Bispecific antibodies against CD277 and tumor antigens

Abstract

The present invention relates to bispecific antibodies that bind to CD277 and human tumor antigen. The invention also relates to polynucleotides encoding such bispecific antibodies as well as vectors and host cells comprising such polynucleotides. In addition, the invention relates to methods for producing such antibodies and methods of using such antibodies to treat disease and therapeutic uses thereof.

Description

[0001] The contents of the electronically submitted sequence listing (name: Evo-PCT sequencelist.xml) submitted together with this patent application are part of the specification. Technical Field

[0002] The present invention relates to bispecific antibodies that bind to CD277 (BTN3A), a member of the butyrophilin 3 family, and a human tumor antigen. The present invention also relates to polynucleotides encoding such bispecific antibodies, and vectors and host cells comprising such polynucleotides. In addition, the present invention relates to methods for producing such antibodies, methods for treating diseases using such antibodies, and therapeutic uses thereof. Background Art

[0003] Vγ9Vδ2T cells are the main subpopulation of γδT cells in peripheral blood, accounting for approximately 60%-95%. Bioinformatic analysis of large metagenomic datasets determined the relative abundance of Vγ9Vδ2T cells within tumors and correlated them with patient outcomes. Tumor-infiltrating γδT lymphocytes (γδTILs) were found in all tumor entities, albeit at low numbers. Importantly, a correlation between the relative abundance of γδTILs and a favorable response to immune checkpoint therapy in multiple cancers was demonstrated. (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 based on Vγ9Vδ2T cells have been tested over the past few decades but have failed to provide consistent clinical efficacy. Other 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] BTN3A (CD277; UniProtKB-00481 (BT3A1_HUMAN), a member of the butyrophilin 3 family, 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, and regulates the proliferation of activated T cells, regulates the release of cytokines and IFNγ by activated T cells, and mediates 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 of the prokaryotic non-mevalonate / 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP) pathway for isoprenoid synthesis. BTN3A can accurately recognize this pathogen-derived molecule, which is comparable to the way TLRs recognize conserved pathogen structures such as LPS or DNA (O'Neill, L.A. J.; 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, DA et 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 between BTN3A1 and HMBPP leads to the binding of BTN3A1 to components of the immune synapse including the γδTCR, and leads to the subsequent activation of Vδ2 T cells. Butyrophilin 3A1 plays an important 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 indispensable compound for every tumor (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) described that BTN3A1 controls anti-tumor responses by coordinating αβ and γδ T cells.

[0007] De Bruin et al. (De Bruin RCG. et al.; Oncoimmunology 2018, VOL. 7, NO. 1, e1375641) described a bispecific nanoantibody approach targeting Vγ9Vδ2T cells and EGFR, which induced Vγ9Vδ2-T cell activation and subsequent tumor cell lysis in in vitro and in vivo mouse xenograft models, demonstrating the cell lytic ability of Vγ9Vδ2T cells.

[0008] 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.

[0009] WO2012080351 and WO2012080769 relate to anti-C277 antibodies (7.2 and 20.1). scFv is mentioned as a possible antibody form. Agonistic anti-C277 antibodies according to the prior art activate the cell lysis function, cytokine production and proliferation of Vγ9Vδ2T cells. According to the description of De Gassart A. et al. in Science Translational Medicine 13, (2021), (https: / / doi.org / 10.1126 / scitranslmed.abj0835), the activation of Vγ9Vδ2T cells in peripheral blood induces a transient decrease in circulating Vγ9Vδ2T cells, which is not due to exhaustion, but due to transport and marginalization. The inventors first recognized the relevance of exhaustion.

[0010] Imbert C. and Olive D. proposed bispecific antibodies targeting both CD277 and tumor antigens for activating Vγ9Vδ2T cells in 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., in Advances in Experimental Medicine and Biology, (2020), Springer, Vol. 1273, 91-104. In WO2020025703, multispecific antibodies, such as bispecific antibodies, are also proposed, which include an arm, the arm includes Fab or scFv, and Fab or scFv includes VH and VL of anti-CD277 antibodies, such as bispecific molecules mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein forms.

[0011] A large number of different formats of bispecific antibodies are known (e.g., reviewed in Brinkmann U. and Kontermann E.; MAbs. 2017 Feb-Mar; 9(2): 182–212; see Fig. 2 of Brinkmann and Kontermann). In 2019, more than 20 different commercial technology platforms are available for bsAb creation and development (reviewed in Lanrijn AF et al.; Nature Reviews; https: / / doi.org / 10.1038 / s41573-019-0028-1). The bispecific antibody format is 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 designated as a main module or parental module, in which scFvs of different specificities are coupled to the C-terminus of the heavy chain (IgG-HC-scFv, "Morrison-type bispecific antibodies"; see Figure 1).

[0012] WO2010112193 (US009382323; EP2414391B1) relates to a multispecific antibody, which comprises a full-length antibody that specifically binds to a first antigen and is composed of two antibody heavy chains and two antibody light chains; and one or more single-chain Fv fragments that bind to one or more other antigens, wherein the single-chain Fv fragment is fused to the full-length antibody at the C- or N-terminus of the heavy chain or light chain of the full-length antibody via a peptide linker.

[0013] Presti et al. (Presti, ELet al., Frontiers in immunology 2017, 8, 975–11) described that antibodies can be used to redirect γδT cells to cancer cells. This can be achieved, for example, by using bispecific antibodies, in which one binding site recognizes tumor-specific cell surface molecules (e.g., EpCAM or HER2 / neu) and the other binding site targets CD3 or the Vγ9 chain of Vγ9Vδ2TCR; such bispecific antibodies have been shown to be effective 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).

[0014] WO2018041827 describes an adenovirus equipped with a bispecific T cell engager (BiTE), wherein one of the binding domains in the BiTE is specific for a non-TCR activating protein such as BTN3A1, and one of the binding domains is specific for the following tumor antigens, such as 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 and ErbB3. WO2012080769 relates to anti-CD277 antibodies (e.g., mAb 7.2, mAb20.1). Antibody fragments and diabodies such as Fv, Fab, F(ab')2, Fab', dsFv, scFv, Sc(Fv)2 are generally referred to.

[0015] WO2020060406 describes an antibody comprising a first binding portion capable of binding to human CD1d and a second binding portion capable of binding to the Vγ9 chain of a T cell receptor on a γδ T cell, for treating chronic lymphocytic leukemia, multiple myeloma or acute myeloid leukemia.

[0016] Tumor antigens are known from various studies, such as comparisons of respective mRNA levels or protein expression levels in tumors and normal tissues or cell lines, or from 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, D. 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,DHet al.,J Hematol Oncol12,61(2019);Wu,M.et al.,Cancer Epidemiology Biomarkers Prev Publ Am AssocCancer Res Cosponsored Am Soc Prev Oncol 8,775–82(1999); Tarn, C. et al., ProcNational Acad Sci 105,8387–8392(2008)). The claudins 18 (CLD18) molecule (UniProtKB-P56856 (CLD18_HUMAN) is an integral transmembrane protein with a molecular weight of approximately 27,9 / 27,72 kD. Claudin is an integral membrane protein located within the tight junctions of epithelial and endothelial cells. Tight junctions organize a network of interconnected chains of intramembrane particles between adjacent cells. Within tight junctions, occludin and claudin are the most prominent transmembrane protein components. Due to their strong cell-cell adhesive properties, they form a first barrier to prevent and control the paracellular transport of solutes and restrict the lateral diffusion of membrane lipids and proteins to maintain cell polarity. Tight junction forming proteins play a key role in organizing the architecture of epithelial tissue. It was hypothesized that this class of proteins might be difficult to reach by antibodies in well-structured epithelial cells but would be exposed on tumor cells.Antibodies against Claudin18 and its splice variant Claudin 18.2 are described in, for example, WO2007059997, WO2008145338, US20150374789, WO2013174403, and US9770487 (US10314890; EP2958945; IMAB362). WO2021024020 describes a combination therapy of anti-Claudin18.2 antibodies and immune checkpoint inhibitors for the treatment of cancer.

[0017] STEAP-1 (six transmembrane epithelial antigen of the prostate-1) is a 339 amino acid cell surface protein that is expressed primarily in 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 overexpressed in other human cancers such as lung cancer and colon cancer. The expression profile of STEAP-1 in normal and cancerous tissues suggests its potential use as a target for immunotherapy. WO 2008 / 052187 reports anti-STEAP-1 antibodies and immunoconjugates thereof. STEAP-1xCD3 bispecific antibodies are described in WO2014165818 and WO2017055388.

[0018] FOLR1 is expressed on epithelial tumor cells of various origins, such as ovarian cancer, lung cancer, breast cancer, kidney cancer, colorectal cancer, endometrial cancer. 10.1517 / 17425247.2012.694863.Epub 2012.WO2012119077 mentions antibodies against FOLR1. Bispecific antibodies targeting FOLR1 and CD3 are described in WO2016 / 079076 and WO2021255143.

[0019] DLL3 is selectively expressed in high-grade pulmonary neuroendocrine tumors, including SCLC and LCNEC. Increased expression of DLL3 was observed in xenograft tumors derived 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 was also observed in extrapulmonary neuroendocrine cancers, including prostate neuroendocrine cancer (Puca et al., Sci Transl Med 11(484):pii:eaav0891(2019). Although DLL3 is expressed on the surface of such tumor cells, it is not expressed in normal tissues. WO2021007371 relates to anti-DLL3 antibodies, and humanized, chimeric or bispecific antibodies are proposed. WO2019195409 mentions multispecific proteins that bind to NKG2D receptors, CD16 and tumor antigens. Summary of the invention

[0020] Agonistic anti-C277 antibodies according to the prior art activate the cytolytic function, cytokine production and proliferation of Vγ9Vδ2 T cells. Agonistic anti-C277 antibodies according to the prior art induce a transient decrease in circulating Vγ9Vδ2 T cells, which is described as a result of trafficking of Vγ9Vδ2 T cells from the circulation to tissues including cancer tissues rather than exhaustion.

[0021] However, the present inventors have recognized that such activation of Vγ9Vδ2T cells by agonistic anti-C277 antibodies according to the prior art in the absence of tumor cells induces self-elimination of Vγ9Vδ2T cells. The present inventors have recognized that bispecific antibodies that specifically and agonistically bind to CD277 (also further referred to as "bispecific anti-CD277 antibodies") and specifically bind to human tumor antigens (also further referred to as "tumor antigens") having the properties described below show excellent killing effects on human tumor cells carrying the tumor antigen and high safety for lysis of non-tumor cells, and do not induce self-elimination of Vγ9Vδ2T cells.

[0022] In one embodiment, the present invention is characterized in that it comprises a first binding moiety that specifically and agonistically binds to human CD277 and a second binding moiety that specifically binds to a tumor antigen, characterized in that the first binding moiety is a full-length bivalent antibody and the second binding moiety is composed of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of the single-chain Fv antibodies being connected to each C-terminus of the first binding moiety via a peptide linker.

[0023] In one embodiment, each of said single chain Fv antibodies is linked at its N-terminus of said variable light chain via a peptide linker to each C-terminus of said first binding moiety.

[0024] In one embodiment, the bispecific antibody according to the invention is characterized in that it comprises in the first binding part as heavy chain CDR sequences CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 3 and CDRH3 of SEQ ID NO: 4, and as light chain CDR sequences CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8.

[0025] In one embodiment the antibody according to the invention is characterized in comprising the substitutions N5S and K10N (also known as N53S, K58N (Kabat) or N185S-K190N) in CDRH2 (SEQ ID NO: 44).

[0026] In one embodiment, the antibody according to the invention is characterized in that, in addition to said CDRH2 substitution, it comprises the substitution L8V (also called L31V) in CDRL1 (SEQ ID NO: 75). In one embodiment, the antibody according to the invention is characterized in that it additionally comprises the substitutions L8V and H1R in CDRL1 (SEQ ID NO: 140).

[0027] In one embodiment, the bispecific antibody according to the present invention is characterized in that it comprises a first binding part that specifically and agonistically binds to human CD277 and a second binding part that specifically binds to a tumor antigen, characterized in that the first binding part is a full-length bivalent antibody, which comprises CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44 and CDRH3 of SEQ ID NO:45 as heavy chain CDR sequences and CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7 and CDRL3 of SEQ ID NO:8 as light chain CDR sequences in the first binding part, and the second binding part consists of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of the single-chain Fv antibodies being connected to each C-terminus of the first binding part via a peptide linker.

[0028] In one embodiment the bispecific antibody according to the invention is characterized in that the CDRH2 is SEQ ID NO:68, SEQ ID NO:72 or SEQ ID NO:110.

[0029] 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.

[0030] In one embodiment the antibody according to the invention is characterized in comprising the substitutions N5S and K10N in CDRH2 (SEQ ID NO: 44).

[0031] In one embodiment, the antibody according to the invention is characterized in that, in addition to said CDRH2 substitution, it comprises the substitution of L8V in CDRL1 (SEQ ID NO: 75).In one embodiment, the antibody according to the invention is characterized in that it additionally comprises the substitutions L8V and H1R in CDRL1 (SEQ ID NO: 140).

[0032] In one embodiment the first binding moiety of the antibody according to the invention is a human, humanized or CDR-grafted antibody.

[0033] In one embodiment, the invention features 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, characterized in comprising CDRH1 of SEQ ID NO: 43, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 45 (CDRH set 1) as heavy chain CDR sequences, and

[0034] b) a CDR set selected from the group consisting of:

[0035] b1) CDRL1 of SEQ ID NO:75, CDRL2 of SEQ ID NO:76 and CDRL3 of SEQ ID NO:77,

[0036] b2) CDRL1 of SEQ ID NO:79, CDRL2 of SEQ ID NO:80 and CDRL3 of SEQ ID NO:81,

[0037] 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,

[0038] b5) CDRL1 of SEQ ID NO: 117, CDRL2 of SEQ ID NO: 118, and CDRL3 of SEQ ID NO: 119,

[0039] b6) CDRL1 of SEQ ID NO: 121, CDRL2 of SEQ ID NO: 122, and CDRL3 of SEQ ID NO: 123,

[0040] b7) CDRL1 of SEQ ID NO: 125, CDRL2 of SEQ ID NO: 126, and CDRL3 of SEQ ID NO: 127,

[0041] b8) CDRL1 of SEQ ID NO: 129, CDRL2 of SEQ ID NO: 130, and CDRL3 of SEQ ID NO: 131,

[0042] b9) CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135,

[0043] b10) CDRL1 of SEQ ID NO: 137, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139,

[0044] b11) CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 139,

[0045] b12) CDRL1 of SEQ ID NO: 140, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135,

[0046] b13) CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 134, and CDRL3 of SEQ ID NO: 135,

[0047] b14) CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 135,

[0048] b15) CDRL1 of SEQ ID NO: 151, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8,

[0049] b16) CDRL1 of SEQ ID NO: 152, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8,

[0050] b17) CDRL1 of SEQ ID NO: 153, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8,

[0051] b18) CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:156,

[0052] b19) CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:157,

[0053] b20) CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7, and CDRL3 of SEQ ID NO:158,

[0054] b21) CDRL1 of SEQ ID NO: 154, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8,

[0055] b22) CDRL1 of SEQ ID NO: 155, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8, and

[0056] c) The second binding moiety consists of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each single-chain Fv antibody being linked to each C-terminus of the first binding moiety.

[0057] 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 chosen 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.

[0058] In one embodiment the bispecific antibody according to the invention is characterized in comprising a humanized form of said variable chain.

[0059] 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). Other useful tumor antigens are described, for example, in Middleburg et al., Cancers (2021) 13, 287, pp 4-6.

[0060] In one embodiment, the antibody according to the invention is characterized in that the first binding moiety comprises a heavy chain and a light chain CDR set selected from the group consisting of: a CDR set shown in compounds EvB#21 to 136 in Table 3, or a variable light chain and a variable heavy chain combination of compounds EvB#21 to 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 characterized in that it is humanized.

[0061] In one embodiment, the antibody according to the invention is characterized in that, for FOLR1 as tumor antigen, the second binding moiety comprises CDRL1 of SEQ ID NO: 11, CDRL2 of SEQ ID NO: 12 and CDRL3 of SEQ ID NO: 13 as light chain CDRs, and CDRH1 of SEQ ID NO: 15, CDRH2 of SEQ ID NO: 16 and CDRH3 of SEQ ID NO: 17 as heavy chain CDRs (FOLR1-CDR set).

[0062] In one embodiment, for STEAP1 as a tumor antigen, the second binding moiety comprises 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 (STEAP1 CDR set).

[0063] In one embodiment, the antibody according to the present invention, for DLL3 as a tumor antigen, comprises 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 as CDRs (DLL3 CDR set) in the second binding portion.

[0064] In one embodiment, the antibody according to the present invention, for CLDN18.2 as a tumor antigen, comprises 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 as CDRs (CLDN18.2 CDR set) in the second binding portion.

[0065] In one embodiment, the antibody according to the invention is characterized in that, for FOLR-1 as tumor antigen, the heavy and light chain variable region combination of SEQ ID NO: 10 and SEQ ID NO: 14 is comprised in the second binding moiety.

[0066] In one embodiment, the antibody according to the invention is characterized in that, for STEAP1 as tumor antigen, the heavy and light chain variable region combination of SEQ ID NO: 18 and SEQ ID NO: 22 is comprised in the second binding moiety.

[0067] In one embodiment, the antibody according to the invention is characterized in that, for DLL3-4 as tumor antigen, the heavy and light chain variable region combination of SEQ ID NO: 26 and SEQ ID NO: 30 is comprised in the second binding moiety.

[0068] In one embodiment, the antibody according to the invention is characterized in that, for CLDN18.2 as tumor antigen, the heavy and light chain variable region combination of SEQ ID NO: 34 and SEQ ID NO: 38 is comprised in the second binding moiety.

[0069] In one embodiment, the antibody according to the invention is characterized in that

[0070] a) the bispecific antibody exhibits an EC50 ratio of 0.001 to 0.2 for lysis of a first tumor antigen-bearing cell line compared to lysis of a reference antibody comprising as heavy chain the heavy chain of SEQ ID NO: 94 and as light chain the light chain of SEQ ID NO: 93,

[0071] b) the bispecific antibody exhibits an EC50 ratio of lysis of a second cell line not bearing the tumor antigen of 5 to 1000 compared to lysis by the reference antibody,

[0072] All were measured in the presence of activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1, in the presence of 12.5 IU / mL interleukin-2, and in the same assay under the same conditions.

[0073] In one embodiment, the bispecific antibody is in the form of a Mab-scFv.

[0074] In one embodiment, the invention features a bispecific antibody comprising a Mab-scFv format 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, characterized in that

[0075] a) the first binding moiety is a full length bivalent antibody,

[0076] b) the second binding moiety specifically binds to the tumor antigen and comprises, as heavy chain CDR and light chain CDR, a set of CDRs selected from the following:

[0077] b1) for FOLR1 as a tumor antigen, 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 (FOLR1 CDR set),

[0078] b2) for STEAP1 as a tumor antigen, 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 (STEAP1 CDR set),

[0079] b3) for DLL3 as a tumor antigen, 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 (DLL3 CDR set),

[0080] b4) for CLDN18.2 as a tumor antigen, 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 (CLDN18.2 CDR set),

[0081] c) the bispecific antibody exhibits an EC50 ratio of 0.001 to 0.2 for lysis of a first tumor antigen-bearing cell line compared to lysis of a reference antibody comprising as heavy chain the heavy chain of SEQ ID NO: 94 and as light chain the light chain of SEQ ID NO: 93,

[0082] d) said bispecific antibody exhibits an EC50 ratio of lysis of a second cell line not bearing said tumor antigen of 5 to 1000 compared to lysis by said reference antibody,

[0083] All were measured in the presence of activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1, in the presence of 12.5 IU / mL interleukin-2, and in the same assay under the same conditions.

[0084] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0085] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 6, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8 (CDRL Set 1) as light chain CDR sequences, and

[0086] b) the CDR sequence is selected from the group consisting of:

[0087] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0088] b2) CDRH1 of SEQ ID NO:47, CDRH2 of SEQ ID NO:48, and CDRH3 of SEQ ID NO:49 (CDRH Set 2),

[0089] b3) CDRH1 of SEQ ID NO:51, CDRH2 of SEQ ID NO:52, and CDRH3 of SEQ ID NO:53 (CDRH Set 3),

[0090] b4) CDRH1 of SEQ ID NO:55, CDRH2 of SEQ ID NO:56, and CDRH3 of SEQ ID NO:57 (CDRH Set 4),

[0091] b5) CDRH1 of SEQ ID NO:59, CDRH2 of SEQ ID NO:60, and CDRH3 of SEQ ID NO:61 (CDRH Set 5),

[0092] b6) CDRH1 of SEQ ID NO:63, CDRH2 of SEQ ID NO:64, and CDRH3 of SEQ ID NO:65 (CDRH Set 6),

[0093] b7) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:68, and CDRH3 of SEQ ID NO:69 (CDRH Set 7),

[0094] b8) CDRH1 of SEQ ID NO:71, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:73 (CDRH Set 8),

[0095] b10) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 106, and CDRH3 of SEQ ID NO: 107 (CDRH Set 10),

[0096] b11) CDRH1 of SEQ ID NO: 109, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 111 (CDRH Set 11),

[0097] b12) CDRH1 of SEQ ID NO: 113, CDRH2 of SEQ ID NO: 114, and CDRH3 of SEQ ID NO: 115 (CDRH Set 12),

[0098] b13) CDRH1 of SEQ ID NO:59, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:4 (CDRH Set 14)

[0099] b14) CDRH1 of SEQ ID NO:59, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:4 (CDRH Set 15)

[0100] b15) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:4 (CDRH Set 20)

[0101] b16) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH Set 18)

[0102] b17) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19), and

[0103] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0104] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0105] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 121, CDRL2 of SEQ ID NO: 7 and CDRL3 of SEQ ID NO: 8 (CDRL Set 2) as light chain CDR sequences, and

[0106] b) the CDR sequence is selected from the group consisting of:

[0107] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0108] b2) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0109] b3) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0110] b4) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:4 (CDRH set 20),

[0111] b5) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:68, and CDRH3 of SEQ ID NO:4 (CDRH Set 7),

[0112] b6) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH Set 18),

[0113] b7) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19), and

[0114] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0115] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0116] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 83, CDRL2 of SEQ ID NO: 84, and CDRL3 of SEQ ID NO: 85 (CDRL Set 3) as light chain CDR sequences, and

[0117] b) the CDR sequence is selected from the group consisting of:

[0118] 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, b12) CDRH Set 12, and

[0119] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0120] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0121] a) the first binding moiety is a full-length bivalent antibody comprising CDRL1 of SEQ ID NO: 133, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL Set 4) as light chain CDR sequences, and

[0122] b) the CDR sequence is selected from the group consisting of:

[0123] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0124] b2) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0125] b3) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0126] b4) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:4 (CDRH Set 1),

[0127] b5) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:4 (CDRH set 21),

[0128] b6) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:68, and CDRH3 of SEQ ID NO:45 (CDRH Set 7),

[0129] b7) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH set 23),

[0130] b8) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:106, and CDRH3 of SEQ ID NO:45 (CDRH set 24),

[0131] b9) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:4 (CDRH set 25),

[0132] b10) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 114, and CDRH3 of SEQ ID NO: 115 (CDRH Set 26),

[0133] b11) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 44, and CDRH3 of SEQ ID NO: 4 (CDRH Set 27)

[0134] b12) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19)

[0135] b13) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 4 (CDRH Set 18), and

[0136] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0137] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0138] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 75, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL Set 5) as light chain CDR sequences, and

[0139] b) the CDR sequence is selected from the group consisting of:

[0140] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0141] b2) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0142] b3) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0143] b4) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 20),

[0144] 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),

[0145] b6) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 4 (CDRH Set 19), and

[0146] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0147] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0148] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 140, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL Set 6) as light chain CDR sequences, and

[0149] b) the CDR sequence is selected from the group consisting of:

[0150] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0151] b2) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0152] b3) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0153] b1) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:44 and CDRH3 of SEQ ID NO:45 (CDRH Set 20),

[0154] b2) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 23)

[0155] b3) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH Set 18),

[0156] b4) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19), and

[0157] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0158] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0159] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 138, and CDRL3 of SEQ ID NO: 8 (CDRL Set 7) as light chain CDR sequences, and

[0160] b) the CDR sequence is selected from the group consisting of:

[0161] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0162] b2) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0163] b3) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0164] b4) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH Set 18),

[0165] b5) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19), and

[0166] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0167] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0168] a) the first binding moiety is a full-length bivalent antibody comprising CDRL1 of SEQ ID NO: 141, CDRL2 of SEQ ID NO: 7, and CDRL3 of SEQ ID NO: 8 (CDRL Set 8) as light chain CDR sequences, and

[0169] b) the CDR sequence is selected from the group consisting of:

[0170] b1) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0171] b2) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0172] b3) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0173] b4) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 20),

[0174] b5) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 23), and

[0175] b6) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH Set 18),

[0176] b7) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19), and

[0177] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0178] In one embodiment, the invention includes 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, characterized in that

[0179] a) the first binding moiety is a full length bivalent antibody comprising as 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), and

[0180] b) the CDR sequence is selected from the group consisting of:

[0181] b1) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:44 and CDRH3 of SEQ ID NO:45 (CDRH Set 20),

[0182] b2) CDRH1 of SEQ ID NO:67, CDRH2 of SEQ ID NO:68, and CDRH3 of SEQ ID NO:45 (CDRH Set 23), and

[0183] b3) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 72, and CDRH3 of SEQ ID NO: 45 (CDRH Set 18),

[0184] b4) CDRH1 of SEQ ID NO: 105, CDRH2 of SEQ ID NO: 110, and CDRH3 of SEQ ID NO: 45 (CDRH Set 19), and

[0185] b5) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 (CDRH Set 1),

[0186] b6) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:72, and CDRH3 of SEQ ID NO:45 (CDRH Set 21),

[0187] b7) CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:110, and CDRH3 of SEQ ID NO:45 (CDRH Set 22),

[0188] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0189] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0190] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 87, CDRL2 of SEQ ID NO: 88, and CDRL3 of SEQ ID NO: 89 (CDRL Set 9) as light chain CDR sequences, and

[0191] b) the CDR sequence is selected from the group consisting of:

[0192] 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

[0193] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0194] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0195] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 79, CDRL2 of SEQ ID NO: 80, and CDRL3 of SEQ ID NO: 81 (CDRL Set 10) as light chain CDR sequences, and

[0196] b) the CDR sequence is selected from the group consisting of:

[0197] 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

[0198] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0199] In one embodiment, the invention comprises 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, characterized in that

[0200] a) the first binding moiety is a full length bivalent antibody comprising CDRL1 of SEQ ID NO: 75, CDRL2 of SEQ ID NO: 76, and CDRL3 of SEQ ID NO: 77 (CDRL Set 11) as light chain CDR sequences, and

[0201] b) the CDR sequence is selected from the group consisting of:

[0202] 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

[0203] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0204] In one embodiment the invention comprises a bispecific antibody according to the invention in the form of a Mab-scFv, characterized in that the first binding moiety comprises as light chain CDR sequence a) a CDR set selected from the group consisting of:

[0205] 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,

[0206] And the second binding moiety comprises a heavy chain and a light chain CDR set selected as a CDR set from the group consisting of: a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set and a CLDN 18.2 CDR set.

[0207] In one embodiment the invention comprises a bispecific antibody according to the invention in the form of a Mab-scFv, characterized in that the first binding moiety comprises as light chain CDR sequence CDRL1 set 2 and as heavy chain CDR sequence a CDR set selected from the group consisting of:

[0208] 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,

[0209] And the second binding moiety comprises a heavy chain and a light chain CDR set selected as a CDR set from the group consisting of: a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set and a CLDN 18.2 CDR set.

[0210] In one embodiment the invention comprises a bispecific antibody according to the invention in the form of a Mab-scFv, characterized in that the first binding moiety comprises as light chain CDR sequence CDRL1 set 3 and as heavy chain CDR sequence a CDR set selected from the group consisting of:

[0211] 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, b12) CDRH Set 12,

[0212] And the second binding moiety comprises a heavy chain and a light chain CDR set selected as a CDR set from the group consisting of: a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set and a CLDN 18.2 CDR set.

[0213] In one embodiment the invention comprises a bispecific antibody according to the invention in the form of a Mab-scFv, characterized in that the first binding moiety comprises as light chain CDR sequence CDRL1 set 4 and as heavy chain CDR sequence a CDR set selected from the group consisting of:

[0214] 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,

[0215] And the second binding moiety comprises a heavy chain and a light chain CDR set selected as a CDR set from the group consisting of: a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set and a CLDN 18.2 CDR set.

[0216] In one embodiment, the present invention comprises a bispecific antibody according to the present invention in the form of a Mab-scFv, characterized in that the first binding moiety comprises as light chain CDR sequence CDRL1 set 5 and as heavy chain CDR sequence 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,

[0217] And the second binding moiety comprises a heavy chain and a light chain CDR set selected as a CDR set from the group consisting of: a FOLR1 CDR set, a STEAP1 CDR set, a DLL3 CDR set and a CLDN 18.2 CDR set.

[0218] One embodiment of the invention is a bispecific antibody according to the invention in the format of Mab-scFv, characterized in that the first binding part is a humanized antibody.

[0219] In one embodiment, the present invention comprises a bispecific antibody in Mab-scFv format, characterized in that the first binding moiety comprises, as variable heavy chain, a variable heavy chain selected from the group consisting of SEQ ID NO: 42, 46, 50, 54, 58, 62, 66 and 70 or a humanized form thereof having at least 95% sequence identity thereto,

[0220] and a sequence selected from the group consisting of:

[0221] a) SEQ ID NO: 5,

[0222] b) SEQ ID NO: 74,

[0223] c) SEQ ID NO: 78,

[0224] d) SEQ ID NO: 82,

[0225] e) SEQ ID NO: 86,

[0226] or a humanized form thereof having at least 95% sequence identity with said sequence,

[0227] And the second binding moiety consists of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of which is connected to each C-terminus of the first binding moiety via a peptide linker. In one embodiment, the second binding moiety comprises a set selected from the group consisting of the following as heavy chain and light chain variable regions:

[0228] e) for FOLR-1 as a tumor antigen, SEQ ID NO: 10 and SEQ ID NO: 14,

[0229] f) For STEAP1 as a tumor antigen, SEQ ID NO: 18 and SEQ ID NO: 22,

[0230] g) for DLL3-4 as a tumor antigen, SEQ ID NO:26 and SEQ ID NO:30, and

[0231] h) CLDN 18.2 as tumor antigen, SEQ ID NO: 34, SEQ ID NO: 38.

[0232] In one embodiment, the present invention comprises a bispecific antibody in the form of Mab-scFv, characterized in that the first binding moiety comprises as variable heavy chain the variable heavy chain of SEQ ID NO: 1 or a humanized form thereof having at least 95% sequence identity thereto, and as light chain sequence a sequence selected from the group consisting of:

[0233] a) SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:86,

[0234] or a humanized form thereof having at least 95% sequence identity with said sequence,

[0235] And the second binding moiety consists of two identical single-chain Fv antibodies that specifically bind to the tumor antigen, each of which is connected to each C-terminus of the first binding moiety via a peptide linker. In one embodiment, the second binding moiety comprises a set selected from the group consisting of the following as heavy chain and light chain variable regions:

[0236] e) SEQ ID NO: 10 and SEQ ID NO: 14 for FOLR-1 as a tumor antigen,

[0237] f) For STEAP1 as a tumor antigen, SEQ ID NO: 18 and SEQ ID NO: 22,

[0238] g) for DLL3-4 as a tumor antigen, SEQ ID NO:26 and SEQ ID NO:30, and

[0239] h) CLDN 18.2 as tumor antigen, SEQ ID NO: 34, SEQ ID NO: 38.

[0240] In one embodiment, the invention comprises a bispecific antibody according to the invention in the form of a Mab-scFv, characterized in that the first binding moiety comprises a variable light chain and a variable heavy chain set selected from the group as described in Table 3,

[0241] and the second binding moiety comprises a variable light chain and a variable heavy chain set selected from the group consisting of:

[0242] a) SEQ ID NO: 10 and SEQ ID NO: 14 for FOLR1 as a tumor antigen,

[0243] b) For STEAP1 as a tumor antigen, SEQ ID NO: 18 and SEQ ID NO: 22,

[0244] c) for DLL3 as a tumor antigen, SEQ ID NO:26 and SEQ ID NO:30, and

[0245] d) CLDN18.2 as tumor antigen, SEQ ID NO: 34, SEQ ID NO: 38.

[0246] In one embodiment, the invention features a bispecific antibody comprising a Mab-scFv format 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, characterized in that

[0247] a) the first binding moiety is a full length bivalent antibody,

[0248] b) the second binding moiety is a single chain Fv antibody (scFv) in the form of Mab-scFv, which specifically binds to the tumor antigen, comprising as heavy and light chain variable regions a set selected from the group consisting of:

[0249] b1) For FOLR1 as a tumor antigen, SEQ ID NO: 10 and SEQ ID NO: 14,

[0250] b2) For STEAP1 as a tumor antigen, SEQ ID NO: 18 and SEQ ID NO: 22,

[0251] b3) for DLL3 as a tumor antigen, SEQ ID NO:26 and SEQ ID NO:30, and

[0252] b4) CLDN18.2 as tumor antigen, SEQ ID NO: 34, SEQ ID NO: 38,

[0253] a) the bispecific antibody exhibits an EC50 ratio of 0.001 to 0.2 for lysis of a first tumor antigen-bearing cell line compared to lysis of a reference antibody comprising as heavy chain the heavy chain of SEQ ID NO: 94 and as light chain the light chain of SEQ ID NO: 93,

[0254] b) the bispecific antibody exhibits an EC50 ratio of lysis of a second cell line not bearing the tumor antigen of 5 to 1000 compared to lysis by the reference antibody,

[0255] All were measured in the presence of activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1, in the presence of 12.5 IU / mL interleukin-2, and in the same assay under the same conditions.

[0256] In one embodiment, the antibody according to the invention is characterized in that the 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 IGKV3-11*01V-KAPPA (X01668) or IGKV1-12*01V-KAPPA (V01577); see IMGT library. In one embodiment, the human VH / VL framework combination is 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 present invention, the framework sequence consists of four parts (FRH1-4 and FRL1-4).

[0257] In one embodiment, the present invention comprises a bispecific antibody in the form of a Mab-scFv 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, characterized in that

[0258] a) the first binding moiety is a full length bivalent antibody comprising a variable light chain of the form FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 and a variable heavy chain of the form FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, wherein 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, wherein 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 set of Tables 6 or 7, and

[0259] c) The second binding moiety consists of two single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen.

[0260] In one embodiment, the antibody according to the invention is characterized in that it is a humanized antibody comprising a variable light chain consisting of the sequence of FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 and a variable heavy chain consisting of the sequence of FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, or a variable light chain consisting of the sequence of FRL1-CDRL1-FRL2-CDRL2-FRL3a-CDRL3-FRL4 and a variable heavy chain consisting of the sequence of FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, and a CDRH / CDRL set selected from the set of Table 6 or 7.

[0261] In one embodiment, the present invention is characterized in that the second binding moiety is composed of two identical single-chain Fv antibodies that specifically bind to the tumor antigen. In one embodiment, the second binding moiety is composed of two identical single-chain Fv antibodies (scFv) that specifically bind to the tumor antigen, each single-chain Fv antibody is connected to each C-terminus of the first binding moiety through its N-terminus. Therefore, for each C-terminus of the Fc portion of the first binding moiety (which is a full-length monospecific anti-CD277 antibody), only one scFv is connected. The format of the bispecific antibody consisting of a full-length bivalent antibody as the first binding moiety and the two scFvs as the second binding moiety is referred to herein as a "Mab-scFv format." Exemplary Mab-scFv formats are shown in Figure 1a middle.

[0262] In one embodiment, each of said scFvs is chemically linked to each C-terminus of the first binding moiety via a first peptide linker (Linker 1).

[0263] One embodiment of the invention comprises a bispecific antibody according to the invention, characterized in that the scFv is bound to the C-terminus in the orientation Peptide Linker 1 -VL-Peptide Linker 2-VH.

[0264] In one embodiment, the peptide linker is selected from the group consisting of peptides of SEQ ID NO: 97, 98, 99, 100 and 101.

[0265] In one embodiment the invention comprises a bispecific antibody according to the invention, characterized in that the first peptide linker consists of 5-25, in one embodiment 10-25 amino acids.

[0266] One embodiment of the invention comprises a bispecific antibody according to the invention, characterized in that the second peptide linker consists of 10-25 amino acids.

[0267] In one embodiment, the bispecific antibody does not induce significant lysis in the second cell line that is 10-fold, in one embodiment 5-fold, in one embodiment two-fold higher than background lysis.

[0268] In one embodiment of the invention, the bispecific antibody exhibits an EC50 ratio for lysis of said first cell line compared to lysis of said reference antibody of in one embodiment no more than 0.2, in one embodiment between 0.001 and 0.2, in one embodiment between 0.005 and 0.2, in one embodiment between 0.01 and 0.2.

[0269] In one embodiment, the bispecific antibody exhibits an EC50 ratio 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, in one embodiment 10 to 5000 for lysis of the second cell line compared to lysis by the reference antibody.

[0270] An EC50 ratio according to the present invention means the ratio of EC50 values ​​as measured for cell lysis. Example 6 describes an exemplary method.

[0271] In one embodiment, said second tumor antigen-negative cell line is said first cell line in which a tumor antigen is inactivated (knockout cell line).

[0272] In one embodiment, the invention comprises a bispecific antibody according to the invention, characterized in that the 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 of the invention, the bispecific antibody shows an Emax ratio of 0.5 to 1.5, in one embodiment 0.8 to 1.5, in one embodiment 0.9 to 1.5 for lysis of the first tumor antigen-positive cell line compared to the Emax of the reference antibody.

[0273] The reference antibody is a full-length bivalent, monospecific and agonistic anti-CD277 antibody comprising as the heavy chain the heavy chain of SEQ ID NO: 94 and as the light chain the light chain of SEQ ID NO: 93. The reference antibody comprises the variable heavy chain of SEQ ID NO: 1 and the variable light chain of SEQ ID NO: 5 and the CDRs of SEQ ID NOs: 2, 3, 4, 6, 7, 8.

[0274] In one embodiment, the invention comprises a bispecific antibody according to the invention, characterized in that the tumor antigen is a tumor antigen that non-internalizes the bispecific antibody of the invention.

[0275] Another embodiment of the invention is a recombinant nucleic acid sequence encoding a bispecific antibody according to the invention.

[0276] Another embodiment of the present invention is a vector comprising a recombinant nucleic acid sequence encoding a bispecific antibody according to the present invention.

[0277] Another 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.

[0278] In one embodiment, the invention comprises a bispecific antibody according to the invention for use in the treatment of a tumor disease.

[0279] In one embodiment, the invention comprises a bispecific antibody according to the invention for use in the treatment of a tumor disease.

[0280] In one embodiment, the tumor disease is selected from the group consisting of colon cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer.

[0281] Another embodiment of the present invention is a pharmaceutical composition comprising said bispecific antibody according to the present invention.

[0282] In one embodiment, the present invention comprises a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a bispecific antibody according to the present invention or a pharmaceutical composition comprising the bispecific antibody.

[0283] The CD277 Mabs according to the present 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 a scFv molecule that specifically binds to a tumor antigen, and the antibody is in the form of a Mab-scFv. BRIEF DESCRIPTION OF THE DRAWINGS

[0285] Figure 1a : One embodiment of the structure of the bispecific antibody according to the present invention.

[0286] Figure 1b: Compared with the monospecific BTN3A antibody of the same sequence, the bispecific antibody against the BTN3A agonist (HC and LC of SEQ ID NO: 94 and 93, "second bispecific antibody") and the tumor antigen showed enhanced efficacy against cells carrying tumor antigens, but still bound to tumor antigen-negative cells with the same efficacy as the monospecific BTN3A antibody of the same BTN3A antibody sequence (see the figure below). This "second" bispecific antibody still causes non-specific activation of Vg9Vd2 cells in circulation and normal tissues. In contrast, compared with the "second" bispecific antibody against the BTN3A agonist, the bispecific antibodies of the present invention showed still strong efficacy against tumor antigen-positive cells, but showed lower efficacy in tumor antigen-negative cells. Therefore, and unexpectedly, the bispecific antibodies of the present invention show lower adverse side effects in treatment.

[0287] Figure 2: Activity of EvB#5 against FOLR1+ and FOLR1- tumor cells.

[0288] 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 and 10% fetal bovine serum). After overnight adhesion of tumor cells, cells were cultured with additional complete medium, in which the indicated concentrations of antibodies and short-term activated Vγ9Vδ2 T cells were in 10 IU / mL rIL-2 at an E / T ratio of 5:1. As a control for spontaneous lysis of tumor cells themselves, tumor cells in additional wells were cultured in medium with 12.5 IU / mL rIL-2 but without the addition of Vγ9Vδ2 T cells or antibodies ("SL", spontaneous lysis control). As a control for maximum lysis, tumor cells in other wells were cultured with short-term activated Vγ9Vδ2T cells at an E / T ratio of 5:1 in medium with 12.5IU / 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 Vγ9Vδ2T cells, tumor cells in other wells were cultured with short-term activated Vγ9Vδ2T cells at an E / T ratio of 5:1 in medium with 12.5IU / mL rIL-2 but without the addition of antibodies ("medium control"). The cell index (CI) was then measured every three minutes for 90h. The lysis of tumor cells at time point tx was calculated by the following formula:

[0289] Tumor cell lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100

[0290] Curve fitting was performed using a sigmoidal dose-response function, with the best fit value (Top value) for the maximum tumor cell lysis (tx) achieved with the reference antibody provided by Graphpad Prism 9. The % tumor cell lysis relative to the maximum tumor cell lysis ("Top") achieved with the reference antibody was calculated by the following formula:

[0291] % tumor cell lysis (tx) = tumor cell lysis (tx) / Top*100.

[0292] Figure 2a ) shows tumor cell lysis % of FOLR1+Ovcar-3 tumor cells at 24 h time point Figure 2b ) % tumor cell lysis of NCI-H1693 WT (FOLR1-) cells ± SD. EC50 values ​​of different constructs are shown. The bispecific antibodies according to the invention showed 50% killing of Ovcar-3 cells at a concentration of 0.12 nM. Background lysis ± SD at the 24 hour time point is indicated by the medium control. Figure 2c ) shows a comparison of the tumor cell lysis % of Ovcar-3 cells of the bispecific antibody in scFv format according to the present invention and the bispecific antibody in reverse format. (EvB#1: full-length bivalent antibody of the VH / VL combination of SEQ ID NO: 1 and 5 linked to two scFvs of the anti-tumor antigen antibody; EvB#8: full-length bivalent antibody of the VH / VL combination of the same anti-tumor antigen antibody linked to two scFvs of SEQ ID NO: 1 and 5 as VH / VL combinations. Figure 2d ) shows both forms.

[0293] Figure 3: Statistical analysis of lysis efficiency.

[0294] Statistical analysis showed that at a concentration of 0.1 nM, the bispecific antibody EvB#5 induced 48% lysis of the FOLR1-bearing cell line Ovcar-3 at an E / T ratio of 5:1 in the presence of activated Vγ9Vδ2 T lymphocytes ( Figure 3a ), whereas the bispecific antibody did not induce lysis significantly above background lysis ("medium control") in the same assay and under the same conditions in NCI-H1693 cells not carrying FOLR1 ( Figure 3b ). The significance of the differences was determined by unpaired t-test using Graphpad Prism 9 software, and the degree of significance is indicated:

[0295]

[0296] Therefore, the bispecific antibody according to the present invention enhances the cytotoxicity of Vγ9Vδ2T cells against FOLR1+Ovcar-3 but not against FOLR1-NCI-H1693 cells, while the reference antibody ("Ref.Ab") does not enhance the cytotoxicity of Vγ9Vδ2γδT cells against FOLR1+Ovcar-3 cells.

[0297] Figure 4: Activity of EvB#2 against NCI-H1693sgNT (WT control) and NCI-H1693sgNT with Antigen 1 knockout (clone 27).

[0298] 10.000 NCI-H1693sgNT (WT control) or clone 27 cells were cultured in complete medium. After overnight adhesion of tumor cells, cells were cultured with additional complete medium, in which antibodies at specified concentrations and short-term activated Vγ9Vδ2 T cells were cultured in 12.5 IU / mL rIL-2 at an E / T ratio of 5:1. As a control for spontaneous lysis of tumor cells themselves, tumor cells in other wells were cultured in medium with 12.5 IU / mL rIL-2 but without the addition of Vγ9Vδ2 T cells or antibodies ("SL", spontaneous lysis control). As a control for maximum lysis, tumor cells in other wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in medium 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 the background lysis of tumor cells by Vγ9Vδ2 T cells, tumor cells in other wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in a medium with 12.5 IU / mL rIL-2 but without the addition of antibodies ("medium control"). The cell index (CI) was then measured every three minutes for 90 h. The lysis of tumor cells at time point tx was calculated by the following formula:

[0299] Tumor cell lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100

[0300] Curve fitting was performed using a sigmoidal dose-response function, with the best fit value (Top value) for the maximum tumor cell lysis (tx) achieved with the reference antibody provided by Graphpad Prism 9. The % tumor cell lysis relative to the maximum tumor cell lysis ("Top") achieved with the reference antibody was calculated by the following formula:

[0301] %Tumor cell lysis (tx) = Tumor cell lysis (tx) / Top * 100

[0302] Figure 4a) shows tumor cell lysis % of Antigen 1+ NCI-H1693 sgNT tumor cells at 24 h time point Figure 4b ) shows % tumor cell lysis ± SD of Antigen 1-Clone 27 cells at 24 hours time point. EC50 values ​​of different constructs are shown. The bispecific antibodies according to the invention showed 50% killing of NCI-H1693sgNT cells at a concentration of 0.012 nM. Background lysis ± SD at 24 hours time point is indicated by the medium control.

[0303] Figure 5: Statistical analysis of lysis efficiency.

[0304] Statistical analysis showed that at a concentration of 0.01 nM, the bispecific antibody EvB#2 induced 61% lysis of the cell line NCI-H1693 sgNT carrying antigen 1 in the presence of activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1 ( Figure 5a ), whereas the bispecific antibody did not induce lysis significantly above background lysis in the same assay and under the same conditions in NCI-H1693 ko cells (clone 27) that do not carry the tumor antigen ( Figure 5b ). The significance of the differences was determined by unpaired t-test using Graphpad Prism 9 software, and the degree of significance is indicated:

[0305]

[0306] Therefore, the bispecific antibodies according to the present invention enhance Vδ2 + Cytotoxicity of γδ T cells against NCI-H1693sgNT but not against ko cells, while the reference antibody ("Ref.Ab") did not enhance Vδ2 + γδ T cell cytotoxicity against NCI-H1693sgNT cells carrying tumor antigen 1.

[0307] FIG. 6 : Activity of EvB#3 against STEAP1+ and STEAP1- tumor cells.

[0308] 10.000UMUC-3 (STEAP1+) or Ovcar-3 (STEAP1-) cells were cultured in complete medium. After overnight adhesion of tumor cells, cells were cultured with additional complete medium, in which the E / T ratio of the indicated concentrations of antibodies and short-term activated Vγ9Vδ2 T cells in 10 IU / mL rIL-2 was 5:1. As a control for spontaneous lysis of tumor cells themselves, tumor cells in other wells were cultured in medium with 12.5 IU / mL rIL-2 but without the addition of Vγ9Vδ2 T cells or antibodies ("SL", spontaneous lysis control). As a control for maximum lysis, tumor cells in other wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in medium 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 the background lysis of tumor cells by Vγ9Vδ2 T cells, tumor cells in other wells were cultured with short-term activated Vγ9Vδ2 T cells at an E / T ratio of 5:1 in a medium with 12.5 IU / mL rIL-2 but without the addition of antibodies ("medium control"). The cell index (CI) was then measured every three minutes for 90 h. The lysis of tumor cells at time point tx was calculated by the following formula:

[0309] Tumor cell lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100

[0310] Curve fitting was performed using a sigmoidal dose-response function, with the best fit value (Top value) for the maximum tumor cell lysis (tx) achieved with the reference antibody provided by Graphpad Prism 9. The % tumor cell lysis relative to the maximum tumor cell lysis ("Top") achieved with the reference antibody was calculated by the following formula:

[0311] %Tumor cell lysis (tx) = Tumor cell lysis (tx) / Top * 100

[0312] Figure 6a ) shows % tumor cell lysis of STEAP1+UMUC-3 tumor cells at 24 h time point Figure 6b ) shows % tumor cell lysis ± SD (tx) of STEAP1-OVCAR-3 tumor cells at 24 hour time point. EC50 values ​​of different constructs are shown. The bispecific antibodies according to the invention showed 50% killing of UMUC-3 cells at a concentration of 0.17 nM. Background lysis ± SD at 24 hour time point is indicated by the medium control.

[0313] Figure 7: Molecular cloning and tumor anchor cassette exchange.

[0314] Figure 8: shows % cell lysis of FOLR1+Ovcar-3 and FOL1R- tumor cells (see Figure 2 description). BTN3A agonist antibodies: reference antibody; EvB#5: bispecific antibody without CDR mutations, EvB#47 and EvB#52: bispecific antibodies with mutations, see e.g. Table 2).

[0315] Figure 9a : Vγ9Vδ2 T cell degranulation assay.

[0316] The degranulation of Vδ2T 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 a concentration 10 times higher than the effective concentration to reflect the higher drug levels in the primary distribution compartment after intravenous administration. The upper figure shows the significant degranulation of Vγ9Vδ2T cells from 4 different donors after activation with reference antibody 20.1 when compared with the surface levels of CD107a in the presence of antibody-free culture medium. The lower figure shows that, in the presence of the antibodies of the present invention, Vγ9Vδ2T cells from 4 different donors did not degranulate significantly when compared with the surface levels of CD107a in the presence of antibody-free culture medium.

[0317] Figure 9b : Vγ9Vδ2 T cell self-elimination assay.

[0318] After staining dead cells with SytoxGreen, self-elimination of Vδ2T cells in the absence of tumor antigen-positive cells was monitored by FACS analysis. Antibodies were applied at a concentration 10 times higher than the effective concentration to reflect higher drug levels in the primary distribution chamber after intravenous administration. The upper figure shows significant killing of Vδ2T cells from 4 different donors after activation with reference antibody 20.1 when compared to the percentage of dead Vδ2T cells in the presence of medium without antibodies. The lower figure shows that in the presence of antibodies of the present invention, Vδ2T cells from 4 different donors were not significantly killed. DETAILED DESCRIPTION OF THE INVENTION

[0320] The inventors have studied the cell lysis of tumor antigen positive and negative cells by agonistic murine anti-CD277 antibodies (parent antibody, 20.1) as described in Imbert C. et al., WO2012080351 and WO2012080769 and bispecific antibodies composed of said anti-CD277 antibodies and exemplary antibodies against tumor antigens in such bispecific antibodies. As shown in FIG9 , such bispecific antibodies in the form of Mab-scFv showed better tumor cell lysis than the corresponding monospecific anti-CD277 antibodies.

[0321] Surprisingly, the inventors have additionally found that a bispecific antibody in Mab-scFv format (comprising said parent anti-CD277 antibody with two point mutations in the CDR heavy chain CDRH2 (SEQ ID NO: 44) (also referred to as N53S, K58N or N5S and K10N in CDRH2 count) 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 and an anti-tumor antigen antibody without these mutations. This surprising effect is further improved by additional point mutations (L31V, L8V in CDRL1 count) in the light chain CDRL1 (e.g., SEQ ID NO: 75, 140, 141) of the anti-CD277 antibody portion. Therefore, the present invention provides such a bispecific antibody and a humanized form thereof.

[0322] In one embodiment, the antibody according to the invention is characterized in that it comprises, in addition to said CDRH2 substitution, a L8V substitution in CDRL1. In one embodiment, the antibody according to the invention is characterized in that it additionally comprises in CDRL1 the substitutions L8V and H1R.

[0323] As used herein, the term "activated Vγ9Vδ2T cells" according to the present invention means that Vγ9Vδ2T cells are activated by stimulation with the aminobisphosphonate (n-BP) zoledronic acid and addition of recombinant IL2 (rIL2); see Example 3.

[0324] 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 (L) chains and two identical heavy (H) chains. A full-length antibody is a monospecific bivalent antibody comprising a variable domain and a constant domain and an Fc portion. Typically, each light chain is connected to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. 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 its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain 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 constant heavy chain 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) in the N-terminal to C-terminal direction, 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) in the N-terminal to C-terminal direction, abbreviated as VL-CL. The antibody light chain constant domain (CL) can be κ (kappa) or λ (lambda). Specific amino acid residues are believed to form an interface between the light chain and 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)]. Based on the amino acid sequence of its constant region, the light chain of antibodies from any vertebrate species can be assigned to one of two clearly different types, called κ and λ. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0325] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0326] The term "humanized antibody or humanized form thereof" refers to an antibody in which the framework or "complementarity determining region" (CDR) has been modified to include a CDR of an immunoglobulin having a different specificity than the CDR of the parent immunoglobulin. In one embodiment, the murine CDR is transplanted into the framework region of a human antibody to prepare a "humanized antibody or form". See, e.g., 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*01V-KAPPA (X01668) or IGKV1-12*01V-KAPPA (V01577). In one embodiment encompassed by the invention, the constant region has been additionally modified or altered from that of the original antibody in order to produce the properties according to the invention, in particular with regard to Clq binding and / or Fc receptor (FcR) binding.

[0327] As used herein, the term "variable domain" refers to the region of an antibody that contains three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called a framework (FR). The variable domains of native heavy and light chains are each composed of four FR regions, most of which adopt a β-sheet configuration, connected by three CDRs, which form a loop connection and in some cases constitute a part of the β-sheet configuration. The CDRs in each chain are kept together in close proximity by the FR region, and together with the CDRs from 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 domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cellular toxicity.

[0328] As used herein, the term "Fc region" 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 human IgG heavy chain Fc region is generally defined as extending from the position of about Cys226 or from the amino acid residue at the position of about Pro230 to the carboxyl terminus of the Fc region (using the numbering system according to Kabat et al. (supra) herein). The Fc region of an immunoglobulin generally includes two constant domains, a CH2 domain and a CH3 domain, and optionally includes a CH4 domain (IgE).

[0329] "Fc region chain" herein means one of the two polypeptide chains of the Fc region.

[0330] The "CH2 domain" (also referred to as the "Cγ2" domain) of the human IgG Fc region generally extends from an amino acid residue at position about 231 to an amino acid residue at position about 340. The CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that carbohydrates can provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol. 22: 161-206 (1985). The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain.

[0331] The "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (from about amino acid residue 341 to about amino acid residue 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "protuberance" in one chain thereof and a corresponding introduced "cavity" in the other chain thereof; see U.S. Pat. No. 5,821,333).

[0332] The "hinge region" is generally defined as extending from about Glu216 or about Cys226 of human IgG1 to about Pro230 (Burton, Mol. Immunol. 22: 161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the SS bond between the heavy chains in the same position. The hinge region herein can 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, so that the two polypeptide chains of the variant hinge region can form a disulfide bond between the two chains. The preferred hinge region herein is a native sequence human hinge region, such as a native sequence human IgG1 hinge region.

[0333] A "functional Fc region" has 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; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be assessed using various assays known in the art for assessing such antibody effector functions.

[0334] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, and preferably from about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, more preferably at least about 95% sequence identity therewith.

[0335] While antibodies of the IgG4 subclass showed reduced Fc receptor (FcyRIIIa) binding, antibodies of other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297, Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues that, if altered, also provide reduced Fc receptor binding (Shields, RL, 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, the antibody according to the invention has reduced FcγR binding compared to an IgG1 antibody, and the full-length antibody is of IgG4 subclass or of IgG1 or IgG2 subclass with mutations in S228, L234, L235 and / or D265, and / or contains a PVA236 mutation. In one embodiment, the mutation in the full-length antibody is S228P, L234A, L235A, L235E and / or PVA236. In another embodiment, the mutation in the full-length antibody is S228P in IgG4 and L234A and L235A in IgG1.

[0336] In another embodiment, the antibody according to the invention is characterized in that the full length antibody is of human IgG1 subclass, or of human IgG1 subclass with mutations L234A and L235A. In another embodiment, the antibody according to the invention is characterized in that the full length antibody is of human IgG4 subclass or of human IgG4 subclass with 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.

[0337] The term "second binding moiety" refers to a single-chain Fv molecule. One identical single-chain Fv molecule is attached to each C-terminus of the Fc portion of the first binding moiety. Thus, the second binding moiety comprises two single-chain Fv molecules.

[0338] 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 connected from its C-terminus to the N-terminus of the variable domain of the heavy chain (VH) via a polypeptide chain. Alternatively, the scFv comprises a polypeptide chain in which the C-terminus of the VH is connected to the N-terminus of the VL via a polypeptide chain.

[0339] The term "peptide linker" or "linker" used in the present invention means a peptide having an amino acid sequence, which is 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, the peptide linker is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5 to 30, more preferably 10 to 20 amino acids in length. In one embodiment, the peptide connector is (GxS)n or (GxS)nGm, wherein 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 and n = 2 or 3, more preferably x = 4, n = 3. In one embodiment, the peptide connector is (G4S)3. Useful peptide linkers are also described in SEQ ID NO: 97-101.

[0340] The variable regions can be linked directly or usually via a linker peptide that allows for the formation of a functional antigen binding moiety. Typical peptide linkers comprise about 100A and are described herein or known in the art.

[0341] The scFv molecule can be further stabilized by a disulfide bridge between the heavy chain and light chain variable domains, for example as described by Reiter et al. (Nat. Biotechnol. 14, 1239-1245 (1996)). Thus, in one embodiment, the T cell activating bispecific antigen binding molecule of the 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 have been replaced with cysteine, so that a disulfide bridge can be formed between the heavy chain and light chain variable domains. 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 have been replaced with cysteine ​​(Kabat numbering).

[0342] 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).

[0343] In one embodiment, scFv can be replaced by a single-chain Fab fragment to improve production. A "single-chain 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, wherein the antibody domain and the linker have one of the following orders in the N-terminal to C-terminal direction: 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 wherein the 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 by a natural disulfide bond between the CL domain and the CH1 domain. 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.

[0344] The antigen-binding constructs described herein are bispecific, and in general embodiments, they comprise at least two antigen-binding polypeptide constructs, each of which is capable of specifically binding to two different antigens. The first binding moiety is a full-length bivalent antibody, and the second binding moiety is composed of two monovalent antibody fragments that do not contain an Fc portion. In a preferred embodiment, the two monovalent antibody fragments are in the form of scFv (i.e., antigen-binding domains composed 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.

[0345] Exemplary heavy chains of preferred Mab-scFv formats are shown (see also Figure 1a ) are shown in SEQ ID NO: 102 (DLL3) and SEQ ID NO: 103 (CLDN18.2).

[0346] "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 an antigen binding moiety to an unrelated protein, as measured by SPR, is less than about 10%, preferably less than 5%, of the binding of the antigen binding moiety to the antigen.

[0347] The term "EC50 ratio" according to the present invention means a ratio wherein the value for the bispecific antibody according to the present invention is the numerator (upper) and the value for the reference antibody is the denominator (lower).

[0348] In one embodiment, said second tumor antigen-negative cell line is said first cell line in which a tumor antigen is inactivated (knockout cell line; ko cell line).

[0349] In one embodiment of the invention, the bispecific antibody shows lysis of the first tumor antigen-positive cell line with an Emax ratio of 0.5 to 1.5 compared to the Emax of the reference antibody. Lysis is measured by monitoring the impedance of tumor cells (see Example 6).

[0350] As used herein, the term "does not induce lysis of human cells that do not carry the tumor antigen" refers to the lysis of tumor cells by the antibodies of the invention measured in the presence of 12.5 IU / mL interleukin-2 in the presence of activated Vγ9Vδ2 T lymphocytes at an E / T ratio of 5:1, which is not significantly different from background lysis (p value>0.05). Background lysis is measured in the same assay under the same conditions but without the addition of antibodies ("medium control").

[0351] As used herein, "CD277 binding" means binding to BTN3A1, BTN3A2 and / or BTN3A3.

[0352] "Affinity" refers to the strength of the interaction between a single binding site of a molecule (e.g., CD277) and its binding partner (e.g., anti-CD277 antibody), which is represented by the dissociation constant (kD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0353] As used herein, "affinity matured antibody" refers to an antibody having one or more changes in one or more CDRs thereof, which, compared to a parent antibody without those changes, results in a reduced affinity of the anti-CD277 antibody. Preferred affinity matured antibodies with reduced affinity will have an affinity in the nanomolar to micromolar range for CD277. 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, e.g., Tabasinezhada M. et al; Immunology Letters Volume 212, August 2019, Pages 106-113; 1. Georgiev, I Set al. J Immunol 192, 1100–1106 (2014).).

[0354] When used herein, the terms "agonist" and "agonism" refer to or describe molecules that can directly or indirectly substantially induce, promote or enhance the biological activity or activation of Vγ9Vδ2T cells (by promoting the formation of immune synapses with γδTCR). Optionally, an "agonist CD277 antibody" is an antibody that has the activity of achieving the above-mentioned Vγ9Vδ2T cell activation by binding and activation of CD277. Preferably, the agonist is a molecule that can activate human and cynomolgus monkey Vγ9Vδ2T cells. Even more preferably, the agonist is an antibody against CD277, and the antibody has an agonist activity that is 5 times less potent than antibody 20.1. The agonist activity of this antibody can be determined by the assay described in Example 6.

[0355] "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 the bispecific antibody (or the second binding moiety) according to the present invention to bind to a specific tumor antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, as measured, for example, by SPR, the degree of binding to an unrelated protein is less than about 10% of the binding of the bispecific antibody (or the second binding moiety) according to the present invention to a tumor antigen.

[0356] As used herein, the term "agonistic antibody that specifically binds to CD277" according to the present invention means that such antibodies activate the cell lysis function, cytokine production and proliferation of Vγ9 / Vδ2T cells. In one embodiment, as measured, for example, by SPR, the degree of binding to an unrelated protein is less than about 10% of the binding of the bispecific antibody (or second binding moiety) according to the present invention to a tumor antigen. In one embodiment, in a cell lysis assay as described in Example 6, in the absence of tumor cells carrying the corresponding tumor antigen, the bispecific antibody according to the present invention does not activate the cell lysis function, cytokine production and proliferation of Vγ9 / Vδ2T cells at a concentration of 5nM or less, in one embodiment 20nM or less.

[0357] As used herein, "tumor antigen knockout cell line or knockout cell line" refers to a tumor cell line that carries the corresponding tumor antigen in its wild-type form and in which the corresponding tumor antigen gene is inactivated. According to the present invention, CRISPR / Cas9 technology can be used to introduce a genetic variant of the gene, thereby inactivating the antigen expression.

[0358] The term "tumor antigen" means an antigen present on the surface of a tumor cell, including a tumor-specific antigen (TSA) and a tumor-associated antigen (TAA). In a preferred embodiment, the tumor antigen is Claudin18.2, FOLR1, STEAP, or DLL3. Other useful tumor antigens are described, for example, in Middleburg et al., Cancers (2021) 13, 287, pp4-6. Some tumor antigens such as FOLR1 are internalized after being bound by their 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).

[0359] It is therefore possible that the availability for recruiting Vγ9Vδ2 T cells is reduced and that the bispecific antibodies according to the invention are co-internalized, which in turn consumes the CD277-receptor on the cell surface. In this case, the receptor will not be further available to form an immunological synapse in contact with the Vγ9Vδ2 T cell receptor of an immune cell. Therefore, it is preferred according to the invention that the bispecific antibodies according to the invention bind to tumor antigens that are not internalized after binding of the corresponding antibodies or only to such an extent that the levels of tumor antigen and CD277 remaining after (co)internalization on the cell surface are still sufficient to trigger Vg9Vd2 T cell activation.

[0360] Preferably, a tumor antigen according to the present invention is selected because cells (tumor cells carrying antigens as well as antigen-negative cells) are treated with the corresponding bispecific antibody or reference antibody for 8 hours. Next, Vγ9Vδ2T cells are added, and tumor cell lysis% by surface-exposed and activated CD277 is measured as described in Example 6. The Emax value is obtained by curve fitting, and the Emax ratio of the bispecific antibody relative to the reference antibody is calculated for each cell line, respectively. The Emax ratio on tumor cells carrying antigens should not be less than half of the Emax ratio on cells that do not carry tumor antigens, indicating that the presence of tumor antigens does not cause more than 50% loss of activity due to co-internalization of CD277 by bispecific antibodies on antigen-carrying tumor cells.

[0361] As used herein, the term "Emax" refers to the response induced by any concentration of an antibody, or antigen-binding portion thereof, in an in vitro or in vivo assay, which is the maximal response.

[0362] As used herein, the term "EC50" refers to the concentration of an antibody, or antigen-binding portion thereof, which induces a response in an in vitro assay that is 50% of the maximal response, ie, halfway between the maximal response and the baseline.

[0363] As used herein, the term "KD or K D ” refers to the equilibrium dissociation constant of the binding reaction between antibody and antigen.

[0364] Antibodies according to the present invention are produced by recombinant means. Therefore, one embodiment of the present invention is a nucleic acid encoding an antibody according to the present invention, and another embodiment is a cell comprising the nucleic acid encoding an antibody according to the present invention. The method for recombinant production is widely known in the prior art, and is included in prokaryotic and eukaryotic cells expressing proteins, followed by separation of antibodies and usually purification to a pharmaceutically acceptable purity. In order to express the antibodies of the present invention in host cells, nucleic acids encoding corresponding modified light chains and heavy chains are inserted into expression vectors by standard methods. Expressed in appropriate prokaryotic or eukaryotic host cells such as Chinese hamster ovary celIs, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C 6 cells, yeast or Escherichia coli cells, and antibodies are recovered from cells (cells after supernatant or lysis). General methods for recombinant production of antibodies are well known in the art and are described in, for example, the literature reviews 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.

[0365] The bispecific antibodies according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography 35. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures.

[0366] The term "host cell" used in the present application refers to any kind of cell system that can be engineered to produce the antibodies according to the present invention. In one embodiment, HEK293 cells and CHO cells are used as host cells.

[0367] 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 preparing a pharmaceutical composition. Another aspect of the invention is a method for making a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the invention provides a composition, such as a pharmaceutical composition, containing an antibody according to the invention formulated with a pharmaceutical carrier.

[0368] One embodiment of the invention is a bispecific antibody according to the invention for use in the treatment of cancer (tumor diseases).

[0369] Another aspect of the present invention is the pharmaceutical composition for use in the treatment of cancer.

[0370] Another aspect of the invention is the use of an antibody according to the invention in the preparation of a medicament for the treatment of cancer.

[0371] 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.

[0372] Another aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention.

[0373] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0374] The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and / or mode will vary according to the desired result. In order to administer the compounds of the present invention by certain routes of administration, it may be necessary to coat the compound with a material to prevent its inactivation or to co-administer the compound with a material to prevent its inactivation. For example, the compound can be administered to the subject in an appropriate carrier (e.g., liposomes or diluents). Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injections or dispersions. It is known in the art to use such media and reagents for pharmaceutically active substances.

[0375] The term cancer as used herein refers to a proliferative disease such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, Hodgkin's disease.

[0376] In one aspect, the cancer (neoplastic disease) is selected from colon cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer and breast cancer.

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384] In the sequence listing, the corresponding CDRs are listed after each VH or VL. Some VHs and VLs contain one or more identical CDRs:

[0385] SEQ ID NO: 2, which is identical to SEQ ID NO: 43, 59, 63, 71, 109.

[0386] SEQ ID NO:4, which is identical to SEQ ID NO:45, 49, 53, 57, 61, 65, 69, 73, 107, 111, 115.

[0387] SEQ ID NO:7, which is identical to SEQ ID NO:80, 84, 88, 118, 122, 126, 130, 134,

[0388] SEQ ID NO:8, which is identical to SEQ ID NO:119, 89, 123, 127, 131, 135, 139.

[0389] SEQ ID NO:44, which is identical to SEQ ID NO:64.

[0390] SEQ ID NO:47, single SEQ.

[0391] SEQ ID NO:51, which is identical to SEQ ID NO:54.

[0392] SEQ ID NO:67, single SEQ.

[0393] SEQ ID NO:75, which is identical to SEQ ID NO:79, 87.

[0394] SEQ ID NO:105, which is identical to SEQ ID NO:113.

[0395] SEQ ID NO: 138, single SEQ.

[0396] Materials and methods

[0397] Cell culture, transfection, antibody production and purification: CHO-S cells (FreeStyle TM , Thermo Fisher Scientific) was maintained at 37°C and 5% CO2 under constant shaking (120 rpm) The cells were maintained in Erlenmeyer flasks (125 mL, Corning). The cells were maintained in CD CHO growth medium (1% (v / v) GlutaMAX 100x and 1% (v / v) HT supplement 100x (Thermo Fisher Scientific). / Thermo Fisher Scientific). Every other day, the cells were adjusted to 0.3×10 6 The day before transfection, cells were plated at a density of 2 × 10 cells / ml to maintain exponential growth. 6 The cells were seeded at a density of 4 × 10 cells / ml on the day of transfection. 6 According to the manufacturer's instructions, use MaxCyte STX TM Transfection was performed using the MaxCyte Transfection Unit (MaxCyte, Gaithersburg, MD, USA). The MaxCyte Processing Assembly OC-400 and an optimized transfection protocol for protein production in CHO-S cells were used. A total of 8 × 10 8 cells and divided into 8×10 7 After washing twice with 4 mL of electroporation (EP) buffer, the cells were resuspended in EP buffer to obtain 8 × 10 7 Cells were plated at a density of 10 cells / 400 μL. A total of 300 μg / ml plasmid DNA or a single vector for expression of surface antigens was added at a 1:1 heavy chain:light chain ratio. After electroporation, CHO-S cells were directly seeded in culture flasks without adding any additional buffer or medium and incubated at 37°C and 5% CO2 for 30 minutes. Post-transfection culture conditions differed between protein production and transient expression of surface markers:

[0398] Surface receptor expression: Cells were maintained in CD CHO growth medium for 48 hours before FACS analysis.

[0399] Antibody production: Add 150 mL of production medium (CD OptiCHO TM +1%(v / v)GlutaMAX 100x+1%(v / v)HT Supplement 100x+1%(v / v)Pluronic TM F-68 100x, both / Thermo Fisher Scientific). One day after transfection, 1 mM sodium butyrate (Thermo Fisher Scientific) was added and 3.5% (v / v) MaxCyte stock solution (28 mL yeast extract 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, / Thermo Fisher Scientific). For the remainder of the production cycle (14 days or until cell viability drops below 50%), the incubation temperature is reduced to 32°C. During the production phase, cell density and viability are measured every other day, and cells are fed daily with MaxCyte feedstock (see above) until production is stopped. The supernatant is harvested by centrifugation and filtered to remove cell debris (final volume approximately 200 ml). In the first step, the supernatant is harvested with CaptureSelect TM Affinity chromatography was performed using CH1-XL (Hu) affinity matrix (Thermo Fisher Scientific). Briefly, 1 ml of beads were added and stirred slowly overnight at 4 ° C. The beads were washed 3 times with 10 ml of PBS in a gravity flow column. 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 a total of 3 times with 2 L of PBS at 4 ° C. Then, size exclusion chromatography ( Pure 25, GE Healthcare Life Science) to isolate monomeric antibodies. 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.

[0400] Flow cytometry: 0.5×10 6Cells were used for individual staining reactions. The cells were washed once in 1 ml PBA (PBS, 1% BSA, 0.05% NaN3). The cell pellet was resuspended with 50 μL purified recombinant protein diluted in PBA at a concentration of 50 μg / ml. The cells were incubated on ice for 30 min. The cells were washed twice in 1 ml PBA. The cell pellet was then resuspended in 25 μL 1:20 diluted anti-human-IgGFITC (Jackson Immuno Research, catalog number: 109-096-098) and incubated on ice for 30 min in the dark. The cells were then washed twice in 1 ml PBA. Finally, the cells were resuspended in 500 μL PBA and immediately analyzed on a Navios flow cytometer (Beckman Coulter). Example

[0401] Example 1: Cloning of bispecific antibodies (Figure 5)

[0402] Bispecific CD277 antibodies were prepared according to the following procedure:

[0403] Expression vectors for producing IgG-scFv molecules were designed by standard procedures [Kellner, CS. et al.; Methods Mol Biol, 2018. 1827: p. 381-397]. pSEC-Tag2-Hygro-C was used as a backbone for producing mammalian expression vectors. IgG-scFv antibody derivatives were designed based on a modified form of the prototype IgG-scFv format originally described by Coloma, MJ et al.; Nat Biotechnol, 1997. 15 (2): p. 159-63.

[0404] Light chain: Light chain design was as described by Kellner, CS et al., supra. For the light chain expression cassette, a secretion 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-κ region was fused at the 3'-end to form a complete κ 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 into the 5'- and 3'-ends, respectively. Cloning in the vector backbone was performed according to standard procedures.

[0405] Heavy chain derivatives: Heavy chain derivatives encode IgG1 heavy chains that carry L234A and L235A amino acid exchanges in the lower hinge region to prevent Fc receptor interactions (Lund, JG. et al.; J Immunol, 1991. 147 (8): p. 2657-62). The heavy chain secretory leader sequence is 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 is added upstream of the start codon to allow optimal initiation of translation. The stop codon of the IgG heavy chain is removed and a sequence encoding a 15 amino acid flexible linker (G4S) 3 (SEQ ID NO: 97) is introduced. The last two codons of the flexible linker (GS) have a BamHI restriction site at the DNA level, followed by a PmeI restriction site. Each scFv fragment was designed into the VL-(G4S)4-VH format as a BamHI-PmeI cloning cassette. Cloning of the final expression construct was performed according to standard procedures.

[0406] In the heavy chain derivatives, additional restriction sites that do not affect the amino acid composition were introduced to allow modular design and exchange of specific parts of the molecule:

[0407] NheI-PpuMI: exchange of VH regions.

[0408] PpuMI-BsrGI: exchange of silent mutations in the CH2 domain.

[0409] BsrGI-BamHI: exchange of linker sequences.

[0410] BamHI-PmeI: Exchange of scFv fragments.

[0411] Table 2a: Bispecific CD277 antibodies comprising a parent anti-CD277 antibody as a first binding moiety

[0412]

[0413] Table 2b: Bispecific CD277 antibodies comprising antibody 47 as the first binding moiety

[0414]

[0415] Table 2c: Bispecific CD277 antibodies comprising antibody 52 as the first binding moiety

[0416]

[0417] Antibody 47: VL parent, VH CDR2 N185S, K190N, all other VH / VL CDRs identical to parent

[0418] Antibody 52: VL CDR1 L31V, VH CDR2 N185S, K190N, all other VH / VL CDRs identical to parent

[0419] Example 2: Generation of humanized bispecific antibodies

[0420] The affinity maturation of antibodies is a stepwise process during the immune response. As described by Rajewsky and colleagues in 1988, by accumulating additional mutations compared to germline in the CDR region and undergoing a rigorous selection process, antibody affinity can be gradually increased (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. Therefore, by gradually restoring the germline configuration in the light chain and heavy chain variable region, humanized antibodies of the present invention can be produced.

[0421] Humanization method

[0422] Humanization of mouse monoclonal antibodies is performed using standard CDR-grafting techniques. The principle of this method is to reshape human antibodies containing only complementary determining regions (CDRs) from mouse monoclonal antibodies, with the aim of reducing immunogenicity when used as a therapeutic agent for people. Humanization by CDR grafting requires that the antigen-binding residues from mouse antibodies be retained in the humanized antibody; therefore, the identification of these residues obviously plays an important role in the scheme. In order to guide the humanization process and help decide whether to retain the parent mouse residues or replace them with their human germline counterparts, the 4F9L X-ray structure of the mouse monoclonal antibody scFv with its antigen BTN3A1 is used.

[0423] The CDR transplantation scheme used is a modernized version of the method that Greg Winter and his colleagues pioneered at the Medical Research Council (Medical Research Council) in Cambridge, UK. The definition of CDR is based on the Kabat nomenclature. The selection of the mouse monoclonal antibody mouse CDR region transplanted to the human framework acceptor region therein is completed by searching the IMGT mouse and human V gene database using IgBLAST, which is developed at NCBI to promote the analysis (http: / / www.ncbi.nlm.nih.gov / igblast / ) of immunoglobulin V region sequences, wherein the mouse monoclonal antibody mouse variable region sequence is used as input. The strategy used is to use human germline sequences, which are natural human sequences that do not contain the specific somatic mutations found in the single human antibody sequence.

[0424] Light chain back mutation: The variable region of the light chain of the first binding moiety from the parental antibody was compared to the germline library of the mouse (IMGT database), and the germline genes showing the closest homology were identified. From this, the IGKV15-103*01 and IGKJ2*01[F] genes were identified and aligned with the parental VL region. Six amino acid residues were identified that differed from the germline. By interpreting the residues identified in the crystal structure of the parental antibody, amino acid residues that were surface exposed and therefore likely to directly contribute to antigen interactions were identified. Single amino acids or amino acid clusters were converted to the germline configuration and used to generate expression constructs.

[0425] Heavy chain back mutation: A similar strategy was applied to identify potential amino acid positions in the heavy chain variable region. IGHV1S81*02[F]v-gene was identified as the closest match. 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 used to reset the VL region to the germline, mutations in the heavy chain CDR1 and CDR2 regions were restored to the germline configuration (single mutation or cluster) in a stepwise manner. In order to identify residues in CDR3, a different strategy was applied because homologous germline gene segments could not be identified. Here, surface exposed residues in the CDR3 region were identified by analyzing the co-crystal structure of 20.1 and 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.; ibid.) and are therefore converted to alanine. Alanine exchange was chosen because alanine scanning has been described to identify residues critical in epitope binding by disrupting antibody / antigen interactions (Parhami-Seren, BM. et al; J Immunol, 2001. 167(9): p.5129-35). Single amino acids or amino acid clusters were converted to germline configuration and used to generate expression constructs.

[0426] Table 3: First binding moiety of the bispecific antibodies according to the invention

[0427]

[0428]

[0429]

[0430] Cluster 1: R162S, Y164W, L165M, Y166H

[0431] Cluster 2: N185S, G188R, K190N, F191Y

[0432] Cluster 1-2: R162S, Y164W, L165M, Y166H, N185S, G188R, K190N, F191Y

[0433] The heavy and light variable chain sets of the bispecific antibodies according to the present invention are defined as two chains in one row in the table. "R162S" means that the amino acid R at position 162 is replaced by the amino acid S. N185S means that the asparagine at position 185 is replaced by serine. N185S and K190N are bold and underlined in SEQ ID NO:44. L31V is bold and underlined in SEQ ID NO:75, with the corresponding meanings for all other similar terms. The counting of other amino acids in the variable chain can start with N185 and L31.

[0434] Example 3: Generation and characterization of an activated Vγ9Vδ2 T cell line.

[0435] To generate expanded γδ T cell lines, 10% of leukocyte concentrate (LRS) from healthy adult blood donors was added to 6 Cells / mL were cultured in 6-well plates in complete medium with 50 IU / mL rIL-2 (Novartis, Basel, Switzerland) and stimulated with 2.5 μM aminobisphosphonate (n-BP) zoledronic acid (Novartis), which induces the selective growth of γδT cells expressing Vγ9Vδ2. Due to rest, the initially stimulated γδT cells only produce very low amounts of IL-2, so 50 IU / mL (15 pg / mL) rIL2 (Oberg et al., Cancer Res. 2014) was added every other day. After two weeks, selective expansion of γδT cells expressing the Vδ2 chain with a purity of >94% was observed. Vδ2T cell activation was indicated by slightly enhanced CD25 expression (Pechhold et al. J Immunol Baltim Md 1950 152, 4984–92 (1994)) and strong upregulation of the activation marker CD69. Additionally, the increased Vδ2 T cell population revealed central memory- (CM, CD27+CD45RA-) or effector memory (EM, CD27-CD45RA-) phenotypes, demonstrating the activation of these expanded γδ T cells.

[0436] Example 4: Screening of tumor cell lines

[0437] To test different antibody constructs, a panel of different tumor cell lines expressing the corresponding tumor antigens was selected based on published information on antigen expression levels or FACS analysis. Briefly, for surface staining, 3 to 5 × 10 5Cells were added twice. After that, cells were stained with fluorescent dye-conjugated or unconjugated antibodies or isotype controls for 25 minutes according to the procedures outlined by the manufacturer, washed twice and resuspended in 1% PFA (paraformaldehyde) solution in PBS buffer or stained with the second step antibody. After incubation with the second step, the antibody cells were washed twice and resuspended in 1% PFA buffer. All samples were analyzed on a LSR-Fortessa flow cytometer (BD Biosciences) using Diva 9 and FlowJo software. The results of the literature and FACS analysis are summarized in Table 4.

[0438] Table 4: Expression of tumor antigens on tumor cells

[0439]

[0440] * = Not disclosed

[0441] Literature: 1. Payne, KKet al. Science 369,942–949 (2020); 2. Ebel, W. et al. Cancer Immun7, 6 (2007); 3. Shivange, G. et al. Cancer Cell 34, 331-345.e11 (2018); 4. Shimizu, T., et al., Oncoimmunology 7, e1424671 (2018); 5. Gomes, IM, et al., Mol Cancer Res 10, 573–587 (2012); 6. Türeci, et al., Oncoimmunology 8, e1523096 (2018); 7. Hipp, S. et al. Clin Cancer Res Official J Am Assoc Cancer Res 26, 5258–5268 (2020); Benyamine, A. et al. Oncoimmunology 7, 00–00 (2017).

[0442] Example 5: Generation of knockout tumor cell lines

[0443] Generation of tumor antigen KO cells by RNP transfection:Guide RNA (gRNA) was prepared by combining the corresponding crRNA and tracrRNA of equimolar concentration (100 μM), annealing at 95 ° C for 5 minutes and renaturing at room temperature. RNPs were then prepared by combining gRNA and recombinant SpCas9 protein in PBS and incubating at room temperature for 15 minutes. RNPs were 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 subsequently generated by FACS sorting (BD Aria), amplified, and verified by flow cytometry staining and amplicon sequencing (NGS).

[0444] Generation of tumor antigen KO cells by lentiviral transfection: First, the parental cells were transduced with Cas9-p2A-blasticidin-lentivirus and selected with blasticidin to achieve stable expression of Cas9. The corresponding guide RNA was then cloned into the CROP-seq-Guide-Puro plasmid (Addgene#86708) and a lentivirus was produced. The cells expressing Cas-9 selected by blasticidin were then transduced with the lentivirus, the cells were selected with puromycin, amplified, and verified by flow cytometry staining and amplicon sequencing (NGS). As a control, the same transfection protocol was applied using a non-targeted guide RNA ("sgNT") that produced the corresponding sgNT cell line. The suitable guide RNA sequence for generating FOLR1 KO cells is SEQ ID NO:96.

[0445] Example 6: Cell lysis assay

[0446] Cytotoxicity against tumor cell lines such as OVCAR-3 (ovarian cancer), NCI-H1693 (NSCLC), or UM-UC-3 (bladder cancer) was determined in triplicate by real-time cell analyzer (RTCA, X-Celligence, ACEA Biosciences, San Diego, CA, USA) as described elsewhere (Oberg et al., 2014 and 2020). Briefly, 7.5-10 × 10 cells / mL in complete culture medium RPMI1640 (supplemented with 25 mM HEPES, 2 mM L-glutamine, 100 μg / mL streptomycin, 100 U / mL penicillin, and 10% fetal bovine serum) were added to 1% paraformaldehyde (P < 0.05). 3Adherent tumor cells / well are added to a 96-well micro-E plate, and the impedance of the tumor cells is monitored by an electronic sensor every five minutes for up to 24-40h. The measured impedance of the tumor cells is expressed as an arbitrary unit called cell index ("CI"), which reflects changes in cell parameters, such as morphological changes (e.g., adhesion, diffusion), cell proliferation, and cell lysis. Since the initial adhesion of tumor cells in different wells may be slightly different, the CI can be normalized to 1 after the tumor cancer cells reach their linear growth phase. When the linear growth rate is reached after 24-40 hours, activated Vγ9Vδ2T lymphocytes with an E / T ratio of 5:1 and culture medium containing 12.5IU / mL rIL-2 and various antibody constructs or various controls at specified concentrations are added ("experimental start", t = 0h). For control, tumor cells are treated with a final concentration of 1% Triton X-100 in several wells as a positive control for complete lysis, and tumor cells are treated with activated Vγ9Vδ2T lymphocytes (same conditions as above) in several other wells as a control for background lysis. Lysis of adherent tumor cells was monitored by measuring the normalized CI at different time points for at least 3 min.

[0447] By using RTCA software (ACEA Biosciences), the raw data files were exported to Microsoft Excel or Graph Pad Prism for further evaluation. The average CI of Triton-X-100 samples and Vγ9Vδ2 T lymphocytes without antibody addition was calculated at the specified time points after the start of the experiment and defined as complete lysis ("TritonX100") and background lysis ("medium control"), respectively. Tumor cell lysis induced by the antibody construct was calculated for each sample at the same time point as the tumor cells ("tx"):

[0448] Lysis (tx) = (CI (tx) - medium control (tx)) / (Triton X100 - medium control (tx)) * 100

[0449] Curve fitting was performed using a sigmoidal dose-response function, with Graphpad Prism 9 providing the best fit value (Top value) for the maximum tumor cell lysis (tx) achieved with the reference antibody. 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

[0450] 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.

[0451] Example 7: SPR assay

[0452] SPR assays were performed according to the prior art. The results for the reference antibodies are shown in Table 5. In brief, recombinant CD277 was immobilized on the surface of a Biacore CM5 optical sensor chip by covalent EDC / NHS coupling following the Biacore amine coupling kit protocol. Antibody samples were applied as serially diluted analytes, allowing for standardized comparison of all antibodies bound to the same target molecule surface. Kinetic analysis data were based on a 1:1 Langmuir curve fitting model and average Langmuir association rate, dissociation rate and KD values: see Table 5.

[0453] Table 5

[0454] VL modification VH modification ka kd KD(nM) none none 3,69±0,54E+4 1,04±0,20E-4 2,80±0,31 none N53S,K58N 1,58±0,22E+4 4,09±0,48E-4 26,30±4,72 L31V N53S,K58N 2,79±0,13E+4 3,85±0,23E-4 13,90±1,33

[0455] E+4 means 10 4 , E-4 means 10 -4

[0456] Example 8: Degranulation and cell death assays

[0457] Principle: Cytotoxic T cells such as γδ T cells store cytotoxic mediators such as granzymes, perforins, and granulysin in secretory lysosomes. Lysosomal-associated membrane glycoproteins (LAMPs) such as LAMP-1 (CD107a) and LAMP-2 (CD107b) are embedded in the lipid bilayer membrane of secretory lysosomes. After T cell activation, secretory lysosomes can move toward the cell membrane and fuse with it. After fusion, LAMPs are transiently expressed on the cell surface of T cells, and secretory lysosomes degranulate their granule contents.

[0458] 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, 10% fetal bovine serum under conventional conditions (5% CO2, humidified, 37°C). γδ T cells supplemented with 12.5 U / mL IL-2 were cultured for six hours in 96-well microtiter plates (Nunc, Wiesbaden) with medium, 300 nM bromohydrin pyrophosphate, different concentrations of constructs or with control construct AV#75. For CD107 assay, 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 three hours after culturing cells. After another 3 h, γδ T cells were washed and stained with PerCP-labeled anti-CD45 mAb (clone 2D1, BD Biosciences), AlexaF700-labeled anti-CD3 mAb (clone SK7, Biolegend), BV510-labeled anti-CD8 mAb (clone SK1, BD Biosciences), PE-Cy7-labeled anti-TCRγδ mAb (clone 11F2, BD Biosciences), and APC-Vio770-labeled anti-Vδ2 (clone REA771, Miltenyi), washed, and incubated in 4% paraformaldehyde containing SYTOX TM Green Dead Cell Stain (1:4000, ThermoScientific, #S34860) was then absorbed in PBS for 20 minutes. The cells were then analyzed by flow cytometry (LSR Fortessa, BD Biosciences). The results for γδ T cells from 4 different donors are shown in FIG9 .

[0459]

[0460]

Claims

1. A bispecific antibody comprising a first binding portion that specifically and agonistically binds to human CD277 and a second binding portion that specifically binds to a tumor antigen, characterized in that 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 which is linked to each C-terminus of the first binding moiety via a peptide linker.

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

3. The bispecific antibody according to claim 1 or 2, characterized in that: The first binding moiety comprises CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44 and CDRH3 of SEQ ID NO:45 as heavy chain CDR sequences and CDRL1 of SEQ ID NO:6, CDRL2 of SEQ ID NO:7 and CDRL3 of SEQ ID NO:8 as light chain CDR sequences.

4. The bispecific antibody according to claim 3, characterized in that The CDRH2 of SEQ ID NO:44 is replaced by SEQ ID NO:68, SEQ ID NO:72 or SEQ ID NO:

110.

5. The bispecific antibody according to claim 3 or 4, characterized in that: The CDRL1 of SEQ ID NO:6 is replaced by SEQ ID NO:75, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:140 or SEQ ID NO:

141.

6. The bispecific antibody according to claim 1, characterized in that comprising CDRH1 of SEQ ID NO:43, CDRH2 of SEQ ID NO:44, and CDRH3 of SEQ ID NO:45 as heavy chain CDR sequences, and b) a CDR set 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.

7. The bispecific antibody according to any one of claims 1 to 6, characterized in that The tumor antigen is selected from the group consisting of: CLDN18.2, FOLR1, STEAP1 and DLL3.

8. The bispecific antibody according to claim 7, characterized in that For the second binding moiety, the variable light chain CDR and the variable heavy chain CDR are a) for FOLR1 as the tumor antigen, CDRL1 of SEQ ID NO: 11, CDRL2 of SEQ ID NO: 12, and CDRL3 of SEQ ID NO: 13, and the variable heavy chain CDRs are CDRH1 of SEQ ID NO: 15, CDRH2 of SEQ ID NO: 16, and CDRH3 of SEQ ID NO: 17, b) for STEAP1 as a tumor antigen, CDRL1 of SEQ ID NO: 19, CDRL2 of SEQ ID NO: 20, 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, c) for DLL3 as a tumor antigen, 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, d) For CLDN18.2 as a tumor antigen, 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.

9. The bispecific antibody according to claim 1, 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:74, SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:

86.

10. The bispecific antibody according to claim 9, characterized in that 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. The bispecific antibody according to any one of claims 1 to 10, characterized in that The first binding portion of the antibody according to the present invention is a humanized antibody or a CDR-grafted antibody.

12. The bispecific antibody according to any one of claims 1 to 11, characterized in that The scFv is bound to the C-terminus in the orientation of peptide linker 1-VL-peptide linker 2-VH.

13. The bispecific antibody according to claim 12, characterized in that: The first peptide linker consists of 5-25 amino acids, and the second peptide linker consists of 10-25 amino acids.

14. The bispecific antibody according to any one of claims 1 to 13, for use in the treatment of tumor diseases.

15. The bispecific antibody according to any one of claims 1 to 13, 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. 16 . A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 13 .

17. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the bispecific antibody according to any one of claims 1 to 13.

18. A recombinant nucleic acid sequence encoding the bispecific antibody according to any one of claims 1 to 13.

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