1, BD Biosciences), PE-Cy7-conjugated anti-TCR γδ mAb (clone 11F2, BD Biosciences) and APC-Vio770-conjugated anti-Vδ2 (clone REA 771, Miltenyi) were added and, after washing, SYTOX in PBS was added before cells were analysed by flow cytometry (LSR Fortessa, BD Biosciences). TM Green Dead Cell Stain (1:4000, Thermo Scientific, # S34860) was added for 20 min. Results for γδ T cells from four different donors are shown in FIG. [Table 6] [Table 7-1] [Table 7-2]

Claims

1. 1. A bispecific antibody comprising a first binding moiety that specifically and agonistically binds to human CD277 and a second binding moiety that specifically binds to a tumor antigen, wherein the first binding moiety is a full-length bivalent antibody, and the second binding moiety consists of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of the single-chain Fv antibodies being linked to each C-terminus of the first binding moiety by a peptide linker.

2. 2. The bispecific antibody of claim 1, wherein each of the single-chain Fv antibodies is characterized in that the N-terminus of the variable light chain is linked to each C-terminus of the first binding moiety by a peptide linker.

3. A bispecific antibody according to claim 1 or 2, characterized in that the first binding portion comprises heavy chain CDR sequences: CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45, and light chain CDR sequences: CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO:

8.

4. A bispecific antibody according to claim 1 or 2, characterized in that the first binding portion comprises heavy chain CDR sequences: CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 68, SEQ ID NO: 72, or SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45, and light chain CDR sequences: CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO:

8. Claim 5: The first binding moiety comprises heavy chain CDR sequences: CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45, and light chain CDR sequences: CDRL1 of SEQ ID NO: 75, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 140, or SEQ ID NO: 141, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8; or 5. The bispecific antibody of claim 4, wherein the first binding portion comprises heavy chain CDR sequences: CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 68, SEQ ID NO: 72, or SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45, and light chain CDR sequences: CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO:

8.

6. a) heavy chain CDR sequences comprising CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45; and b) as a light chain CDR sequence: b1) a CDRL1 of SEQ ID NO: 75, a CDRL2 of SEQ ID NO: 76, and a CDRL3 of SEQ ID NO: 77; b2) a CDRL1 of SEQ ID NO: 79, a CDRL2 of SEQ ID NO: 80, and a CDRL3 of SEQ ID NO: 81; b3) a CDRL1 of SEQ ID NO: 83, a CDRL2 of SEQ ID NO: 84, and a CDRL3 of SEQ ID NO: 85; b4) a CDRL1 of SEQ ID NO: 87, a CDRL2 of SEQ ID NO: 88, and a CDRL3 of SEQ ID NO: 89; b5) a CDRL1 of SEQ ID NO: 117, a CDRL2 of SEQ ID NO: 118, and a CDRL3 of SEQ ID NO: 119; b6) a CDRL1 of SEQ ID NO: 121, a CDRL2 of SEQ ID NO: 122, and a CDRL3 of SEQ ID NO: 123; b7) a CDRL1 of SEQ ID NO: 125, a CDRL2 of SEQ ID NO: 126, and a CDRL3 of SEQ ID NO: 127; b8) CDRL1 of SEQ ID NO: 129, CDRL2 of SEQ ID NO: 130, and CDRL3 of SEQ ID NO: 131; b9) CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135; b10) CDRL1 of SEQ ID NO: 137, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139; b11) CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139; b12) CDRL1 of SEQ ID NO: 140, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135; b13) CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135; b14) CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 135; b15) a CDRL1 of SEQ ID NO: 151, a CDRL2 of SEQ ID NO: 7, and a CDRL3 of SEQ ID NO: 8; b16) a CDRL1 of SEQ ID NO: 152, a CDRL2 of SEQ ID NO: 7, and a CDRL3 of SEQ ID NO: 8; b17) CDRL1 of SEQ ID NO: 153, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8; b18) a CDRL1 of SEQ ID NO: 6, a CDRL2 of SEQ ID NO: 7, and a CDRL3 of SEQ ID NO: 156; b19) a CDRL1 of SEQ ID NO: 6, a CDRL2 of SEQ ID NO: 7, and a CDRL3 of SEQ ID NO: 157; b20) a CDRL1 of SEQ ID NO: 6, a CDRL2 of SEQ ID NO: 7, and a CDRL3 of SEQ ID NO: 158; b21) a CDRL1 of SEQ ID NO: 154, a CDRL2 of SEQ ID NO: 7, and a CDRL3 of SEQ ID NO: 8; b22) CDRL1 of SEQ ID NO: 155, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 a set of CDRs selected from the group consisting of 2. The bispecific antibody of claim 1, comprising:

7. 2. The bispecific antibody of claim 1, wherein the tumor antigen is selected from the group consisting of CLDN18.2, FOLR1, STEAP1, and DLL3.

8. 8. The bispecific antibody of claim 7, characterized in that for the second binding moiety, the variable light and heavy chain CDRs are: a) CDRL1 of SEQ ID NO: 11, CDRL2 of SEQ ID NO: 12, and CDRL3 of SEQ ID NO: 13, and CDRH1 of SEQ ID NO: 15, CDRH2 of SEQ ID NO: 16, and CDRH3 of SEQ ID NO: 17, whose heavy chain variable region CDRs are directed against FOLR1 as a tumor antigen; b) CDRL1 of SEQ ID NO: 19, CDRL2 of SEQ ID NO: 20, and CDRL3 of SEQ ID NO: 21, and heavy chain variable region CDRs of CDRH1 of SEQ ID NO: 23, CDRH2 of SEQ ID NO: 24, and CDRH3 of SEQ ID NO: 25 directed against STEAP1 as a tumor antigen; c) CDRL1 of SEQ ID NO: 27, CDRL2 of SEQ ID NO: 28, and CDRL3 of SEQ ID NO: 29, and heavy chain variable region CDRs of CDRH1 of SEQ ID NO: 31, CDRH2 of SEQ ID NO: 32, and CDRH3 of SEQ ID NO: 33 of DLL3 as a tumor antigen; d) CDRL1 of SEQ ID NO: 35, CDRL2 of SEQ ID NO: 36, and CDRL3 of SEQ ID NO: 37, and the heavy chain variable region CDR is CDRH1 of SEQ ID NO: 39, CDRH2 of SEQ ID NO: 40, CDRH3 of SEQ ID NO: 41 of CLDN18.2 as a tumor antigen.

9. 2. The bispecific antibody of claim 1, wherein the variable heavy chain of the first binding moiety is SEQ ID NO: 42 and the variable light chain is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO:

86.

10. 10. The bispecific antibody of claim 9, wherein for the second binding moiety the variable light chain is SEQ ID NO: 10 and the variable heavy chain is SEQ ID NO:

14.

11. 2. The bispecific antibody of claim 1, wherein the first binding portion of the antibody is a humanized antibody or a CDR-grafted antibody.

12. The scFv is characterized in that peptide linker 1-VL-peptide linker 2-VH are linked to the C-terminus in this order; 2. The bispecific antibody of claim 1, wherein the first peptide linker preferably consists of 5 to 25 amino acids and the second peptide linker preferably consists of 10 to 25 amino acids.

13. 2. The bispecific antibody according to claim 1 for use in the treatment of a tumor disease, preferably for use in the treatment of a tumor disease selected from the group consisting of colon cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer.

14. A pharmaceutical composition comprising the bispecific antibody of claim 1.

15. 15. A pharmaceutical composition comprising the bispecific antibody of claim 14 for use in a method for treating cancer in an individual, the method comprising administering to the individual an effective amount of the bispecific antibody.

16. A recombinant nucleic acid sequence encoding the bispecific antibody of claim 1.

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