Multispecific binding proteins for cancer therapy

Abstract

The present invention relates to novel multispecific binding proteins for cancer therapy, i.e. B7H6 / CD3 binding proteins. The invention further relates to nucleic acids encoding said proteins; methods for the production of said proteins; host cells expressing or capable of expressing said proteins; compositions comprising said proteins; and uses of said proteins or said compositions, in particular in the field of cancer diseases for therapeutic purposes.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080068909.4, filed on October 1, 2020, entitled "Multispecific binding protein for cancer treatment". Technical Field

[0002] This invention relates to a multispecific binding protein comprising a first antigen-binding unit specific to B7H6 (also referred to herein as "B7-H6") and a second antigen-binding unit specific to CD3. The invention also relates to nucleic acids encoding said binding protein, methods for preparing said binding protein; host cells expressing or capable of expressing said binding protein, compositions comprising said binding protein, and uses of said binding protein or said compositions, particularly for therapeutic purposes in the field of cancer. Background Technology

[0003] B7H6 is a tumor-selective member of the B7 family, described for attracting the innate immune system to target cells and exhibiting similar functions to other B7 family members. It possesses two Ig-like domains within its extracellular domain: an N-terminal IgV-like domain and a C-terminal IgC1-like domain. B7H6 triggers NKp30-mediated activation of human natural killer (NK) cells, leading to degranulation and IFNγ secretion (Brandt et al., J. Exp. Med. 2009 206(7); 1495-1503). Currently available data suggest a role for B7H6 in inflammatory responses to infectious conditions and in solid tumors.

[0004] It has been shown that, due to the inflammatory process in this acute illness state, B7H6 is present in CD14 isolated from the peripheral blood of patients with sepsis. + CD16 + cellular expression. These findings have been confirmed by in vitro analysis of CD14 expression upon stimulation with IL-1β and TNFα. + CD16 + The upregulation of B7H6 on the cell surface of pro-inflammatory monocytes and neutrophils has been confirmed (Matta et al., Blood 2013 122(3)), indicating the role of B7H6 in the inflammatory response to sepsis.

[0005] In addition to the sepsis condition mentioned above, B7H6 is selectively expressed in tumor cells in other ways and cannot be detected in normal human tissues at homeostasis. For example, B7H6 has been described as effective against T-cell lymphoma, myeloid leukemia, colon cancer, breast cancer, and ovarian cancer cell lines (Brandt et al., J. Exp. Med. 2009 206(7):1495-1503; Li et al., J. Exp. Med. 2011 208(4); Greaves et al., Blood 2013 121(5); Zhang et al., Oncology Letters 2018 16:91-96), non-small cell lung cancer tissues (Zhang et al., Int J clin Exp Pathol 2014; 7(10):6936-6942), gastrointestinal tumor tissues (Chen et al., Pathol. Oncol. Res. 2014 20:203-207; Zhao et al., Cell Proliferation 2018; e12468), and ovarian cancer tissues (Zhou et al., Int clin Exp Pathol). The expression of B7H6 in tumors has been observed in 2015 8(8), oral squamous cell carcinoma (Wang et al., J Oral Pathol Med. 2017;46:766-772) and hepatocellular carcinoma (Li et al., Int. J. Mol. Sci. 2019, 20, 156), but the function of B7H6 in tumors is not fully understood.

[0006] Therapeutic applications, including the use of anti-B7H6 antibodies or anti-B7H6 antibody-drug conjugates involved in the ADCC / CDC pathway, are described in WO2009 / 046407A2 and WO2011 / 07044A2 for the treatment of cancer.

[0007] However, B7H6-targeted therapy based on ADCC / CDC activity is not the optimal mode of action because B7H6 is poorly expressed on the cell surface and the success rate of using conventional antibodies with ADCC / CDC activity in solid tumors is low.

[0008] Targeted therapies based on B7H6-specific antibody-drug conjugates (ADCs) may also have limitations, as most patients relapse after chemotherapy due to low expression of B7H6 on the cell surface. Furthermore, ADC methods often exhibit off-target toxicity caused by free drug, a result of connective instability or degradation.

[0009] CAR-T cells and T-cell adaptor antibodies are alternative approaches to targeted therapy for solid tumors expressing B7H6 (Wu et al., Gene 2015 22, 675-684; Hua et al., Protein Engineering, Design & Selection 2017 30(10), 713-721; WO 2017 / 181001). For example, Wu et al. (J Immunol. 2015 1 6 1; 194(11):5305-11) described preclinical data for B7H6-specific BiTE, which stands for bispecific T-cell adaptor, a fusion protein of approximately 55 kDa consisting of two single-chain variable fragments (scFv). In this context, B7H6-specific BiTE was engineered based on the OKT3-CD3-adhesive and the previously disclosed B7H6 antibody (Zhang et al., J Immunol. 2012 Sep 1; 189(5):2290-9; WO 2013 / 169691). However, the OKT3 antibody does not cross-react with cynomolgus monkey CD3, and therefore preclinical toxicology testing is not permitted in cynomolgus monkeys, which are the preferred test species for preparing clinical trials (Chatenoud et al., The Rev Diabet Stud 2012; 9(4):372-381). Another challenge is the short half-life of the relatively small and easily degraded BiTE molecule, which requires continuous intravenous administration in clinical trials. Therefore, the success of this approach has not been demonstrated. To date, there are no targeted therapies for tumors expressing B7-H6, and unmet needs remain that are not addressed by current approaches.

[0010] For example, colorectal cancer (CRC) shows a high prevalence and predictable expression of B7-H6. It is one of the leading causes of cancer morbidity and mortality worldwide. Approximately 25% of CRC patients initially present with obvious metastases, and metastatic disease develops in 40-50% of newly diagnosed patients. Although recent improvements in chemotherapy and targeted therapy have prolonged the survival of metastatic CRC patients, most patients will still die from the disease.

[0011] Given the poor prognosis for cancer patients with advanced disease, there is a need to identify more effective treatments, especially effective treatments with improved tolerability.

[0012] Therefore, the object of the present invention is to provide pharmacologically active agents, compositions, and / or methods that offer certain advantages compared to currently used and / or known agents, compositions, and / or methods in the art. These advantages include, particularly compared to candidate drugs known in the art, improved therapeutic and pharmacological properties, such as in vivo efficacy, fewer side effects, reduced immunogenicity, improved therapeutic window, reduced frequency of administration (e.g., infusion), lower dosage, extended half-life to allow for less frequent dosing, and other advantageous properties such as improved ease of preparation, stability, compatibility with conventional antibody methods, or reduced commercial costs. Summary of the Invention

[0013] This invention is based on a bispecific T-cell adaptor method, which employs a multispecific binding protein having a binding arm for CD3 expressed on T cells and a binding arm for B7H6 expressed on the cell surface of tumor cells. By simultaneously binding to T cells and tumor cells, the T-cell adaptor of this invention forces the formation of a cell lysis synapse between the two cell types, thereby selectively redirecting T-cell activity to target tumor cells.

[0014] In one aspect, the present invention provides a multispecific binding protein comprising a first antigen-binding unit specifically binding to B7H6 and a second antigen-binding unit specifically binding to CD3, wherein the first antigen-binding unit specifically binding to B7H6 is selected from the group consisting of i) to xxiv).

[0015] i) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2) and SEQ ID NO:3 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2) and SEQ ID NO:6 (CDR3);

[0016] ii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2) and SEQ ID NO:9 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2) and SEQ ID NO:12 (CDR3);

[0017] iii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2) and SEQ ID NO:15 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:16 (CDR1), SEQ ID NO:17 (CDR2) and SEQ ID NO:18 (CDR3);

[0018] iv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2) and SEQ ID NO:21 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:24 (CDR3);

[0019] v) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2) and SEQ ID NO:27 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2) and SEQ ID NO:30 (CDR3);

[0020] vi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2) and SEQ ID NO:33 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2) and SEQ ID NO:36 (CDR3);

[0021] vii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2) and SEQ ID NO:39 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2) and SEQ ID NO:42 (CDR3);

[0022] viii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2) and SEQ ID NO:45 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2) and SEQ ID NO:48 (CDR3);

[0023] ix) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2) and SEQ ID NO:51 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2) and SEQ ID NO:54 (CDR3);

[0024] x) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2) and SEQ ID NO:57 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2) and SEQ ID NO:60 (CDR3);

[0025] xi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2) and SEQ ID NO:63 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2) and SEQ ID NO:66 (CDR3);

[0026] xii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2) and SEQ ID NO:69 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2) and SEQ ID NO:72 (CDR3);

[0027] xiii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2) and SEQ ID NO:75 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2) and SEQ ID NO:78 (CDR3);

[0028] xiv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2) and SEQ ID NO:81 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2) and SEQ ID NO:84 (CDR3);

[0029] xv) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2) and SEQ ID NO:87 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2) and SEQ ID NO:90 (CDR3);

[0030] xvi) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2) and SEQ ID NO:93 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2) and SEQ ID NO:96 (CDR3);

[0031] xvii) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2) and SEQ ID NO:99 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2) and SEQ ID NO:102 (CDR3);

[0032] xviii) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:103 (CDR1), SEQ ID NO:104 (CDR2) and SEQ ID NO:105 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2) and SEQ ID NO:108 (CDR3);

[0033] xix) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:109 (CDR1), SEQ ID NO:110 (CDR2) and SEQ ID NO:111 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:112 (CDR1), SEQ ID NO:113 (CDR2) and SEQ ID NO:114 (CDR3);

[0034] xx) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:115 (CDR1), SEQ ID NO:116 (CDR2) and SEQ ID NO:117 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:118 (CDR1), SEQ ID NO:119 (CDR2) and SEQ ID NO:120 (CDR3);

[0035] xxi) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:121 (CDR1), SEQ ID NO:122 (CDR2) and SEQ ID NO:123 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:124 (CDR1), SEQ ID NO:125 (CDR2) and SEQ ID NO:126 (CDR3);

[0036] xxii) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:127 (CDR1), SEQ ID NO:128 (CDR2) and SEQ ID NO:129 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:130 (CDR1), SEQ ID NO:131 (CDR2) and SEQ ID NO:132 (CDR3);

[0037] xxiii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3); and

[0038] xxiv) Antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:139 (CDR1), SEQ ID NO:140 (CDR2) and SEQ ID NO:141 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:142 (CDR1), SEQ ID NO:143 (CDR2) and SEQ ID NO:144 (CDR3).

[0039] In some embodiments of the binding protein of the present invention, the first antigen-binding unit that specifically binds to B7H6 is selected from the group consisting of i) to xxiv):

[0040] i) A light chain variable domain containing the amino acid sequence of SEQ ID NO:145 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:146.

[0041] ii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:147 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:148;

[0042] iii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:149 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:150;

[0043] iv) A light chain variable domain containing the amino acid sequence of SEQ ID NO:151 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:152;

[0044] v) The light chain variable domain containing the amino acid sequence of SEQ ID NO:153 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:154.

[0045] vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:155 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:156.

[0046] vii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:157 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:158.

[0047] viii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:159 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:160.

[0048] ix) The light chain variable domain containing the amino acid sequence of SEQ ID NO:161 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:162;

[0049] x) The light chain variable domain containing the amino acid sequence of SEQ ID NO:163 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:164.

[0050] xi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:165 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:166;

[0051] xii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:167 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:168.

[0052] xiii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:169 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:170.

[0053] xiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:171 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:172.

[0054] xv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:173 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:174;

[0055] xvi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:175 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:176.

[0056] xvii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:177 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:178;

[0057] xviii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:179 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:180;

[0058] xix) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:181 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:182.

[0059] xx) The light chain variable domain containing the amino acid sequence of SEQ ID NO:183 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:184;

[0060] xxi) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:185 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:186.

[0061] xxii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:187 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:188;

[0062] xxiii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:189 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:190; and

[0063] xxiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:191 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:192.

[0064] In some embodiments of the binding protein of the present invention, the second antigen-binding unit that specifically binds to CD3 is selected from the group consisting of i)-vi):

[0065] i) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2) and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2) and SEQ ID NO:262 (CDR3);

[0066] ii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:263 (CDR1), SEQ ID NO:264 (CDR2) and SEQ ID NO:265 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:266 (CDR1), SEQ ID NO:267 (CDR2) and SEQ ID NO:268 (CDR3);

[0067] iii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:269 (CDR1), SEQ ID NO:270 (CDR2) and SEQ ID NO:271 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:272 (CDR1), SEQ ID NO:273 (CDR2) and SEQ ID NO:274 (CDR3);

[0068] iv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:275 (CDR1), SEQ ID NO:276 (CDR2) and SEQ ID NO:277 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:278 (CDR1), SEQ ID NO:279 (CDR2) and SEQ ID NO:280 (CDR3);

[0069] v) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:281 (CDR1), SEQ ID NO:282 (CDR2), and SEQ ID NO:283 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:284 (CDR1), SEQ ID NO:285 (CDR2), and SEQ ID NO:286 (CDR3); and

[0070] vi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:287 (CDR1), SEQ ID NO:288 (CDR2) and SEQ ID NO:289 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:290 (CDR1), SEQ ID NO:291 (CDR2) and SEQ ID NO:292 (CDR3).

[0071] In some embodiments of the binding protein of the present invention, the second antigen-binding unit that specifically binds to CD3 is selected from the group consisting of i) to vi):

[0072] i) The light chain variable domain containing the amino acid sequence of SEQ ID NO:293 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:294;

[0073] ii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:295 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:296;

[0074] iii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:297 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:298;

[0075] iv) A light chain variable domain containing the amino acid sequence of SEQ ID NO:299 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:300;

[0076] v) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:301 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:302; and

[0077] vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:303 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:304.

[0078] In some embodiments of the binding protein of the present invention, the first antigen-binding unit specifically binding to B7H6 comprises, from its N-terminus to its C-terminus, a first light chain variable domain, a first light chain constant domain, a first peptide linker, a first heavy chain variable domain, and a first heavy chain constant CH1 domain; and the second antigen-binding unit specifically binding to CD3 comprises, from its N-terminus to its C-terminus, a second light chain variable domain, a second light chain constant domain, a second peptide linker, a second heavy chain variable domain, and a second heavy chain constant CH1 domain. In some embodiments of the binding protein of the present invention, the first and / or second peptide linker comprises 26 to 42 amino acids, preferably any one of 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, more preferably 38 amino acids. In some embodiments of the present invention, the first linker and / or the second linker is a Gly-Ser linker, preferably comprising the amino acid sequence of SEQ ID NO:250, more preferably the first and second peptide linkers comprising the same sequence (e.g., SEQ ID NO:250). In some embodiments of the present invention, the first light chain constant structural domain and the second light chain constant structural domain independently contain human κ or λ structural domains.

[0079] In some embodiments, a first antigen-binding unit specific to B7H6 of the binding protein of the present invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, and SEQ ID NO:216, and a second antigen-binding unit specific to CD3 ... SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309 and SEQ ID NO:310, with SEQ ID NO:305 being preferred.

[0080] In some embodiments, the binding protein of the present invention further includes a first and a second Fc domain, wherein the first Fc domain is covalently linked to the first antigen-binding unit, preferably the C-terminus of the first antigen-binding unit, and the second Fc domain is covalently linked to the second antigen-binding unit, preferably the C-terminus of the second antigen-binding unit.

[0081] In some embodiments of the present invention

[0082] i) The first Fc domain contains tyrosine (Y) at position 366 [T366Y], and the second Fc domain contains threonine (T) at position 407 [Y407T], or

[0083] ii) The first Fc domain contains tryptophan (W) at position 366 [T366W], and the second Fc domain contains serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V], or

[0084] iii) The second Fc domain contains tyrosine (Y) at position 366 [T366Y], and the first Fc domain contains threonine (T) at position 407 [Y407T], or

[0085] iv) The second Fc domain contains tryptophan (W) at position 366 [T366W], and the first Fc domain contains serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V].

[0086] Preferably, the first or second Fc domain further comprises arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436. In some embodiments, the first and / or second Fc domain comprises alanine [L234A] at position 234 and alanine [L235A] at position 235.

[0087] In some embodiments, the binding protein of the present invention comprises a first polypeptide chain specifically binding to B7H6, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 and SEQ ID NO:240; and a second polypeptide chain specifically binding to CD3, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 and SEQ ID NO:240; and a second polypeptide chain specifically binding to CD3, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:220, SEQ ID NO:220, SEQ ID NO:230, SEQ ID NO:230, SEQ ID NO:230, SEQ ID NO:2 SEQ ID NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315 and SEQ ID NO:316, with SEQ ID NO:311 being preferred.

[0088] In another aspect, the present invention provides isolated nucleic acid molecules that i) encode a first antigen-binding unit and / or a second antigen-binding unit of the binding protein of the present invention, optionally further encoding a first and / or a second Fc domain, or ii) encode a first and / or a second polypeptide chain of the binding protein of the present invention. In other aspects, this document provides expression vectors comprising the nucleic acid molecules of the present invention, host cells transfected with said expression vectors, and methods for manufacturing the proteins of the present invention.

[0089] In another aspect of the invention, a multispecific binding protein is provided, comprising a first polypeptide chain specifically binding to B7H6 and a second polypeptide chain specifically binding to CD3, wherein the first polypeptide chain comprises a first light chain, a first linker, and a first heavy chain, and the second polypeptide chain comprises a second light chain, a second linker, and a second heavy chain. Preferably, the C-terminus of the first light chain is covalently bound to the N-terminus of the first heavy chain via a first peptide linker, and the C-terminus of the second light chain is covalently bound to the N-terminus of the second heavy chain via a second peptide linker. Those skilled in the art will understand that any reference to “light chain” or “heavy chain” herein refers to the antibody light chain or the antibody heavy chain, respectively.

[0090] In some embodiments of the protein of the present invention, the first polypeptide chain specifically binding to B7H6 includes light chain variable and heavy chain variable domains, comprising a CDR sequence, a VH / VL sequence and / or a single-chain Fab sequence as defined by the antigen-binding unit of any of the following B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24 described herein. In some embodiments, the second polypeptide chain that specifically binds to CD3 includes light chain variable and heavy chain variable domains, which include a CDR sequence, a VH / VL sequence and / or a scFab sequence as defined for an antigen-binding unit such as CD3#1 as described herein.

[0091] Other aspects, embodiments, uses, and methods relating to the binding proteins of the present invention will become apparent from the following embodiments of the invention and the appended claims.

[0092] This invention provides a novel binding protein that allows for more effective treatment of cancers expressing B7H6, such as (metastatic) colorectal cancer ((m)CRC), non-small cell lung cancer (NSCLC), or head and neck squamous cell carcinoma (HNSCC). Attached Figure Description

[0093] Figure 1 : Schematic diagram of the bispecific binding protein of the present invention.

[0094] Figure 2 Schematic diagram of extracellular B7H6 protein expressed on the cell surface of CHO-K1 cells.

[0095] Figure 3 : Binding of 34 exemplary B7H6 / CD3 binding proteins to recombinant human B7H6 extracellular protein.

[0096] Figure 4 : Binding of 34 exemplary B7H6 / CD3 binding proteins to recombinant human alanine-mutant B7H6 extracellular protein.

[0097] Figure 5 : Binding of 34 exemplary B7H6 / CD3 binding proteins to HCT-15 cells expressing endogenous human B7H6.

[0098] Figure 6 : Binding of 23 exemplary B7H6 / CD3 binding proteins to recombinant CHO-K1 cells expressing cynomolgus monkey B7H6.

[0099] Figure 7 : Binding of 23 exemplary B7H6 / CD3 binding proteins to CD3-expressing human T cells.

[0100] Figure 8 : Binding of 23 exemplary B7H6 / CD3 binding proteins to B7H6-negative CHO-K1 cells.

[0101] Figure 9 Inhibitory activity of 17 exemplary B7H6 / CD3 binding proteins secreted by B7H6-dependent IFNγ from NK-92MI cells.

[0102] Figure 10 The efficacy of 11 exemplary B7H6 / CD3 binding proteins in lysing target cells to redirect unstimulated T cells to human HCT-15 cells.

[0103] Figure 11 The efficacy of 23 exemplary B7H6 / CD3 binding proteins in lysing target cells to redirect unstimulated T cells to human HCT-15 cells.

[0104] Figure 12 Efficacy of exemplary B7H6 / CD3 binding proteins in lysing target cells at various effector-to-target (E:T) cell ratios.

[0105] Figure 13 The efficacy of 23 exemplary B7H6 / CD3 binding proteins in lysed cells in redirecting unstimulated T cells to recombinant CHO cells transfected with B7H6 and Cho wt cells.

[0106] Figure 14 The efficacy of six exemplary B7H6 / CD3 binding proteins in CD25 upregulation on T cells in the presence of HCT-15 cells.

[0107] Figure 15 The efficacy of upregulating the expression of six exemplary B7H6 / CD3 binding proteins in T cells in the presence of HCT-15 cells.

[0108] Figure 16 The efficacy of upregulating intracellular granzyme B expression in T cells by six exemplary B7H6 / CD3 binding proteins in the presence of HCT-15 cells.

[0109] Figure 17 The efficacy of six exemplary B7H6 / CD3 binding proteins in T cell proliferation in the presence of HCT-15 cells.

[0110] Figure 18The potency of five exemplary B7H6 / CD3 binding proteins in T cell secretion of IFNγ in the presence of HCT-15 cells.

[0111] Figure 19 : Pharmacokinetic profile of an exemplary B7H6 / CD3 binding protein.

[0112] Figure 20 Antitumor activity of an exemplary B7H6 / CD3 binding protein in a mouse xenograft model with T cell implantation.

[0113] Figure 21 T cell infiltration in NCI-H716 xenograft tumor tissue using exemplary B7H6 / CD3 binding protein.

[0114] Figure 22 Pharmacokinetics of four exemplary B7H6-binding proteins.

[0115] Figure 23 Antitumor activity of four exemplary B7H6 / CD3 binding proteins in a mouse xenograft model with T cell implantation.

[0116] Figure 24 Antitumor activity of the exemplary B7H6 / CD3 binding protein administered as a single dose in a mouse xenograft model with T-cell implantation, q7d or as a single dose. Detailed Implementation

[0117] Terms and definitions used

[0118] The above and other aspects and embodiments of the present invention will become apparent from the further description herein, wherein:

[0119] Unless otherwise specified or defined, all terms used have their common meaning in the art, as will be apparent to those skilled in the art. References are made to, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and the general background art cited herein. Furthermore, unless otherwise specified, all methods, steps, techniques, and manipulations not explicitly described in detail may be practiced and implemented in a manner known per se, as will be apparent to those skilled in the art. References are again made to, for example, standard manuals, the general background art mentioned above, and other references cited therein.

[0120] When used in this document, the term “comprising” and its variations (e.g., “comprises and comprise”) may be replaced by the terms “containing”, “including”, or “having”.

[0121] Unless the context requires a more limited interpretation, the term “sequence” as used herein (e.g., in terms such as “heavy / light chain sequence,” “antibody sequence,” “variable domain sequence,” “constant domain sequence,” or “protein sequence”) should generally be understood to include the associated amino acid sequence as well as the nucleic acid or nucleotide sequence that encodes it.

[0122] As used herein, the term "antigen-binding unit" encompasses the minimum structural requirements derived from an antibody that allow binding to its specific target or antigen (i.e., the minimum structural requirements typically present in antibodies). Therefore, an antigen-binding unit comprises at least three light chain and three heavy chain CDR sequences; preferably, it comprises at least a light chain variable domain and a heavy chain variable domain.

[0123] The general structure of antibodies or immunoglobulins is well known to those skilled in the art. The molecule is a heterotetrameric glycoprotein, typically about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains, and is commonly referred to as a full-length antibody. Each light chain is covalently linked to the heavy chain by a disulfide bond to form a heterodimer, and the heterotetrameric molecule is formed via covalent disulfide bonds between the two identical heavy chains of the heterodimer. Although the light and heavy chains are linked together by a single disulfide bond, the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at its N-terminus, followed by three or four (in the case of IgE) constant domains (CH1, CH2, CH3, and CH4), and a hinge region between CH1 and CH2. Each light chain has two domains: an N-terminal variable domain (VL) and a C-terminal constant domain (CL). The VL and VH domains are non-covalently associated, while the CL domain is typically covalently linked to the CH1 domain via disulfide bonds. It is believed that specific amino acid residues form interfaces between the light and heavy chain variable domains (Chothia et al., 1985, J. Mol. Biol. 186:651-663). Variable domains are also referred to herein as variable regions or Fv and represent the portion that confers antibody specificity for the antigen by carrying antigen-binding sites.

[0124] As used herein, the “light chain variable domain” (or “light chain variable region”) and the “heavy chain variable domain” (or “heavy chain variable region”) have the same general structure, and each domain is essentially composed of four frame (FR) regions whose sequences are widely conserved. These frame regions are referred to in the art and hereinafter as “Frame Region 1” or “FR1”, “Frame Region 2” or “FR2”, “Frame Region 3” or “FR3”, and “Frame Region 4” or “FR4”, respectively. The frame regions are interrupted by three hypervariable regions (HVRs) (or CDRs), which are referred to in the art and hereinafter as “Complementarity Determinant Region 1” or “CDR1”, “Complementarity Determinant Region 2” or “CDR2”, and “Complementarity Determinant Region 3” or “CDR3”, respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The frame regions adopt a β-sheet conformation, and the CDRs can form loops connecting the β-sheet structures. The CDR in each chain maintains its three-dimensional structure through the framework region and together with the CDR of the other chain, forms an antigen binding site.

[0125] Various definitions of CDR are known in the art, for example, based on CCG, also known as IMGT (Lefranc MP, Pommié C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G. “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.” Dev Comp Immunol. January 2003; 27(1):55-77; Giudicelli V, Brochet X, Lefranc MP. “IMGT / V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences”. Cold Spring Harb Protoc. 2011; 2011(6):695-715) or based on Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196: 901-917) and Kabat (EAKabat, TT Wu, H. Bilofsky, M. Reid-Miller and H. Perry, Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983)). Within the context of this invention, references to CDR are based on the definition of CCG (IMGT).

[0126] As used in this application, the terms "constant domain" or "constant region" refer to the sum of the domains in an antibody excluding the variable region. These constant domains and regions are well known in the state of the art and, for example, described by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91-3242 (1991)). Antibodies or immunoglobulins are classified into several classes based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively, based on their heavy chain constant regions. Some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2.

[0127] The "Fc moiety" or "Fc domain" of an antibody does not directly participate in antibody-antigen binding, but exhibits various effector functions. The term "Fc moiety / domain" is well-known to those skilled in the art and is defined based on the papain cleavage of antibodies. The Fc moiety of an antibody directly participates in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding, and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc moiety of most IgG antibody subclasses. Although the effect of an antibody on the complement system depends on certain conditions, binding to C1q is caused by the defined binding sites in the Fc moiety. The binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbered according to Eu designations (Edelman et al., ProcNatl Acad Sci US A. May 1969; 63(1):78-85)). Among the residues mediating the binding of C1q and Fcγ receptors in IgG1, L234 and L235 are the most critical (Hezareh et al., J. Virology 75 (2001) 12161-12168, Shields et al. (2001) JBC, 276 (9): 6591-6604). Antibodies against subclasses IgG1 and IgG3 typically show complement activation and binding to C1q and C3, while IgG2 and IgG4 do not activate the complement system and do not bind to C1q and C3.

[0128] The term “antibody” or “antibody molecule” (used synonymously herein) encompasses monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, sequence-optimized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of antibodies (specifically Fv, Fab, Fab', or F(ab')2 fragments), single-chain antibodies (specifically single-chain variable fragments (scFv), single-chain Fab fragments (scFab)), small modular immunopharmaceuticals (SMIPs), domain antibodies, nanobodies®, and bivalent antibodies. Antibodies may have effector functions, such as ADCC or CDC, which are typically mediated by the Fc portion of the antibody, or they may not have effector functions, such as lacking an Fc portion or having a blocked, masked Fc portion that is inherently not or insufficiently recognized by immune cells or components of the immune system (e.g., the complement system).

[0129] Monoclonal antibodies (mAbs) are monospecific antibodies with a consistent amino acid sequence. They can be produced from hybridoma cell lines (called hybridomas) using hybridoma technology, which represent a pure line of a fusion of B cells producing specific antibodies and myeloma (B-cell carcinoma) cells (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495-7.). Alternatively, monoclonal antibodies can be produced through recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I. (May 1997). "Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells." JImmunol Methods 204 (1): 77-87; see also below). "Recombinant antibody" or "recombinant binding protein" is an antibody or binding protein that has been produced by recombinantly engineered host cells. It may optionally be isolated or purified.

[0130] The antibody molecules of the present invention also include fragments of immunoglobulins that retain antigen-binding properties, such as Fab, Fab', or F(ab')2 fragments. These fragments can be obtained by fragmentation of the immunoglobulin, for example by proteolytic digestion, or by recombinant expression of the fragments. For example, immunoglobulin digestion can be performed using conventional techniques, such as papain or pepsin (WO 94 / 29348). Papain digestion of antibodies typically produces two identical antigen-binding fragments (Fab). The Fab fragment consists of a constant domain and a variable domain of each of the heavy and light chains. Pepsin treatment produces F(ab')2. In the Fab fragment, each variable domain is fused to a constant domain of an immunoglobulin, preferably of human origin. Thus, the heavy chain variable domain is fused to the CH1 domain (the so-called Fd fragment), and the light chain variable domain is fused to the CL domain. The Fab fragment can be produced by recombinant expression of individual nucleic acids in host cells, see below.

[0131] Numerous techniques have been developed for placing variable domains of immunoglobulins, or molecules derived from said variable domains, into different molecular environments. These should also be considered as “antibodies” or “antibody molecules” according to the present invention. Generally, said antibody molecules are smaller in size than immunoglobulins and may contain a single amino acid chain or several amino acid chains. For example, a “single-chain variable fragment (scFv)” is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked together by short linkers (usually serine (S) or glycine (G)) (WO 88 / 01649; WO 91 / 17271; Huston et al.; International Reviews of Immunology, Vol. 10, 1993, 195-217). A “single-domain antibody” or “nanobody®” has an antigen-binding site in a single Ig-like domain (WO 94 / 04678; WO 03 / 050531, Ward et al., Nature. Oct. 12, 1989; 341(6242):544-6; Revets et al., Expert Opin Biol Ther. 5(1):111-24, 2005). One or more single-domain antibodies with binding specificity to the same or different antigens can be linked together. “Bivalent antibodies” are bivalent antibody molecules composed of two amino acid chains containing two variable domains (WO 94 / 13804, Holliger et al., Proc Natl Acad Sci US A. July 15, 1993; 90(14):6444-8). Other examples of antibody-like molecules are “immunoglobulin superfamily antibodies” (IgSF; Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). Different concepts lead to the so-called “small modular immunopharmaceuticals (SMIPs)”, which contain an Fv domain linked to a single-chain hinge and an effector domain lacking the constant CH1 domain (WO 02 / 056910). "Single-chain Fab" or "scFab" is a fusion of a light chain Fab domain (i.e., a light chain variable domain (VL) connected to a light chain constant domain (CL)) and a heavy chain Fab domain (i.e., a heavy chain variable domain (VH) connected to a heavy chain constant domain (CH1)). Single-chain Fab can recognize and bind antigens. scFab may also optionally contain a linker (e.g., a peptide linker) located between the CL and VH domains (Hust et al., BMC Biotechnology 2007, 7:14).

[0132] For human applications, it is generally desirable to reduce the immunogenicity of therapeutic molecules, such as antibodies or binding proteins containing antigen-binding units as described herein, which are originally derived from other species, such as mice. This can be accomplished by constructing chimeric antibodies / binding proteins or by a method known as “humanization.” In this context, “chimeric antibody” or “chimeric antigen-binding unit” should be understood as an antibody or antigen-binding unit containing a sequence portion (e.g., a variable domain) derived from a species (e.g., mice) fused with a sequence portion (e.g., a constant domain) derived from a different species (e.g., humans). In this context, “humanized antibody,” “humanized antigen-binding unit,” or “humanized VL / VH domain” contains an antibody, antigen-binding unit, or VH / VL domain that originally originates from a non-human species, wherein certain amino acids have been mutated to make the overall sequence of the variable domain more closely resemble the sequence of a human variable domain. Humanization methods for antibodies are well known in the field (Billetta R, Lobuglio AF. “Chimeric antibodies”. IntRev Immunol. 1993; 10(2-3):165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). “Reshaping human antibodies for therapy”. Nature: 332:323).

[0133] As used herein, the terms “human antibody,” “human antigen-binding unit,” or “human VH / VL domain” include antibodies, antigen-binding units, or VH / VL domains having variable (and, where applicable, constant) regions derived from human germline immunoglobulin sequences. As used herein, the terms “human antibody,” “human antigen-binding unit,” or “human VH / VL domain” are not intended to include antibodies whose CDR sequences derived from another (mammal) species (e.g., mouse, rat, or rabbit) have been grafted onto human frame sequences. Therefore, as used herein, the terms “human antibody,” “human antigen-binding unit,” or “human VH / VL domain” refer to antibodies, antigen-binding units, or VH / VL domains in which each part of the protein (e.g., CDR, frame, CL, CH domains (e.g., CH1, CH2, CH3), hinge, VL, VH) is substantially non-immunogenic in humans and has only minimal sequence alterations or variations, as further described below.

[0134] Techniques for generating the aforementioned “human antibodies,” “human antigen-binding units,” or “human VH / VL domains” have been described, including but not limited to phage display or the use of transgenic animals (WWW.Ablexis.com / technology-alivamab.php; WO 90 / 05144; D. Marks, HR Hoogenboom, TP Bonnert, J. McCafferty, AD Griffiths, and G. Winter (1991) “By-passing immunisation. Human antibodies from V-gene libraries displayed on phage.” J. Mol. Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus, “Concepts in antibody phage display”. Briefings in Functional Genomics and Proteomics 2002). 1(2):189-203; Lonberg N, Huszar D. "Human antibodies from transgenic mice". Int Rev Immunol. 1995; 13(1):65-93.; Brüggemann M, TaussigMJ. "Production of human antibody repertoires in transgenic mice". Curr OpinBiotechnol. 1997 Aug;8(4):455-8.).

[0135] Therefore, human antibodies, human antigen-binding units, or human VH / VL domains differ from, for example, chimeric or humanized antibodies. It should be noted that human antibodies, human antigen-binding units, or human VH / VL domains can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes.

[0136] The chimeric, humanized, or human antibody, antigen-binding unit, or VH / VL domain of the present invention can be further optimized; referred herein as “optimized” or “sequence-optimized” antibody, antigen-binding unit, or VH / VL domain. This optimization includes, but is not limited to, the removal or exchange of undesired amino acids, for example, to reduce immunogenicity in humans, or to avoid deamidation, undesired charges or lipophilicity, or nonspecific binding. For example, this removal or exchange of undesired amino acids can be introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. Furthermore, regarding chimeric or humanized antibodies, antigen-binding units, or VH / VL domains, it should be understood that certain mouse FR residues may be important for the function of the optimized antibody, antigen-binding unit, and VH / VL domain. Therefore, these important amino acid residues may be retained in the optimized antibody, antigen-binding unit, and VH / VL domain.

[0137] The term "monomer" refers to a homologous form of an antibody or multispecific protein as described herein. For example, for a full-length antibody, a monomer means a monomeric antibody having two identical heavy chains and two identical light chains. In the context of this invention, a monomer means the protein of this invention having a single antigen-binding unit specific to B7H6 and a single antigen-binding unit specific to CD3, as described herein. For example, the monomer of the binding protein described herein may have two polypeptide chains: a first polypeptide chain containing a single-chain Fab and a first Fc domain specific to B7H6, and a second polypeptide chain containing a single-chain Fab and a second Fc domain specific to CD3.

[0138] An epitope is a region of an antigen that is bound by an antibody or antigen-binding moiety (such as an antigen-binding unit of a protein as described herein). The term "epitaph" includes any polypeptide determinant capable of specifically binding to an antibody or antigen-binding moiety. In some embodiments, epitope determinants include chemically active surface groups of a molecule (e.g., amino acids, glycan side chains, phosphoryl groups, or sulfonyl groups), and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. The difference between conformational and non-conformational epitopes lies in the fact that they lose binding to the former in the presence of denaturing solvents, but do not lose binding to the latter.

[0139] The terms "bind," "bind to," "specifically bind," or "specifically bind to," "bind (to)," or "specifically bind to," and "have an affinity," "are specific to," and / or "are specific to" an antigen-binding molecule / protein (e.g., immunoglobulin, antibody, antigen-binding unit, or fragment of such antigen-binding molecule / protein) for a specific epitope, antigen, or protein (or at least a portion, fragment, or epitope), are referred to as "against or directed against" that epitope, antigen, or protein, or "binding" molecule / protein involving that epitope, antigen, or protein. These terms are used interchangeably herein.

[0140] As used herein, the terms “binding” and “specific binding” refer to the binding of an antigen-binding molecule / protein (e.g., immunoglobulin, antibody, antigen-binding unit, or fragment thereof) to an antigenic epitope using purified wild-type antigen in an in vitro analysis, preferably in a plasmon resonance analysis (Malmqvist M., “Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.”, Curr Opin Immunol. April 1993; 5(2):282-6.). Antibody affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, RJ and Brault PA., “Kinetic exclusion assay technology: Characterization of Molecular Interactions.” ASSAY and Drug Development Technologies. Dec 2004 2(6): 647-657). For example, the binding protein or protein of the present invention binds to the epitope of B7H6 with its first antigen-binding unit / first polypeptide chain and binds to the epitope of CD3 with its second antigen-binding unit / second polypeptide chain.

[0141] Generally, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule / protein (e.g., immunoglobulin, antibody, antigen-binding unit, or fragment of the antigen-binding molecule / protein) can bind to. Binding specificity to B7H6 means that the antigen-binding protein / molecule of the present invention (e.g., the first antigen-binding unit of the binding protein) has a significantly higher binding affinity to B7H6 than to structurally unrelated molecules. Binding specificity to CD3 means that the antigen-binding protein / molecule of the present invention (e.g., the second antigen-binding unit of the binding protein) has a significantly higher binding affinity to CD3 than to structurally unrelated molecules. The specificity of an antigen-binding molecule / protein can be determined based on its affinity and / or binding strength. The equilibrium constant (Ki) for the dissociation of the antigen from the antigen-binding protein... D Affinity, expressed as K, is a measure of the binding strength between an epitope on an antigen-binding molecule / protein and an antigen-binding site. D The smaller the value, the stronger the binding strength between the epitope and the antigen binding site (or, affinity can also be expressed as the affinity constant (K)). A ), which is 1 / K D As will be apparent to those skilled in the art (e.g., based on further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding molecule / protein (e.g., an immunoglobulin, antibody, antigen-binding unit, or fragment of such an antigen-binding molecule / protein) and the associated antigen. Affinity is related to both the affinity between an epitope on the antigen-binding molecule / protein and its antigen-binding site, and the number of associated binding sites present on the antigen-binding molecule / protein.

[0142] When referring to antigen-binding units / antigens, ligands / receptors, or other binding pairs, the terms "specific binding" or "selective binding" indicate a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological products. Therefore, under the conditions of nomenclature, an antigen-binding unit is designated to bind to a specific antigen and not to other proteins present in the sample in significant quantities. Under the conditions of nomenclature, the antigen-binding unit binds to its antigen with an affinity at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than that with unrelated antigens.

[0143] As used herein, the term "separated" refers to material removed from its original or natural environment (e.g., the natural environment if it is natural). For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not separated, but the same polynucleotide or polypeptide separated from some or all of the coexisting material in a natural system through human intervention is separated. The polynucleotide may be part of a carrier and / or the polynucleotide or polypeptide may be part of a composition and is still separated because the carrier or composition is not part of the environment in which it is found in nature. For example, when compared with the natural biological source and / or reaction or culture medium from which nucleic acid, protein / polypeptide molecules are obtained, they are considered to be "substantially separated" when they are separated from at least one other component (e.g., another nucleic acid, another protein / polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component) that is normally bound to them in that source or medium. Specifically, nucleic acids or proteins / peptides are considered "substantially separated" if they are purified at least 2-fold, specifically at least 10-fold, more specifically at least 100-fold, and up to 1000-fold or more. Nucleic acid or protein / peptide molecules that are "substantially separated" are preferably substantially homogeneous, as determined using suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0144] As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term "consistent" or "consistency percentage" refers to two or more sequences or subsequences being identical or having a specified percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence. To determine the consistency percentage, sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the sequence of a first amino acid or nucleic acid to facilitate optimal alignment with a second amino acid sequence or second nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is consistent at that position. The consistency percentage between two sequences is a function of the number of consistent positions shared by the sequences (i.e., consistency % = number of consistent positions / total number of positions (e.g., overlapping positions) × 100). In some embodiments, if appropriate (e.g., excluding additional sequences extending beyond the compared sequences), the two compared sequences have the same length after a gap is introduced within the sequence. For example, when comparing variable region sequences, leader (signal peptide) and / or constant domain sequences are not considered. For sequence comparisons between two sequences, the “corresponding” CDR refers to the CDR at the same position in both sequences (e.g., CDR-H1 for each sequence).

[0145] The determination of the percentage of identity or similarity between two sequences can be accomplished using mathematical algorithms. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST procedure (score=100, word length=12) to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST protein searches can be performed using the XBLAST procedure (score=50, word length=3) to obtain amino acid sequences homologous to the protein of interest. To obtain gap alignments for comparative purposes, gap BLAST can be used, as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches to detect long-range relationships between molecules (ibid.). When using BLAST, gap BLAST, and PSI-Blast programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Roboti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option for setting the sensitivity and speed of the search. If ktup = 2, similar regions in the two compared sequences are found by observing and comparing residue pairs; if ktup = 1, single-aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or 1 to 6 for DNA sequences. If ktup is not specified, the default value is 2 for proteins and 6 for DNA.Alternatively, protein sequence alignment can be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0146] As used herein, the terms “covalent link” or “covalent bond” refer to a direct covalent bond or indirect link / bond between residues, where the two residues are not directly bonded but are covalently bonded to an intermediate molecule or domain, such as the intermediate domain or linker of an immunoglobulin.

[0147] The multispecific binding protein of the present invention

[0148] This invention provides a multispecific binding protein comprising at least one antigen-binding unit specifically binding to B7H6 (a first antigen-binding unit) and at least one antigen-binding unit specifically binding to CD3 (a second antigen-binding unit). By simultaneously binding to tumor cell antigens and CD3 on T cells, the binding protein functions as a T cell activating protein and is also referred to herein as a T cell adaptor. The term "(multispecific) binding protein" is used interchangeably with the term "(multispecific) binding molecule" herein. Other terms used herein to refer to the multispecific binding protein of this invention are "protein of this invention," "binding protein of this invention," "antigen-binding protein," and "multispecific protein."

[0149] The inventors have surprisingly discovered that the multispecific binding protein of the present invention induces potent and selective lysis of B7H6-positive colorectal cancer cell lines in the presence of T cells, and is active even at low effector-target cell ratios. Importantly, the binding protein of the present invention does not lyse B7H6-negative cells and does not induce T cell activation, T cell proliferation, or cytokine secretion in the absence of B7H6-positive cells. Notably, the protein of the present invention, which does not inhibit B7H6-dependent NK cell activation via NKp30 in vitro, is more potent in lysing B7H6-positive tumor cells. This activity is described in, for example, the in vitro analysis in Example 11.

[0150] To avoid ambiguity, B7H6 as used herein refers to human B7H6 of UniProt Q68D85, and the nucleic acid sequence encoding that protein. Similarly, CD3 as used herein refers to the human CD3ε (UniProt P07766) and CD3γ (UniProt: P09693) complex (human CD3εγ complex). Those skilled in the art will understand that the terms B7H6 and B7-H6 are used interchangeably herein.

[0151] In one aspect, the multispecific binding protein of the present invention comprises a first antigen-binding unit specifically binding to B7H6 and a second antigen-binding unit specifically binding to CD3, wherein the first binding unit is selected from the group consisting of i) to xxiv).

[0152] i) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2) and SEQ ID NO:3 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2) and SEQ ID NO:6 (CDR3) (antigen-binding unit B7H6#1);

[0153] ii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2) and SEQ ID NO:9 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2) and SEQ ID NO:12 (CDR3) (antigen-binding unit B7H6#2);

[0154] iii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2) and SEQ ID NO:15 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:16 (CDR1), SEQ ID NO:17 (CDR2) and SEQ ID NO:18 (CDR3) (antigen-binding unit B7H6#3);

[0155] iv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2) and SEQ ID NO:21 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:24 (CDR3) (antigen-binding unit B7H6#4);

[0156] v) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2) and SEQ ID NO:27 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2) and SEQ ID NO:30 (CDR3) (antigen-binding unit B7H6#5);

[0157] vi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2) and SEQ ID NO:33 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2) and SEQ ID NO:36 (CDR3) (antigen-binding unit B7H6#6);

[0158] vii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2) and SEQ ID NO:39 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2) and SEQ ID NO:42 (CDR3) (antigen-binding unit B7H6#7);

[0159] viii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2) and SEQ ID NO:45 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2) and SEQ ID NO:48 (CDR3) (antigen-binding unit B7H6#8);

[0160] ix) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2) and SEQ ID NO:51 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2) and SEQ ID NO:54 (CDR3) (antigen-binding unit B7H6#9);

[0161] x) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2) and SEQ ID NO:57 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2) and SEQ ID NO:60 (CDR3) (antigen-binding unit B7H6#10);

[0162] xi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2) and SEQ ID NO:63 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2) and SEQ ID NO:66 (CDR3) (antigen-binding unit B7H6#11);

[0163] xii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2) and SEQ ID NO:69 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2) and SEQ ID NO:72 (CDR3) (antigen-binding unit B7H6#12);

[0164] xiii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2) and SEQ ID NO:75 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2) and SEQ ID NO:78 (CDR3) (antigen-binding unit B7H6#13);

[0165] xiv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2) and SEQ ID NO:81 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2) and SEQ ID NO:84 (CDR3) (antigen-binding unit B7H6#14);

[0166] xv) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2) and SEQ ID NO:87 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2) and SEQ ID NO:90 (CDR3) (antigen-binding unit B7H6#15);

[0167] xvi) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2) and SEQ ID NO:93 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2) and SEQ ID NO:96 (CDR3) (antigen-binding unit B7H6#16);

[0168] xvii) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2) and SEQ ID NO:99 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2) and SEQ ID NO:102 (CDR3) (antigen-binding unit B7H6#17);

[0169] xviii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:103 (CDR1), SEQ ID NO:104 (CDR2) and SEQ ID NO:105 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2) and SEQ ID NO:108 (CDR3) (antigen-binding unit B7H6#18);

[0170] xix) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:109 (CDR1), SEQ ID NO:110 (CDR2) and SEQ ID NO:111 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:112 (CDR1), SEQ ID NO:113 (CDR2) and SEQ ID NO:114 (CDR3) (antigen-binding unit B7H6#19);

[0171] xx) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:115 (CDR1), SEQ ID NO:116 (CDR2) and SEQ ID NO:117 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:118 (CDR1), SEQ ID NO:119 (CDR2) and SEQ ID NO:120 (CDR3) (antigen-binding unit B7H6#20);

[0172] xxi) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:121 (CDR1), SEQ ID NO:122 (CDR2) and SEQ ID NO:123 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:124 (CDR1), SEQ ID NO:125 (CDR2) and SEQ ID NO:126 (CDR3) (antigen-binding unit B7H6#21);

[0173] xxii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:127 (CDR1), SEQ ID NO:128 (CDR2) and SEQ ID NO:129 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:130 (CDR1), SEQ ID NO:131 (CDR2) and SEQ ID NO:132 (CDR3) (antigen-binding unit B7H6#22);

[0174] xxiii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3) (antigen-binding unit B7H6#23); and

[0175] xxiv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:139 (CDR1), SEQ ID NO:140 (CDR2) and SEQ ID NO:141 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:142 (CDR1), SEQ ID NO:143 (CDR2) and SEQ ID NO:144 (CDR3) (antigen-binding unit B7H6#24).

[0176] In some embodiments of the binding protein of the present invention, the second antigen-binding unit that specifically binds to CD3 is selected from the group consisting of i)-vi):

[0177] i) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2) and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2) and SEQ ID NO:262 (CDR3) (antigen-binding unit CD3#1);

[0178] ii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:263 (CDR1), SEQ ID NO:264 (CDR2) and SEQ ID NO:265 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:266 (CDR1), SEQ ID NO:267 (CDR2) and SEQ ID NO:268 (CDR3) (antigen-binding unit CD3#2);

[0179] iii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:269 (CDR1), SEQ ID NO:270 (CDR2) and SEQ ID NO:271 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:272 (CDR1), SEQ ID NO:273 (CDR2) and SEQ ID NO:274 (CDR3) (antigen-binding unit CD3#3);

[0180] iv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:275 (CDR1), SEQ ID NO:276 (CDR2) and SEQ ID NO:277 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:278 (CDR1), SEQ ID NO:279 (CDR2) and SEQ ID NO:280 (CDR3) (antigen-binding unit CD3#4);

[0181] v) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:281 (CDR1), SEQ ID NO:282 (CDR2), and SEQ ID NO:283 (CDR3) and a heavy chain CDR (antigen-binding unit CD3#5) containing the amino acid sequences of SEQ ID NO:284 (CDR1), SEQ ID NO:285 (CDR2), and SEQ ID NO:286 (CDR3); and

[0182] vi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:287 (CDR1), SEQ ID NO:288 (CDR2) and SEQ ID NO:289 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:290 (CDR1), SEQ ID NO:291 (CDR2) and SEQ ID NO:292 (CDR3) (antigen-binding unit CD3#6).

[0183] The first antigen-binding units i) to xxiv) as described above are referred to as B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24, respectively, and the second antigen-binding units i) to vi) as described above are referred to as CD3#1, CD3#2, CD3#3, CD3#4, CD3#5 and CD3#6, respectively. This document provides a sequence listing that readily allows for the identification of individual amino acid sequences of the specific antigen-binding unit and the full-length binding protein against which the invention is derived. An overview is provided in Table 1 of Example 2.

[0184] As used herein, the terms "first" and "second" in relation to antigen-binding units are intended only to indicate that the unit is two distinct units (due to their binding to different target antigens). Therefore, these terms should not be construed as referring to the exact order or sequence of units within the binding protein of this invention.

[0185] In some embodiments, the binding protein of the present invention comprises a first antigen-binding unit selected from the group consisting of: B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#1 ...6#4, B7H6#6#5, B7H6#6#6, B7H6#6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#6#6#6#7, B7H6#6#6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#6#6#6#6#7#8, B7H6#14, B7H6#15, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7H6#16, B7 14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24; and second antigen-binding units selected from the group consisting of: CD3#1, CD3#2, CD3#3, CD3#4, CD3#5 and CD3#6 as defined by the respective CDR sequences shown in Table 1.

[0186] In some embodiments, the binding protein of the present invention comprises a first antigen-binding unit selected from the group consisting of: B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24 as defined by the respective CDR sequences shown in Table 1; and a second antigen-binding unit CD3#1 as defined by the respective CDR sequences shown in Table 1. In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit selected from the group consisting of: B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24 as defined by the respective CDR sequences shown in Table 1; and a second antigen-binding unit CD3#1 as defined by the respective CDR sequences shown in Table 1. In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit selected from the group consisting of: B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24 as defined by the respective CDR sequences shown in Table 1; and a second antigen-binding unit CD3#1 as defined by the respective CDR sequences shown in Table 1. In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit selected from the group consisting of: B7H6#12, B7H6#14, B7H6#15, B7H6#16 and B7H6#23 as defined by the respective CDR sequences shown in Table 1; and a second antigen-binding unit CD3#1 as defined by the respective CDR sequences shown in Table 1.

[0187] In addition to the CDR sequence as described herein, the antigen-binding unit of the binding protein of the present invention also includes an immunoglobulin framework region (FR) sequence. This sequence is preferably non-immunogenic in humans, and therefore preferably a human, humanized, or optimized FR sequence. Suitable human, humanized, or optimized FR sequences are known in the art. Particularly preferred FR sequences may be derived from the embodiments shown herein, which disclose the complete antigen-binding unit, and thereby disclose both the CDR and FR sequences. In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit specifically binding to B7H6, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2), and SEQ ID NO:69 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2), and SEQ ID NO:72 (CDR3); and a second antigen-binding unit specifically binding to CD3, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3). This antigen-binding protein is referred to herein as B7H6#14 / CD3#1. In a particularly preferred embodiment, the antigen-binding units that specifically bind to B7H6 and CD3 respectively each include a VL / VH domain, such as a sequence-optimized VL / VH domain containing a CDR (B7H6#12 / CD3#1) as defined above. In a particularly preferred embodiment, the antigen-binding units (B7H6#12 / CD3#1) that specifically bind to B7H6 and CD3 respectively are each formed from scFab and optionally each is linked to an Fc domain.

[0188] In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit specifically binding to B7H6, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2), and SEQ ID NO:81 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2), and SEQ ID NO:84 (CDR3); and a second antigen-binding unit specifically binding to CD3, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3). This antigen-binding protein is referred to herein as B7H6#14 / CD3#1. In a particularly preferred embodiment, the antigen-binding units that specifically bind to B7H6 and CD3 respectively each include a VL / VH domain, such as a sequence-optimized VL / VH domain containing a CDR (B7H6#14 / CD#1) as defined above. In a particularly preferred embodiment, the antigen-binding units (B7H6#14 / CD3#1) that specifically bind to B7H6 and CD3 respectively are each formed from scFab and optionally each is linked to an Fc domain.

[0189] In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit specifically binding to B7H6, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3); and a second antigen-binding unit specifically binding to CD3, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3). This antigen-binding protein is referred to herein as B7H6#15 / CD3#1. In a particularly preferred embodiment, the antigen-binding units that specifically bind to B7H6 and CD3 respectively each include a VL / VH domain, such as a sequence-optimized VL / VH domain containing a CDR (B7H6#15 / CD3#1) as defined above. In a particularly preferred embodiment, the antigen-binding units (B7H6#15 / CD3#1) that specifically bind to B7H6 and CD3 respectively are each formed from scFab and optionally each is linked to an Fc domain.

[0190] In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit specifically binding to B7H6, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2), and SEQ ID NO:93 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2), and SEQ ID NO:96 (CDR3); and a second antigen-binding unit specifically binding to CD3, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3). This antigen-binding protein is referred to herein as B7H6#16 / CD3#1. In a particularly preferred embodiment, the antigen-binding units that specifically bind to B7H6 and CD3 respectively each include a VL / VH domain, such as a sequence-optimized VL / VH domain containing a CDR (B7H6#16 / CD3#1) as defined above. In a particularly preferred embodiment, the antigen-binding units (B7H6#16 / CD3#1) that specifically bind to B7H6 and CD3 respectively are each formed from scFab and optionally each is linked to an Fc domain.

[0191] In a preferred embodiment, the binding protein of the present invention comprises a first antigen-binding unit specifically binding to B7H6, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3); and a second antigen-binding unit specifically binding to CD3, comprising a light chain CDR comprising the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR comprising the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3). This antigen-binding protein is referred to herein as B7H6#14 / CD3#1. In a particularly preferred embodiment, the antigen-binding units that specifically bind to B7H6 and CD3 respectively each include a VL / VH domain, such as a sequence-optimized VL / VH domain containing a CDR (B7H6#23 / CD3#1) as defined above. In a particularly preferred embodiment, the antigen-binding units (B7H6#23 / CD3#1) that specifically bind to B7H6 and CD3 respectively are each formed from scFab and optionally each is linked to an Fc domain.

[0192] In a preferred embodiment of the binding protein of the present invention, the first and second binding units each include a light chain variable domain and a heavy chain variable domain. For the first antigen binding unit, the light / heavy chain variable domains are composed of B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, and B7H6#6#8. The CDR sequence of any one of #14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, or B7H6#24 defines the light / heavy chain variable domain for the second antigen-binding unit, and the CDR sequence of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 defines the light / heavy chain variable domain for the second antigen-binding unit. In some embodiments of the binding protein of the present invention, B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H The VH and / or VL domains of the antigen-binding units of any one of B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, B7H6#24, CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 are human, humanized, or optimized VH and / or VL domains.

[0193] In a preferred embodiment of the binding protein of the present invention, the light / heavy chain variable domain of the first antigen-binding unit that specifically binds to B7H6 is further defined as follows.

[0194] i) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:145 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:146 (antigen-binding unit B7H6#1); or

[0195] ii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:147 and a heavy chain variable domain (antigen-binding unit B7H6#2) comprising the amino acid sequence of SEQ ID NO:148; or

[0196] iii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:149 and a heavy chain variable domain (antigen-binding unit B7H6#3) comprising the amino acid sequence of SEQ ID NO:150; or

[0197] iv) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:151 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:152 (antigen-binding unit B7H6#4); or

[0198] v) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:153 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:154 (antigen-binding unit B7H6#5); or

[0199] vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:155 and the heavy chain variable domain (antigen-binding unit B7H6#6) containing the amino acid sequence of SEQ ID NO:156; or

[0200] vii) a light chain variable domain containing the amino acid sequence of SEQ ID NO:157 and a heavy chain variable domain (antigen-binding unit B7H6#7) containing the amino acid sequence of SEQ ID NO:158; or

[0201] viii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:159 and a heavy chain variable domain (antigen-binding unit B7H6#8) comprising the amino acid sequence of SEQ ID NO:160; or

[0202] ix) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:161 and a heavy chain variable domain (antigen-binding unit B7H6#9) comprising the amino acid sequence of SEQ ID NO:162; or

[0203] x) A light chain variable domain containing the amino acid sequence of SEQ ID NO:163 and a heavy chain variable domain (antigen-binding unit B7H6#10) containing the amino acid sequence of SEQ ID NO:164; or

[0204] xi) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:165 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:166 (antigen-binding unit B7H6#11); or

[0205] xii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:167 and a heavy chain variable domain (antigen-binding unit B7H6#12) comprising the amino acid sequence of SEQ ID NO:168; or

[0206] xiii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:169 and a heavy chain variable domain (antigen-binding unit B7H6#13) comprising the amino acid sequence of SEQ ID NO:170; or

[0207] xiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:171 and the heavy chain variable domain (antigen-binding unit B7H6#14) containing the amino acid sequence of SEQ ID NO:172; or

[0208] xv) a light chain variable domain containing the amino acid sequence of SEQ ID NO:173 and a heavy chain variable domain (antigen-binding unit B7H6#15) containing the amino acid sequence of SEQ ID NO:174; or

[0209] xvi) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:175 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:176 (antigen-binding unit B7H6#16); or

[0210] xvii) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:177 and a heavy chain variable domain (antigen-binding unit B7H6#17) comprising the amino acid sequence of SEQ ID NO:178; or

[0211] xviii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:179 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:180 (antigen-binding unit B7H6#18); or

[0212] xix) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:181 and a heavy chain variable domain (antigen-binding unit B7H6#19) comprising the amino acid sequence of SEQ ID NO:182; or

[0213] xx) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:183 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:184 (antigen-binding unit B7H6#20); or

[0214] xxi) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:185 and a heavy chain variable domain (antigen-binding unit B7H6#21) comprising the amino acid sequence of SEQ ID NO:186; or

[0215] xxii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:187 and a heavy chain variable domain (antigen-binding unit B7H6#22) comprising the amino acid sequence of SEQ ID NO:188; or

[0216] xxiii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:189 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:190 (antigen-binding unit B7H6#23); or

[0217] xxiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:191 and the heavy chain variable domain (antigen-binding unit B7H6#24) containing the amino acid sequence of SEQ ID NO:192.

[0218] In a preferred embodiment of the binding protein of the present invention, the light / heavy chain variable domain of the second antigen-binding unit that specifically binds to CD3 is further defined as follows.

[0219] i) A light chain variable domain containing the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain (antigen-binding unit CD3#1) containing the amino acid sequence of SEQ ID NO:294; or

[0220] ii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:295 and a heavy chain variable domain (antigen-binding unit CD3#2) comprising the amino acid sequence of SEQ ID NO:296; or

[0221] iii) A light chain variable domain containing the amino acid sequence of SEQ ID NO:297 and a heavy chain variable domain (antigen-binding unit CD3#3) containing the amino acid sequence of SEQ ID NO:298; or

[0222] iv) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:299 and a heavy chain variable domain (antigen-binding unit CD3#4) comprising the amino acid sequence of SEQ ID NO:300; or

[0223] v) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:301 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:302 (antigen-binding unit CD3#5); or

[0224] vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:303 and the heavy chain variable domain (antigen-binding unit CD3#6) containing the amino acid sequence of SEQ ID NO:304.

[0225] In a preferred embodiment, the binding protein of the present invention comprises a combination of first and second antigen-binding units selected from the group consisting of: B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7H6#1 3 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, and B7H6#24 / CD3#1, wherein the first and second antigen-binding units are defined by the CDR and / or VH and VL sequences of the antigen-binding units as shown in Table 1.

[0226] In a preferred embodiment, the binding protein of the present invention comprises a combination of first and second antigen-binding units selected from the group consisting of: B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6 #16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, and B7H6#24 / CD3#1, wherein the first and second antigen-binding units are defined by the CDR and / or VH and VL sequences of the antigen-binding units as shown in Table 1.

[0227] In a preferred embodiment, the binding protein of the present invention comprises a combination of first and second antigen-binding units selected from the group consisting of: B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, and B7H6#24 / CD3#1, wherein the first and second antigen-binding units are defined by the CDR and / or VH and VL sequences of the antigen-binding units as shown in Table 1.

[0228] In a preferred embodiment, the binding protein of the present invention comprises a combination of first and second antigen-binding units selected from the group consisting of: B7H6#12 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, and B7H6#23 / CD3#1, wherein the first and second antigen-binding units are defined by the CDR and / or VH and VL sequences of the antigen-binding units as shown in Table 1.

[0229] In a preferred embodiment, the binding protein of the present invention comprises (i) a first antigen-binding unit specifically binding to B7H6, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:167 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:168, and (ii) a second antigen-binding unit specifically binding to CD3, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294. This binding protein is referred to herein as B7H6#12 / CD3#1. In a particularly preferred embodiment, the antigen-binding units (B7H6#12 / CD3#1) specifically binding to B7H6 and CD3 respectively, as defined above, are each formed from scFab, which is optionally covalently linked to an Fc domain.

[0230] In a preferred embodiment, the binding protein of the present invention comprises (i) a first antigen-binding unit specifically binding to B7H6, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:171 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:172, and (ii) a second antigen-binding unit specifically binding to CD3, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294. This binding protein is referred to herein as B7H6#14 / CD3#1. In a particularly preferred embodiment, the antigen-binding units (B7H6#14 / CD3#1) specifically binding to B7H6 and CD3 respectively, as defined above, are each formed from scFab, which is optionally covalently linked to an Fc domain.

[0231] In a preferred embodiment, the binding protein of the present invention comprises (i) a first antigen-binding unit specifically binding to B7H6, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:173 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:174, and (ii) a second antigen-binding unit specifically binding to CD3, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294. This binding protein is referred to herein as B7H6#15 / CD3#1. In a particularly preferred embodiment, the antigen-binding units (B7H6#15 / CD3#1) specifically binding to B7H6 and CD3 respectively, as defined above, are each formed from scFab, which is optionally covalently linked to an Fc domain.

[0232] In a preferred embodiment, the binding protein of the present invention comprises (i) a first antigen-binding unit specifically binding to B7H6, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:175 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:176, and (ii) a second antigen-binding unit specifically binding to CD3, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294. This binding protein is referred to herein as B7H6#16 / CD3#1. In a particularly preferred embodiment, the antigen-binding units (B7H6#16 / CD3#1) specifically binding to B7H6 and CD3 respectively, as defined above, are each formed from scFab, which is optionally covalently linked to an Fc domain.

[0233] In a preferred embodiment, the binding protein of the present invention comprises (i) a first antigen-binding unit specifically binding to B7H6, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:189 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:190, and (ii) a second antigen-binding unit specifically binding to CD3, comprising a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294. This binding protein is referred to herein as B7H6#23 / CD3#1. In a particularly preferred embodiment, the antigen-binding units (B7H6#23 / CD3#1) specifically binding to B7H6 and CD3 respectively, as defined above, are each formed from scFab, which is optionally covalently linked to an Fc domain.

[0234] In some embodiments, the binding protein of the present invention comprises i) a first antigen-binding unit that specifically binds to B7H6 (e.g., B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18 as defined by the respective CDR or VH / VL sequences shown in Table 1). B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, B7H6#24), comprising a first light chain variable domain covalently linked to a first heavy chain variable domain via a first peptide linker, and / or ii) a second antigen-binding unit specifically bound to CD3 (e.g., any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as defined by the respective CDR or VH / VL sequences shown in Table 1), comprising a second light chain variable domain covalently linked to a second heavy chain variable domain via a second peptide linker. Optionally, the first and second antigen-binding units are covalently linked to each other via peptide linkers.

[0235] In some embodiments of the binding protein of the present invention, the first and / or second antigen-binding unit further comprises CL and CH1 domains, as in the light / heavy Fab fragments of conventional antibody molecules. Therefore, the first binding unit comprises a) a VL domain covalently linked (preferably directly bound) to the first CL domain (e.g., by B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6). Light chain CDRs of any of the following: B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, or B7H6#24 (as defined by the LCCDR or VL sequence), and b) covalently linking (preferably directly binding) to the VH domain of the first CH1 domain (e.g., the heavy chain CDR of any one of B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, or B7H6#24). (defined by an HCCDR or VH sequence), and / or the second antigen-binding unit comprises a) a VL domain covalently linked (preferably directly bound) to the second CL domain (e.g., defined by an LCCDR or VL sequence of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6), and b) a VH domain covalently linked (preferably directly bound) to the second CH1 domain (e.g., defined by an HCCDR or VH sequence of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6).

[0236] In the context of this invention, the CL domain is a constant domain of an antibody light chain, such as a kappa (κ) or lambda (λ) light chain. An example of a constant region of the κ light chain is shown in SEQ ID NO:247. An example of a constant region of the λ light chain is shown in SEQ ID NO:248. In some embodiments, the first and second CL domains are identical; for example, both the first and second CL domains are constant domains of the κ light chain, or both the first and second CL domains are constant domains of the λ light chain. In a preferred embodiment, the first and second CL domains are different; for example, the first CL domain is a constant κ domain and the second CL domain is a constant λ domain, or vice versa.

[0237] In the context of this invention, the CH1 domain is the first constant domain of the antibody heavy chain. An example of a constant CH1 domain is shown in SEQ ID NO:249.

[0238] In a preferred embodiment of the binding protein of the present invention, a first antigen-binding unit specific to B7H6 (e.g., B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#17, B7H6#18, B7H6#19, B7H6#19, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#19, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#19, B7H6#10, B7H6#11, B7H6#12, B7H6#19, B7H6#10, 21, B7H6#22, B7H6#23, or B7H6#24) comprises, from the N-terminus to the C-terminus: a first light chain variable domain, a first CL domain, a first adaptor peptide, a first VH domain, and a first CH1 domain, and / or the second binding unit of the binding protein of the present invention (e.g., CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 defined by the CDR and / or VH / VL sequences shown in Table 1) comprises, from the N-terminus to the C-terminus: a second light chain variable domain, a second CL domain, a second adaptor peptide, a second VH domain, and a second CH1 domain. In the embodiments described, the first and / or second binding units have a single-chain Fab structure. For both the first and / or second antigen-binding units, the order is reversible when forming a single-chain Fab, such that the antigen-binding unit comprises, from the N-terminus to the C-terminus: VH-CH1-[adaptor peptide]-VL-CL. In some embodiments of the protein of the present invention, when the first and / or second antigen-binding units comprise Fab or single-chain Fab, the constant domains may be of the same type (e.g., both CL domains are κ or λ light chain constant domains) or of different types (the first CL domain is κ and the second CL domain is λ light chain constant domain, or vice versa). Preferably, the first and second CL domains are of different types. In a preferred embodiment, the first antigen-binding unit consists of a first single-chain Fab specific to B7H6 (preferably any one of B7H6#12, B7H6#14, B7H6#15, B7H6#16, or B7H6#23 as defined by the CDR and / or VH / VL sequences shown in Table 1), and the second antigen-binding unit consists of a second single-chain Fab specific to CD3 (e.g., CD3#1 as defined by the CDR and / or VH / VL sequences shown in Table 1).

[0239] The linker sequence of the B7H6 / CD3 binding protein (e.g., the B7H6 / CD3 scFab described above) can be a natural or non-natural sequence. For therapeutic purposes, the linker preferably does not have immunogenicity in the individual to which the binding protein of the present invention is administered. Preferably, the linker comprises 26 to 42 amino acids, for example, 30 to 40 amino acids. In another aspect, the linker used in the protein of the present invention comprises 34 to 40 amino acids, for example, 36 to 39 amino acids, for example, 38 amino acids.

[0240] One useful group in the linker sequence is a linker derived from the hinge region of a heavy chain antibody as described in WO1996 / 34103 and WO1994 / 04678. Other examples are poly-alanine linker sequences, such as Ala-Ala-Ala.

[0241] Other preferred examples of connector sequences are Gly / Ser connectors of different lengths, such as (glyxsery)z connectors, including (for example) (gly4ser)3, (gly4ser)5, (gly4ser)7, (gly3ser)3, (gly3ser)5, (gly3ser)7, (gly3ser2)3, (gly3ser2)5 and (gly3ser2)7; or any one of SEQ ID NO: 250, 251, 252, 253, 254, 255 or 256, preferably the connector of SEQ ID NO: 250.

[0242] In some embodiments of the binding protein of the present invention, the VL domain of the first antigen-binding unit (e.g., a light chain CDR of any one of B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, B7H6#24 shown in Table 1) (as defined by the LCCDR or VL sequence) is covalently linked via a first Gly / Ser linker (e.g., a Gly / Ser linker of any one of 26 to 42 amino acids, 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, preferably 38 amino acids) to the VH domain of the first antigen-binding unit (e.g., by B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18 as shown in Table 1) to the VH domain of the first antigen-binding unit (e.g., by B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#15, B7H6#16, B7H6#17, B7H6#18 as shown in Table 1). The heavy chain CDR (HCCDR) or VH sequence of any one of B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, and B7H6#24 is defined; and the VL domain of the second antigen-binding unit (e.g., defined by the light chain CDR of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as shown in Table 1) is also defined. (LCCDR) or VL sequence defined) is covalently linked via a second Gly / Ser linker (e.g., a Gly / Ser linker of any one of 26 to 42 amino acids, 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, preferably 38 amino acids) to the VH domain of the second antigen-binding unit (e.g., a heavy chain CDR (HCCDR) or VH sequence defined by any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as shown in Table 1). More preferably, the first and second linkers are identical. Even more preferably, the first and second linkers each comprise the amino acid sequence of SEQ ID NO:250.

[0243] In a preferred embodiment of the binding protein of the present invention, the first antigen-binding unit specifically binding to B7H6 includes, from the N-terminus to the C-terminus, i) a VL domain (e.g., defined by the light chain CDR (LCCDR) or VL sequence of any one of B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, B7H6#24 as shown in Table 1), ii) iii) via a first CL domain, iv) via a first Gly / Ser linker (e.g., a Gly / Ser linker of any one of 26 to 42 amino acids, 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, preferably 38 amino acids). VH domains (e.g., defined by the heavy chain CDR (HCCDR) or VH sequence of any of B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, B7H6#24 as shown in Table 1), and v) The first CH1 domain, and / or the second antigen-binding unit that specifically binds to CD3, comprises from the N-terminus to the C-terminus i) a VL domain (e.g., defined by the light chain CDR (LCCDR) or VL sequence of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as shown in Table 1), ii) a second CL domain, iii) a second Gly / Ser linker (e.g., a Gly / Ser linker of any one of 26 to 42 amino acids, 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, preferably 38 amino acids), iv) a VH domain of the second antigen-binding unit (e.g., defined by the heavy chain CDR (HCCDR) or VH sequence of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as shown in Table 1) and v) a second CH1 domain.Preferably, i) to v) are respectively linked from the N-terminus to the C-terminus of the antigen-binding unit via direct covalent bonds in the order i) to v) (each antigen-binding unit thus having an scFab structure). More preferably, the first and second adapters are identical. Even more preferably, the first and second adapters each contain the amino acid sequence of SEQ ID NO:250.

[0244] In a preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab forming a first antigen-binding unit specific to B7H6 and comprising a sequence selected from the group consisting of SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, and SEQ ID NO:216; and a second single-chain Fab forming a second antigen-binding unit specific to CD3 and comprising SEQ ID NO:216. The sequence of NO:305.

[0245] In a preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab forming a first antigen-binding unit specific to B7H6 and comprising a sequence selected from the group consisting of SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215 and SEQ ID NO:216; and a second single-chain Fab forming a second antigen-binding unit specific to CD3 and comprising the sequence of SEQ ID NO:305.

[0246] In a preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab forming a first antigen-binding unit specific to B7H6 and comprising a sequence selected from the group consisting of SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215 and SEQ ID NO:216; and a second single-chain Fab forming a second antigen-binding unit specific to CD3 and comprising the sequence of SEQ ID NO:305.

[0247] In one preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab comprising the sequence of SEQ ID NO:204 and a second single-chain Fab comprising the sequence of SEQ ID NO:305, wherein optionally each single-chain Fab is further linked to an Fc domain, thereby forming a first polypeptide chain (“B7H6 chain”) and a second polypeptide chain (“CD3 chain”). In one preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab comprising the sequence of SEQ ID NO:206 and a second single-chain Fab comprising the sequence of SEQ ID NO:305, wherein optionally each single-chain Fab is further linked to an Fc domain, thereby forming a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain). In one preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab comprising the sequence of SEQ ID NO:207 and a second single-chain Fab comprising the sequence of SEQ ID NO:305, wherein optionally each single-chain Fab is further linked to an Fc domain, thereby forming a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain). In a preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab comprising the sequence of SEQ ID NO:208 and a second single-chain Fab comprising the sequence of SEQ ID NO:305, wherein optionally each single-chain Fab is further linked to an Fc domain, thereby forming a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain). In a preferred embodiment, the binding protein of the present invention comprises a first single-chain Fab comprising the sequence of SEQ ID NO:215 and a second single-chain Fab comprising the sequence of SEQ ID NO:305, wherein optionally each single-chain Fab is further linked to an Fc domain, thereby forming a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain).

[0248] In some implementations, the first antigen-binding unit (e.g., B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18 as defined by the CDR and / or VH / VL sequences shown in Table 1) B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, or B7H6#24) and / or the second antigen-binding unit (e.g., any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as defined by the CDR and / or VH / VL sequences shown in Table 1) comprises a VL domain covalently linked (preferably directly bound) to the CL domain and a VH domain linked to the CH1 domain (together forming a Fab fragment), and the CH1 domain is further covalently linked (e.g., directly bound) to the Fc domain, thereby forming an arm of a conventional Y-shaped antibody molecule having one light chain and one heavy chain. In some embodiments, the first and second antigen-binding units each form Fab fragments, namely the first and second Fab fragments, which are covalently linked (preferably directly bound) to the first and second Fc domains, thereby forming conventional heterotetramer bispecific and divalent (monovalent for B7H6 and CD3, respectively) antibody molecules.

[0249] In a preferred embodiment, the binding protein of the present invention comprises (i) a first antigen-binding unit comprising a first single-chain Fab that specifically binds to B7H6, i.e., via a peptide linker (e.g., a Gly / Ser linker of any one of 26 to 42 amino acids, 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, preferably 38 amino acids, or even more preferably SEQ ID NO. 1). The NO:250 connector is covalently attached to the VH-CH1 domain of the heavy chain (as defined by the CDR and / or VH / VL sequences shown in Table 1: B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18). (ii) An antibody light chain (VL-CL) comprising the VL and VH domains of any one of B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 or B7H6#24, wherein the first antigen-binding unit is covalently linked (e.g., directly bound) to the first Fc domain, and (ii) a second antigen-binding unit comprising an antibody light chain (VL-CL) specifically bound to a second single-chain Fab that is covalently linked to the VH-CH1 domain of the heavy chain (such as the VL and VH domains of any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, CD3#6 as defined by the respective CDR or VH / VL sequences shown in Table 1), wherein the second antigen-binding unit is covalently linked (e.g., directly bound) to the second Fc domain. Therefore, in a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain comprising (a) a first antigen-binding unit specific to B7H6, the first antigen-binding unit comprising a first single-chain Fab specific to B7H6 (preferably any one of B7H6#12, B7H6#14, B7H6#15, B7H6#16 or B7H6#23 as defined by the CDR and / or VH / VL sequences shown in Table 1), and (b) a first Fc domain (this first polypeptide chain is also referred to herein as the “B7H6 chain”), and (ii) a second polypeptide chain specific to CD3 comprising (a) a second antigen-binding unit comprising a second single-chain Fab specific to CD3 (preferably CD3#1 as defined by the CDR and / or VL / VH sequences shown in Table 1), and (b) a second Fc domain (this second polypeptide chain is also referred to herein as the “CD3 chain”). Therefore, as used herein, the term "peptide chain" includes at least the scFab and Fc domains. In some embodiments, the first and second Fc domains are identical.In a preferred embodiment, the first and second Fc domains are different. The binding protein obtained by the present invention comprises two different polypeptide chains, each with an intact Fc and two independent binding sites: a first antigen-binding unit formed by a first scFab specific to B7H6 and a second binding unit formed by a second scFab specific to CD3.

[0250] In a preferred embodiment, the binding protein of the present invention comprises two distinct polypeptide chains, each containing an antigen-binding unit formed by scFab. These two distinct polypeptide chains have different specificities and are covalently linked to an Fc domain. The polypeptide chains are covalently linked to each other via disulfide bonds or potentially via peptide linkers. In a preferred embodiment, the binding protein of the present invention is a bispecific, divalent (monovalent for B7H6 and CD3, respectively) heterodimeric protein comprising two polypeptide chains. One polypeptide chain (the first polypeptide chain or the B7H6 chain) contains an antigen-binding unit formed by scFab that specifically binds to B7H6 (e.g., B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6# ... 5, any one of B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 or B7H6#24) and the Fc structural domain (preferably SEQ) The first antigen-binding unit comprises an antigen-binding unit (e.g., any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6) formed by scFab that specifically binds to CD3, and an Fc domain (preferably the Fc domain of SEQ ID NO:243). In some embodiments, the first antigen-binding unit is composed of a first single-chain Fab and the second antigen-binding unit is composed of a second single-chain Fab. In some embodiments of the binding protein, the first polypeptide chain (B7H6 chain) specific to B7H6 consists of: a) a first antigen-binding unit composed of scFab (preferably any one of B7H6#12, B7H6#14, B7H6#15, B7H6#16 or B7H6#23 as defined by the CDR, VH / VL and / or scFab sequences shown in Table 1) and b) a first Fc domain. The second polypeptide chain (CD3 chain) specific to CD3 consists of: a) a second antigen-binding unit composed of scFab (preferably CD3#1 as defined by the CDR, VH / VL and / or scFab sequences shown in Table 1) and b) a second Fc domain. Preferably, the C-terminus of scFab is directly covalently linked to the N-terminus of the Fc domain. Preferably, the first and second polypeptide chains are covalently linked to each other via disulfide bonds, forming an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0251] In the context of this invention, the Fc domain is, for example, a heavy chain derived from IgG, such as IgG1, IgG2, or IgG4. For instance, the Fc domain of this invention is the Fc domain of a heavy chain of IgG1 or IgG4 and includes a hinge region and two constant domains (C... H2 and C H3 Examples of Fc structural domains (including hinge regions) are shown in SEQ ID NO:241 and 244.

[0252] Unless otherwise specified, the amino acid numbers in the amino acid chain of the proteins of this invention are assigned herein according to the Eu numbering system (Edelman et al., PNAS USA May 1969, 63(1):78-85; Cunningham et al., PNAS USA November 1969, 64(3):997-1003). This means that, unless otherwise specified, according to the Eu numbering system, the amino acid numbers indicated herein correspond to positions in the heavy chain of the corresponding isotype (e.g., IgG1 or IgG4).

[0253] In some embodiments, the first and second Fc domains of the protein of the present invention each contain one or more amino acid variations that reduce the formation of homodimers of the first or second polypeptide chain, rather than heterodimers of the first and second polypeptide chains. Through these variations, a “protrusion” is created in one of the Fc domains by replacing one or more small amino acid side chains at a heavy chain interface with a larger side chain (e.g., tyrosine or tryptophan). A compensatory “cavity” of the same or similar size is created at the interface of the other Fc domain by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers beyond other unwanted end products (e.g., homodimers, specifically homodimers with “protruding” Fc domains) (see, for example, Ridgway et al., Protein Eng, 1996. 9(7): p. 617-21; Atwell et al., JMB, 1997, 270, 26-35). In some embodiments, the amino acid is varied to be tyrosine (Y) [T366Y] at position 366 of the first Fc domain and threonine (T) [Y407T] at position 407 of the second Fc domain. In some embodiments, the first Fc domain comprises serine (S) [T366S] at position 366 and the second Fc domain comprises tryptophan (W) [T366W] at position 366, alanine (A) [L368A] at position 368 and valine (V) [Y407V] at position 407. In a preferred embodiment, the first Fc domain comprises tryptophan (W) [T366W] at position 366 and the second Fc domain comprises serine (S) [T366S] at position 366, alanine (A) [L368A] at position 368 and valine (V) [Y407V] at position 407. For example, according to the Eu number corresponding to amino acid position 146 in the human IgG1 Fc sequence of SEQ ID NO:241, position 366 of the Fc domain changes from T at position 146 in SEQ ID NO:241 to W at position 146 in SEQ ID NO:242; and according to the Eu number corresponding to amino acid positions 146, 148, and 187 in SEQ ID NO:241, positions 366, 368, and 407 change from T, L, and Y at the positions described in SEQ ID NO:241 to S, A, and V at the positions described in SEQ ID NO:243. In any of the embodiments, the amino acid changes for the first Fc domain may be located in the second Fc domain, and individual amino acid changes in the second Fc domain may be located in the first Fc domain. In other words, the terms "first" and "second" are interchangeable in the embodiments. In some embodiments, the Fc domain is an Fc domain derived from the heavy chain of IgG1 or IgG4.

[0254] In some embodiments, in addition to tryptophan (W) at position 366 [T366W], the first Fc domain also includes cysteine ​​(C) at position 354 [S354C], and in addition to serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V], the second Fc domain also includes cysteine ​​(C) at position 349 [Y349C]. In one aspect, the Fc domain is an Fc domain derived from the heavy chain of IgG4.

[0255] In some embodiments, the first or second Fc domain of the binding protein of the present invention further comprises one or more amino acid changes that reduce the binding of the Fc domain to protein A. In some embodiments, the amino acid change is arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436 of one of the Fc domains. Both changes are derived from the sequence of human IgG3 (IgG3 does not bind to protein A). These two mutations are located in the CH3 domain and incorporated into one of the Fc domains to reduce binding to protein A (see, for example, Jendeberg et al., J Immunol Methods, 1997. 201(1): pp. 25-34). These two changes facilitate the removal of homodimers of the heavy chain containing the changes during protein purification.

[0256] In some embodiments, in the binding protein of the present invention, the Fc domain comprising threonine (T) [Y407T] at position 407 further comprises arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436. In this case, the other heavy chain comprises tyrosine (Y) [T366Y] at position 366, but does not include the two variations at positions 435 and 436. Alternatively, in some embodiments, in the protein of the present invention, the Fc domain comprising serine (S) [T366S] at position 366, alanine (A) [L368A] at position 368, and valine (V) [Y407V] at position 407 further comprises arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436. In this case, the other Fc domain comprises tryptophan (W) [T366W] at position 366, but does not include the two variations at positions 435 and 436. Therefore, the Fc domain containing the amino acid changes that produce the "cavity" described above also contains amino acid changes that reduce binding to protein A. By reducing binding to protein A, the homodimer containing this Fc domain is removed. The presence of the "protrusion" reduces the formation of the homodimer containing another Fc domain containing the "protrusion".

[0257] In some embodiments, the Fc domain of the protein of the present invention may or may not further include the YTE mutation (M252Y / S254T / T256E, Eu number (Dall'Acqua et al. J. Biol. Chem. 2006, 281(33):23514-24). This mutation has been shown to improve the pharmacokinetic properties of the Fc domain by preferentially enhancing binding affinity to nascent FcRn receptors at pH 6.0.

[0258] In some embodiments, the first and / or second Fc domains of the present invention derived from IgG1 also include “KO” mutations (L234A, L235A) (Xu et al., Cellular Immunology, February 25, 2000, 200(1):16-26). In another embodiment, the first and / or second Fc domains of the present invention derived from IgG4 also include Pro hinge mutations (S228P) (Angal et al., Molecular Immunology 1993, 30(1):105-108; Labrijn et al., Nature Biotechnology 2009, 27:767-771).

[0259] In a preferred embodiment of the binding protein of the present invention, the first Fc domain comprises the amino acid sequence of SEQ ID NO:242 and the second Fc domain comprises the amino acid sequence of SEQ ID NO:243.

[0260] In a preferred embodiment of the present invention, the binding protein comprises i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:217 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#1 / CD3#1), or ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:218 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#2 / CD3#1), or iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:219 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#3 / CD3#1), or iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:220 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#4 / CD3#1), or v) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:221 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#5 / CD3#1), or vi) The first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#6 / CD3#1); or vii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:223 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#7 / CD3#1); or viii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:224 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#8 / CD3#1); or ix) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:225 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#9 / CD3#1); or x) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:226 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6 #10 / CD3#1); or xi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6 #10 / CD3#1); or xi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6 #10 / CD3#1); or xi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6 #10 / CD3#1). The first polypeptide chain containing the amino acid sequence of SEQ ID NO:227 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#11 / CD3#1); or xii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:228 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#12 / CD3#1); or xiii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:229 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#13 / CD3#1);Or xiv) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:230 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#14 / CD3#1); or xv) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:231 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#15 / CD3#1); or xvi) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:232 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#16 / CD3#1); or xvii) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:233 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#17 / CD3#1); or xviii) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:234 ​​and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311. The second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#18 / CD3#1); or xix) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:235 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#19 / CD3#1); or xx) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:236 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#20 / CD3#1); or xxi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:237 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#21 / CD3#1); or xxii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:238 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#22 / CD3#1); or xxiii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:239 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:235 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311. The second polypeptide chain (B7H6#23 / CD3#1) containing the amino acid sequence of SEQ ID NO:311; or xxiv) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:240 and the second polypeptide chain (B7H6#24 / CD3#1) containing the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked by one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0261] In a preferred embodiment, the first polypeptide chain comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 217, 218, 219, 220, 221, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 240, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 311. Even more preferably, the first polypeptide chain comprises an amino acid sequence consisting of any one of SEQ ID NO: 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 240, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 311, the first and second polypeptide chains being linked by one or more disulfide bonds and forming an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0262] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:228 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311.

[0263] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:230 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311.

[0264] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:231 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311.

[0265] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:232 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311.

[0266] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:239 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311.

[0267] For all embodiments described herein, it should be understood that the use of the term "comprising" is intended to also include embodiments in which the individual protein, molecule, antigen-binding unit, or polypeptide chain "consists of the indicated amino acid sequence":

[0268] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6, consisting of the amino acid sequence of SEQ ID NO:228, and a second polypeptide chain specific to CD3, consisting of the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0269] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6, consisting of the amino acid sequence of SEQ ID NO:230, and a second polypeptide chain specific to CD3, consisting of the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked by one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0270] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6, consisting of the amino acid sequence of SEQ ID NO:231, and a second polypeptide chain specific to CD3, consisting of the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0271] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6, consisting of the amino acid sequence of SEQ ID NO:232, and a second polypeptide chain specific to CD3, consisting of the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0272] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6, consisting of the amino acid sequence of SEQ ID NO:239, and a second polypeptide chain specific to CD3, consisting of the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0273] In another aspect, the present invention provides a binding protein comprising a first polypeptide chain (B7H6 chain) specifically binding to B7H6 and a second polypeptide chain (CD3 chain) specifically binding to CD3, wherein the first polypeptide chain specifically binding to B7H6 comprises a first light chain covalently linked (preferably directly bound) to a first adapter, the first adapter itself being covalently linked (e.g., directly bound) to a first heavy chain, and wherein the second polypeptide chain specifically binding to CD3 comprises a second light chain covalently linked (preferably directly bound) to a second adapter, the second adapter itself being covalently linked (e.g., directly bound) to a second heavy chain.

[0274] Unless otherwise defined herein, all definitions and preferred embodiments provided herein regarding the binding proteins of the present invention having specifically enumerated antigen-binding units are also applicable mutatis mutandis to such binding proteins of the present invention comprising first and second polypeptide chains.

[0275] In some embodiments, the first polypeptide chain (also referred to herein as the B7H6 chain) includes, starting from its N-terminus, a first light chain variable domain specifically binding to B7H6, a first light chain constant domain, a first linker, a first heavy chain variable domain specific to B7H6, and a first heavy chain constant region. In some embodiments, the second polypeptide chain (also referred to herein as the CD3 chain) includes, starting from its N-terminus, a second light chain variable domain specifically binding to CD3, a second light chain constant domain, a second linker, a second heavy chain variable domain specific to CD3, and a second heavy chain constant domain.

[0276] The resulting protein possesses an intact Fc, which is larger than IgG (due to the linker between the light and heavy chains) and has two independent binding sites (e.g., each binding site is monovalent for a specific antigen), with the first binding site targeting B7H6 and the second binding site targeting CD3. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds. Therefore, the protein of the present invention has an antibody-like structure, possessing the Y-shaped structure of a conventional full-length antibody (see...). Figure 1 The protein comprises two polypeptide chains, each containing an scFab and an Fc domain. In a preferred embodiment, the protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specific to B7H6, consisting of a first scFab and a first Fc domain specific to B7H6, and (ii) a second polypeptide chain (CD3 chain) specific to CD3, consisting of a second single-stranded Fab and a second Fc domain specific to CD3.

[0277] Preferably, the first scFab is directly covalently linked to the first Fc domain and the second scFab is directly covalently linked to the second Fc domain. This bispecific format typically reduces heterogeneity after expression and purification (e.g., by avoiding mismatches between light and medium variable domains with different binding specificities) while maintaining the functional property that the binding portion within the structure is unlikely to generate unwanted immunogenic responses. This also allows for good expression of the heterodimeric protein in, for example, mammalian cells.

[0278] In a preferred embodiment of the protein of the present invention, the first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprises a first light chain variable domain and a first heavy chain variable domain, which includes a CDR sequence selected from the group consisting of i) to xxiv):

[0279] i) The light chain CDR containing the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2) and SEQ ID NO:3 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2) and SEQ ID NO:6 (CDR3);

[0280] ii) Light chain CDRs containing the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2) and SEQ ID NO:9 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2) and SEQ ID NO:12 (CDR3);

[0281] iii) The light chain CDR containing the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2) and SEQ ID NO:15 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:16 (CDR1), SEQ ID NO:17 (CDR2) and SEQ ID NO:18 (CDR3);

[0282] iv) Light chain CDR containing the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2) and SEQ ID NO:21 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:24 (CDR3);

[0283] v) The light chain CDR containing the amino acid sequences of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2) and SEQ ID NO:27 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2) and SEQ ID NO:30 (CDR3);

[0284] vi) The light chain CDR containing the amino acid sequences of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2) and SEQ ID NO:33 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2) and SEQ ID NO:36 (CDR3);

[0285] vii) The light chain CDR containing the amino acid sequences of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2) and SEQ ID NO:39 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2) and SEQ ID NO:42 (CDR3);

[0286] viii) The light chain CDR containing the amino acid sequences of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2) and SEQ ID NO:45 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2) and SEQ ID NO:48 (CDR3);

[0287] ix) light chain CDR containing the amino acid sequences of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2) and SEQ ID NO:51 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2) and SEQ ID NO:54 (CDR3);

[0288] x) The light chain CDR containing the amino acid sequences of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2) and SEQ ID NO:57 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2) and SEQ ID NO:60 (CDR3);

[0289] xi) light chain CDR containing the amino acid sequences of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2) and SEQ ID NO:63 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2) and SEQ ID NO:66 (CDR3);

[0290] xii) The light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2) and SEQ ID NO:69 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2) and SEQ ID NO:72 (CDR3);

[0291] xiii) The light chain CDR containing the amino acid sequences of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2) and SEQ ID NO:75 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2) and SEQ ID NO:78 (CDR3);

[0292] xiv) The light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2) and SEQ ID NO:81 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2) and SEQ ID NO:84 (CDR3);

[0293] xv) light chain CDR containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2) and SEQ ID NO:87 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2) and SEQ ID NO:90 (CDR3);

[0294] xvi) The light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2) and SEQ ID NO:93 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2) and SEQ ID NO:96 (CDR3);

[0295] xvii) light chain CDR containing the amino acid sequences of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2) and SEQ ID NO:99 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2) and SEQ ID NO:102 (CDR3);

[0296] xviii) The light chain CDR containing the amino acid sequences of SEQ ID NO:103 (CDR1), SEQ ID NO:104 (CDR2) and SEQ ID NO:105 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2) and SEQ ID NO:108 (CDR3);

[0297] xix) light chain CDR containing the amino acid sequences of SEQ ID NO:109 (CDR1), SEQ ID NO:110 (CDR2) and SEQ ID NO:111 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:112 (CDR1), SEQ ID NO:113 (CDR2) and SEQ ID NO:114 (CDR3);

[0298] xx) light chain CDR containing the amino acid sequences of SEQ ID NO:115 (CDR1), SEQ ID NO:116 (CDR2) and SEQ ID NO:117 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:118 (CDR1), SEQ ID NO:119 (CDR2) and SEQ ID NO:120 (CDR3);

[0299] xxi) light chain CDR containing the amino acid sequences of SEQ ID NO:121 (CDR1), SEQ ID NO:122 (CDR2) and SEQ ID NO:123 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:124 (CDR1), SEQ ID NO:125 (CDR2) and SEQ ID NO:126 (CDR3);

[0300] xxii) The light chain CDR containing the amino acid sequences of SEQ ID NO:127 (CDR1), SEQ ID NO:128 (CDR2) and SEQ ID NO:129 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:130 (CDR1), SEQ ID NO:131 (CDR2) and SEQ ID NO:132 (CDR3);

[0301] xxiii) The light chain CDR containing the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3); and

[0302] xxiv) Light chain CDR containing the amino acid sequences of SEQ ID NO:139 (CDR1), SEQ ID NO:140 (CDR2) and SEQ ID NO:141 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:142 (CDR1), SEQ ID NO:143 (CDR2) and SEQ ID NO:144 (CDR3).

[0303] The individual light / heavy chain variable structural domains defined by the CDR sequence are respectively named B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24.

[0304] In a preferred embodiment of the binding protein of the present invention, the second polypeptide chain (CD3 chain) that specifically binds to CD3 includes a second light chain variable domain and a second heavy chain variable domain, and includes a CDR sequence selected from the group consisting of:

[0305] i) The light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2) and SEQ ID NO:259 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2) and SEQ ID NO:262 (CDR3);

[0306] ii) Light chain CDRs containing the amino acid sequences of SEQ ID NO:263 (CDR1), SEQ ID NO:264 (CDR2) and SEQ ID NO:265 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:266 (CDR1), SEQ ID NO:267 (CDR2) and SEQ ID NO:268 (CDR3);

[0307] iii) Light chain CDRs containing the amino acid sequences of SEQ ID NO:269 (CDR1), SEQ ID NO:270 (CDR2) and SEQ ID NO:271 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:272 (CDR1), SEQ ID NO:273 (CDR2) and SEQ ID NO:274 (CDR3);

[0308] iv) Light chain CDRs containing the amino acid sequences of SEQ ID NO:275 (CDR1), SEQ ID NO:276 (CDR2) and SEQ ID NO:277 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:278 (CDR1), SEQ ID NO:279 (CDR2) and SEQ ID NO:280 (CDR3);

[0309] v) The light chain CDR containing the amino acid sequences of SEQ ID NO:281 (CDR1), SEQ ID NO:282 (CDR2), and SEQ ID NO:283 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:284 (CDR1), SEQ ID NO:285 (CDR2), and SEQ ID NO:286 (CDR3); and

[0310] vi) Light chain CDR containing the amino acid sequences of SEQ ID NO:287 (CDR1), SEQ ID NO:288 (CDR2) and SEQ ID NO:289 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:290 (CDR1), SEQ ID NO:291 (CDR2) and SEQ ID NO:292 (CDR3).

[0311] The individual light / heavy chain variable structural domains defined by the CDR sequence are referred to as CD3#1, CD3#2, CD3#3, CD3#4, CD3#5 and CD3#6, respectively.

[0312] Preferably, the light chain and heavy chain CDR sequences are selected from the group consisting of the following: B7H6#1, B7H6#2, B7H6#, B7H6#4, B7H6#5, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24 as defined above.

[0313] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically binding to B7H6, comprising a first light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2), and SEQ ID NO:69 (CDR3) and a first heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2), and SEQ ID NO:72 (CDR3); and (ii) a second polypeptide chain specifically binding to CD3, comprising a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3). The second heavy chain variable domain of the heavy chain CDR (CDR3) amino acid sequence.

[0314] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically binding to B7H6, comprising a first light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2), and SEQ ID NO:81 (CDR3) and a first heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2), and SEQ ID NO:84 (CDR3); and (ii) a second polypeptide chain specifically binding to CD3, comprising a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262. The second heavy chain variable domain of the heavy chain CDR (CDR3) amino acid sequence.

[0315] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain specifically binding to B7H6, comprising a first light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3) and a first heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3); and (ii) a second polypeptide chain specifically binding to CD3, comprising a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a second heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:87 (CDR3). The second heavy chain variable domain of the heavy chain CDR of the amino acid sequence NO:262(CDR3).

[0316] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain specifically binding to B7H6, comprising a first light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2), and SEQ ID NO:93 (CDR3) and a first heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2), and SEQ ID NO:96 (CDR3); and (ii) a second polypeptide chain specifically binding to CD3, comprising a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a second heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:96 (CDR3). The second heavy chain variable domain of the heavy chain CDR of the amino acid sequence NO:262(CDR3).

[0317] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain specifically binding to B7H6, comprising a first light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and a first heavy chain variable domain having a heavy chain CDR containing the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3); and (ii) a second polypeptide chain specifically binding to CD3, comprising a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a second light chain variable domain having a light chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262. The second heavy chain variable domain of the heavy chain CDR (CDR3) amino acid sequence.

[0318] In a preferred embodiment of the protein of the present invention, the first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprises a light chain variable domain (first light chain variable domain) and a heavy chain variable domain (first heavy chain variable domain), selected from the group consisting of i) to xiv):

[0319] i) A light chain variable domain containing the amino acid sequence of SEQ ID NO:145 and a heavy chain variable domain (B7H6#1) containing the amino acid sequence of SEQ ID NO:146;

[0320] ii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:147 and the heavy chain variable domain (B7H6#2) containing the amino acid sequence of SEQ ID NO:148;

[0321] iii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:149 and the heavy chain variable domain (B7H6#3) containing the amino acid sequence of SEQ ID NO:150;

[0322] iv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:151 and the heavy chain variable domain (B7H6#4) containing the amino acid sequence of SEQ ID NO:152;

[0323] v) The light chain variable domain containing the amino acid sequence of SEQ ID NO:153 and the heavy chain variable domain (B7H6#5) containing the amino acid sequence of SEQ ID NO:154.

[0324] vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:155 and the heavy chain variable domain (B7H6#6) containing the amino acid sequence of SEQ ID NO:156.

[0325] vii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:157 and the heavy chain variable domain (B7H6#7) containing the amino acid sequence of SEQ ID NO:158;

[0326] viii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:159 and the heavy chain variable domain (B7H6#8) containing the amino acid sequence of SEQ ID NO:160;

[0327] ix) The light chain variable domain containing the amino acid sequence of SEQ ID NO:161 and the heavy chain variable domain (B7H6#9) containing the amino acid sequence of SEQ ID NO:162;

[0328] x) The light chain variable domain containing the amino acid sequence of SEQ ID NO:163 and the heavy chain variable domain (B7H6#10) containing the amino acid sequence of SEQ ID NO:164.

[0329] xi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:165 and the heavy chain variable domain (B7H6#11) containing the amino acid sequence of SEQ ID NO:166.

[0330] xii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:167 and the heavy chain variable domain (B7H6#12) containing the amino acid sequence of SEQ ID NO:168.

[0331] xiii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:169 and the heavy chain variable domain (B7H6#13) containing the amino acid sequence of SEQ ID NO:170.

[0332] xiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:171 and the heavy chain variable domain (B7H6#14) containing the amino acid sequence of SEQ ID NO:172.

[0333] xv) contains a light chain variable domain of the amino acid sequence of SEQ ID NO:173 and a heavy chain variable domain (B7H6#15) containing the amino acid sequence of SEQ ID NO:174.

[0334] xvi) contains a light chain variable domain of the amino acid sequence of SEQ ID NO:175 and a heavy chain variable domain (B7H6#16) of the amino acid sequence of SEQ ID NO:176.

[0335] xvii) contains a light chain variable domain of the amino acid sequence of SEQ ID NO:177 and a heavy chain variable domain (B7H6#17) of the amino acid sequence of SEQ ID NO:178.

[0336] xviii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:179 and the heavy chain variable domain (B7H6#18) containing the amino acid sequence of SEQ ID NO:180;

[0337] xix) contains a light chain variable domain of the amino acid sequence of SEQ ID NO:181 and a heavy chain variable domain (B7H6#19) containing the amino acid sequence of SEQ ID NO:182.

[0338] xx) The light chain variable domain containing the amino acid sequence of SEQ ID NO:183 and the heavy chain variable domain (B7H6#20) containing the amino acid sequence of SEQ ID NO:184;

[0339] xxi) contains a light chain variable domain of the amino acid sequence of SEQ ID NO:185 and a heavy chain variable domain (B7H6#21) containing the amino acid sequence of SEQ ID NO:186.

[0340] xxii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:187 and the heavy chain variable domain (B7H6#22) containing the amino acid sequence of SEQ ID NO:188;

[0341] xxiii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:189 and a heavy chain variable domain (B7H6#23) comprising the amino acid sequence of SEQ ID NO:190; and

[0342] xxiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:191 and the heavy chain variable domain (B7H6#24) containing the amino acid sequence of SEQ ID NO:192.

[0343] Preferably, the light chain variable and heavy chain variable structural domain sequences are selected from the group consisting of: B7H6#1, B7H6#2, B7H6#, B7H6#4, B7H6#5, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24 as defined above.

[0344] In a preferred embodiment of the protein of the present invention, the second polypeptide chain (CD3 chain) that specifically binds to CD3 includes a light chain variable domain (second light chain variable domain) and a heavy chain variable domain (second heavy chain variable domain), selected from the group consisting of:

[0345] i) A light chain variable domain containing the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:294 (CD3#1);

[0346] ii) A light chain variable domain containing the amino acid sequence of SEQ ID NO:295 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:296 (CD3#2);

[0347] iii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:297 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:298 (CD3#3);

[0348] iv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:299 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:300 (CD3#4);

[0349] v) A light chain variable domain containing the amino acid sequence of SEQ ID NO:301 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:302 (CD3#5);

[0350] vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:303 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:304 (CD3#6).

[0351] In some embodiments, the binding protein of the present invention comprises first and second polypeptide chains comprising CDR and / or VH and VL sequences of a light / heavy chain variable domain selected from the list of the following: B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3 #1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, B7H6#24 / CD3#1. In a preferred embodiment, the binding protein of the present invention comprises first and second polypeptide chains, which include CDR and / or VH and VL sequences of light / heavy chain variable domains selected from the list of the following: B7H6#12 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#23 / CD3#1. Even more preferably, the first polypeptide chain includes an Fc domain comprising the amino acid sequence of SEQ ID NO:242 and the second polypeptide chain includes an Fc domain comprising the amino acid sequence of SEQ ID NO:243.

[0352] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically bound to B7H6, which includes the light chain variable domain of SEQ ID NO:167 and the heavy chain variable domain of SEQ ID NO:168; and (ii) a second polypeptide chain (CD3 chain) specifically bound to CD3, which includes the light chain variable domain of SEQ ID NO:293 and the heavy chain variable domain of SEQ ID NO:294.

[0353] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically bound to B7H6, which includes a light chain variable domain of SEQ ID NO:171 and a heavy chain variable domain of SEQ ID NO:172; and (ii) a second polypeptide chain (CD3 chain) specifically bound to CD3, which includes a light chain variable domain of SEQ ID NO:293 and a heavy chain variable domain of SEQ ID NO:294.

[0354] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically bound to B7H6, which includes the light chain variable domain of SEQ ID NO:173 and the heavy chain variable domain of SEQ ID NO:174; and (ii) a second polypeptide chain (CD3 chain) specifically bound to CD3, which includes the light chain variable domain of SEQ ID NO:293 and the heavy chain variable domain of SEQ ID NO:294.

[0355] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically bound to B7H6, which includes the light chain variable domain of SEQ ID NO:175 and the heavy chain variable domain of SEQ ID NO:176; and (ii) a second polypeptide chain (CD3 chain) specifically bound to CD3, which includes the light chain variable domain of SEQ ID NO:293 and the heavy chain variable domain of SEQ ID NO:294.

[0356] In a preferred embodiment, the binding protein of the present invention comprises (i) a first polypeptide chain (B7H6 chain) specifically bound to B7H6, which includes the light chain variable domain of SEQ ID NO:189 and the heavy chain variable domain of SEQ ID NO:190; and (ii) a second polypeptide chain (CD3 chain) specifically bound to CD3, which includes the light chain variable domain of SEQ ID NO:293 and the heavy chain variable domain of SEQ ID NO:294.

[0357] In a preferred embodiment, the first polypeptide chain specific to B7H6 comprises a single-chain Fab having an amino acid sequence of any one of SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, or SEQ ID NO:216, and the second polypeptide chain specific to CD3 comprises a single-chain Fab having an amino acid sequence of SEQ ID NO:305.

[0358] Preferably, the first polypeptide chain comprises a single-chain Fab containing an amino acid sequence selected from the group consisting of: SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215 and SEQ ID NO:216, more preferably an amino acid sequence selected from the group consisting of: SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:216, ...7, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:216, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:208, SEQ ID NO: The second polypeptide chain comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, and SEQ ID NO:216, and the second polypeptide chain includes a single-chain Fab containing the amino acid sequence of SEQ ID NO:305.

[0359] In a preferred embodiment, the first polypeptide chain (B7H6 chain) specific to B7H6 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:204, and the second polypeptide chain (CD3 chain) specific to CD3 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:305.

[0360] In a preferred embodiment, the first polypeptide chain (B7H6 chain) specific to B7H6 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:206, and the second polypeptide chain (CD3 chain) specific to CD3 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:305.

[0361] In a preferred embodiment, the first polypeptide chain (B7H6 chain) specific to B7H6 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:207, and the second polypeptide chain (CD3 chain) specific to CD3 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:305.

[0362] In a preferred embodiment, the first polypeptide chain (B7H6 chain) specific to B7H6 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:208, and the second polypeptide chain (CD3 chain) specific to CD3 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:305.

[0363] In a preferred embodiment, the first polypeptide chain (B7H6 chain) specific to B7H6 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:215, and the second polypeptide chain (CD3 chain) specific to CD3 comprises a single-chain Fab containing the amino acid sequence of SEQ ID NO:305.

[0364] In addition, regarding this specific implementation scheme for scFabs, the terminology is also intended to include "composed of the amino acid sequence defined above," which has a more general meaning.

[0365] In some embodiments of the binding protein of the present invention, the first and second polypeptide chains comprise an Fc domain derived from a heavy chain of IgG (e.g., IgG1, IgG2, or IgG4). For example, the Fc domain of the present invention is the Fc domain of the heavy chain of IgG1 or IgG4 and includes a hinge region and two constant domains (CH2 and CH3). Examples of the Fc domain of human IgG are shown in SEQ ID NO:241 and SEQ ID NO:244.

[0366] In some embodiments of the binding protein of the present invention, the heavy chain comprises one or more amino acid variations. For example, the amino acid variation is tyrosine (Y) at position 366 of the first heavy chain [T366Y] and threonine (T) at position 407 of the second heavy chain [Y407T]. In some embodiments, the first heavy chain comprises serine (S) at position 366 [T366S], and the second heavy chain comprises tryptophan (W) at position 366 [T366W], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V]. In a preferred embodiment, the first heavy chain comprises tryptophan (W) at position 366 [T366W], and the second heavy chain comprises serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V]. For example, according to the Eu number corresponding to amino acid position 146 in the human IgG1 Fc sequence of SEQ ID NO:241, position 366 of the Fc domain changes from T at position 146 in SEQ ID NO:241 to W at position 146 in SEQ ID NO:242; and according to the Eu number corresponding to amino acid positions 146, 148, and 187 in SEQ ID NO:241, positions 366, 368, and 407 change from T, L, and Y at the positions described in SEQ ID NO:241 to S, A, and V at the positions described in SEQ ID NO:243. In any of the embodiments, the amino acid changes for the first heavy chain may be located in the second heavy chain, and individual amino acid changes in the second heavy chain may be located in the first heavy chain. In other words, the terms "first" and "second" are interchangeable in the embodiments. In some embodiments, the heavy chain is derived from the heavy chain of IgG1 or IgG4.

[0367] In some embodiments, the first or second heavy chain of the binding protein of the present invention further comprises one or more amino acid variations that reduce the binding of the heavy chain to protein A. In some embodiments, the amino acid variation is arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436 of one of the heavy chains.

[0368] In some embodiments, in the protein of the present invention, the heavy chain comprising threonine (T) [Y407T] at position 407 further comprises arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436. In this case, another heavy chain comprises tyrosine (Y) [T366Y] at position 366, but does not include the two variations at positions 435 and 436. Alternatively, in some embodiments, in the protein of the present invention, the heavy chain comprising serine (S) [T366S] at position 366, alanine (A) [L368A] at position 368, and valine (V) [Y407V] at position 407 further comprises arginine [H435R] at position 435 and phenylalanine [Y436F] at position 436. In this case, another heavy chain comprises tryptophan (W) [T366W] at position 366, but does not include the two variations at positions 435 and 436. Therefore, the heavy chain containing the amino acid changes that produce the "cavity" described above also contains amino acid changes that reduce binding to protein A. By reducing binding to protein A, the homodimer containing the heavy chain is removed. The presence of the "protrusion" reduces the formation of a homodimer of another heavy chain containing the "protrusion".

[0369] In some embodiments, the heavy chain of the protein of the present invention may or may not further contain the YTE mutation (M252Y / S254T / T256E, Eu number (Dall'Acqua et al. J. Biol. Chem. 2006, 281(33):23514-24). This mutation has been shown to improve the pharmacokinetic properties of the heavy chain by preferentially enhancing binding affinity to the nascent FcRn receptor at pH 6.0.

[0370] In some embodiments, the first and / or second heavy chains of the present invention derived from IgG1 also include “KO” mutations (L234A, L235A) (Xu et al., Cellular Immunology, February 25, 2000, 200(1):16-26). In another embodiment, the first and / or second heavy chains of the present invention derived from IgG4 also include Pro hinge mutations (S228P) (Angal et al., Molecular Immunology 1993, 30(1):105-108; Labrijn et al., Nature Biotechnology 2009, 27:767-771).

[0371] In a preferred embodiment of the binding protein of the present invention, the first polypeptide chain includes an Fc domain comprising the amino acid sequence of SEQ ID NO:242 and the second polypeptide chain includes an Fc domain comprising the amino acid sequence of SEQ ID NO:243.

[0372] In a preferred embodiment of the present invention, the binding protein comprises i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:217 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#1 / CD3#1), or ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:218 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#2 / CD3#1), or iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:219 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#3 / CD3#1), or iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:220 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#4 / CD3#1), or v) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:221 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:311 (B7H6#5 / CD3#1), or vi) The first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#6 / CD3#1); or vii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:223 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#7 / CD3#1); or viii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:224 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#8 / CD3#1); or ix) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:225 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#9 / CD3#1); or x) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:226 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#10 / CD3#1); or xi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#10 / CD3#1); or xi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#10 / CD3#1); or xi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:222 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#6 / CD3#1). The first polypeptide chain containing the amino acid sequence of SEQ ID NO:227 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#11 / CD3#1); or xii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:228 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#12 / CD3#1); or xiii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:229 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#13 / CD3#1);Or xiv) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:230 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#14 / CD3#1); or xv) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:231 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#15 / CD3#1); or xvi) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:232 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#16 / CD3#1); or xvii) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:233 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#17 / CD3#1); or xviii) a first polypeptide chain containing the amino acid sequence of SEQ ID NO:234 ​​and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:311. The second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#18 / CD3#1); or xix) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:235 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#19 / CD3#1); or xx) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:236 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#20 / CD3#1); or xxi) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:237 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#21 / CD3#1); or xxii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:238 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311 (B7H6#22 / CD3#1); or xxiii) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:239 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:235 and the second polypeptide chain containing the amino acid sequence of SEQ ID NO:311. The second polypeptide chain (B7H6#23 / CD3#1) containing the amino acid sequence of SEQ ID NO:311; or xxiv) the first polypeptide chain containing the amino acid sequence of SEQ ID NO:240 and the second polypeptide chain (B7H6#24 / CD3#1) containing the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked by one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0373] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence SEQ ID NO:228 and a second polypeptide chain specific to CD3 containing the amino acid sequence SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0374] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:230 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0375] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence of SEQ ID NO:231 and a second polypeptide chain specific to CD3 containing the amino acid sequence of SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0376] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific to B7H6 containing the amino acid sequence SEQ ID NO:232 and a second polypeptide chain specific to CD3 containing the amino acid sequence SEQ ID NO:311. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0377] In another aspect, the protein of the present invention comprises a first antigen-binding unit or polypeptide chain having specificity for B7H6, wherein the affinity for human and cynomolgus monkey B7H6 is preferably ≤ 10 nM, more preferably ≤ 1 nM, and even more preferably ≤ 0.1 nM. Affinity can be measured using recombinant B7H6-protein in an SPR (BIAcore® SPR system (GE Healthcare Life Sciences)) analysis, as described, for example, in the examples or other methods well known to those skilled in the art. The protein comprises a second antigen-binding unit or polypeptide chain having an affinity for the human and cynomolgus monkey CD3Ɛγ complex preferably ≤ 500 nM, more preferably ≤ 100 nM, and even more preferably ≤ 10 nM.

[0378] In another aspect, the B7H6 / CD3 binding protein of the present invention does not bind to B7H6-negative cells and does not cross-react with B7H1 (see, for example, Examples 10 and 4, respectively).

[0379] In a preferred embodiment, the B7H6 / CD3 binding proteins of the present invention (e.g., B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, and B7H6#24 / CD3#1 does not inhibit the activation of natural killer cells. It is noteworthy that the B7H6 / CD3 binding protein of this invention, which does not inhibit the activation of natural killer cells in vitro, binds to B7H6, wherein the NKp30 interaction site is substituted with an alanine residue.

[0380] B7H6 on the cell surface binds to NKp30 on the cell surface of NK cells, triggering NKp30-mediated NK cell activation, NK cell cytotoxicity, and cytokine secretion (Brandt et al., J. Exp. Med. 2009, 206(7):1495-503). This can be simulated in vitro by culturing NK cell lines (e.g., NK92MI) or primary NK cells on plates coated with recombinant B7H6 extracellular domain proteins, followed by analysis of upregulation of activation markers (e.g., CD25 or CD69) or NK cell cytokine secretion. This assay setup was used to evaluate whether our B7H6 / CD3 binding protein inhibits the interaction between B7H6 and NKp30, thereby leading to inhibition of IFNγ secretion (Example 11).

[0381] Using recombinant Ala-mutated B7H6 extracellular protein (with the NKp30 interaction site substituted with alanine), two groups of binding proteins were observed: 1) binding proteins that strongly bind to wild-type B7H6 but do not bind or only weakly bind to recombinant Ala-mutated B7H6 extracellular protein inhibited B7H6-dependent IFNγ secretion from NK cells in vitro (“inhibitors of B7H6-dependent NK cell activation”), and 2) binding proteins that strongly bind to wild-type B7H6 and maintain the ability to bind to recombinant Ala-mutated B7H6 extracellular protein do not inhibit B7H6-dependent activation and associated IFNγ secretion from NK cells in vitro (“non-inhibitors of B7H6-dependent NK cell activation”) (see Examples 6 and 11). Figure 4 and Figure 9 Surprisingly, the binding protein of the present invention, as a non-inhibitor of B7H6-dependent NK cell activation, is more potent in tumor cells expressing B7H6 through T cell redirection and lysis (see Example 12). Figure 10 and Figure 11 Unbound by theory, non-inhibitors of B7H6-dependent NK cell activation may allow B7H6-NKp30 interactions without affecting the natural role of B7H6 in mediating innate immunity.

[0382] On the other hand, the B7H6 / CD3 binding protein of the present invention can mediate T cell-redirected cytotoxicity against tumor cells, independent of NK cell activity (as shown in a mouse xenograft model lacking NK cells, see Example 19). Figure 20 and Figure 23 And as shown in the cell lysis analysis in the absence of NK cells, see Example 12. Figure 10 and Figure 11 ).

[0383] Various methods can be used to measure the cytotoxicity mediated by the B7H6 / CD3 binding protein of the present invention. For example, cytotoxicity can be measured using the method described in Example 12. Effector cells can be, for example, stimulated or unstimulated (human or cynomolgus monkey) T cells or their subgroups (e.g., CD4, CD8) or unstimulated (human or cynomolgus monkey) peripheral blood mononuclear cells (PBMCs). Target cells should express at least the extracellular domain of (human or cynomolgus monkey) B7H6 and can be cells expressing endogenous (natural) B7H6, such as human small cell lung cancer cell lines SHP77 and NCI-H82, or can be recombinant cells expressing full-length B7H6 or the extracellular domain of B7H6. The effector-to-target cell ratio (E:T) is typically about 10:1, but can vary. The cytotoxic activity of the B7H6 / CD3 binding molecule can be determined, for example, in an LDH release assay after 48 or 72 hours of incubation. The incubation time and readout modifications used to determine cytotoxicity are possible and known to those skilled in the art. Cytotoxicity readout systems may include MTT / MTS analysis, ATP-based analysis, FACS-based analysis, 51-chromium release analysis, sulfonyl rose red B (SRB) analysis, colorimetric (WST) analysis, homologous line formation analysis, ECIS technology, and bioluminescence analysis.

[0384] The cytotoxic activity mediated by the B7H6 / CD3 binding protein of the present invention is preferably measured in a cell-based cytotoxicity assay. Cytotoxicity is measured by EC50 in the cytotoxicity assay. 90 Values ​​are expressed. Those skilled in the art will understand that when purified T cells are used as effector cells, compared to PBMCs, EC is expected to... 90 Lower, as those skilled in the art will also understand, when stimulated T cells are used, EC 90 Even lower. Furthermore, it can be expected that when target cells express high levels of B7H6 on their cell surface, EC2 levels will be significantly lower compared to cells expressing low levels of B7H6 molecules on their cell surface. 90 The value is low. EC50 of B7H6 / CD3 binding protein 90 Preferred < 10nM, more preferably < 5 nM or even better < 1 nM.

[0385] Preferably, the multispecific binding protein of the present invention does not induce / mediate the lysis of B7H6-negative cells. The term "does not induce / mediate the lysis of B7H6-negative cells" means that the B7H6 / CD3 binding molecule does not induce or mediate the lysis of more than 30%, preferably no more than 20%, more preferably no more than 10%, and especially no more than 5% of B7H6-negative cells, while the lysis of B7H6-positive colorectal cell lines is set at 100%. This generally applies to binding protein concentrations up to 1000 nM.

[0386] Furthermore, the B7H6 / CD3 binding protein of the present invention exhibits a monomer content of over 95% during a two-step purification process (see Example 20), possesses favorable pharmacokinetic properties and good downstream manufacturability, and is further expected to have good biodistribution (e.g., see Example 18). The protein of the present invention further exhibits favorable immunogenicity (see Example 22) and good in vitro and in vivo stability (e.g., see Examples 21 and 18). Moreover, the B7H6 / CD3 binding protein of the present invention shows favorable efficacy in a humanized in vivo xenograft mouse model. The B7H6 / CD3 binding protein induces strong tumor regression, which begins immediately after the first administration of the B7H6 / CD3 binding protein (e.g., see Example 19). Furthermore, the B7H6 / CD3 binding protein of the present invention induces tumor regression at an extremely low dose of 0.05 mg / kg administered once weekly (q7d), further supporting its therapeutic applicability. Specifically, the B7H6 / CD3 binding protein of the present invention induces selective T cell proliferation, T cell activation, T cell degranulation and cytokine secretion only in the presence of B7H6 positive target cells and in the absence of B7H6 negative target cells (see Examples 16, 14, 15 and 17, respectively), and further significantly increases T cell infiltration into tumor tissue (see Example 24).

[0387] Another aspect of the present invention provides isolated nucleic acid molecules encoding first and / or second antigen-binding units of the multispecific binding protein of the present invention (respectively antigen-binding units B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H...). The sequence of any one of B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, and B7H6#24 and / or any one of the antigen-binding units CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, and CD3#6, as defined by the CDR, VH / VL, or scFab sequences as shown in Table 1. In some embodiments, the nucleic acid molecule further encodes a first and / or a second Fc domain as described herein, the first and / or the second Fc domain being respectively linked to the 3' end of the nucleic acid molecule encoding the first and / or the second antigen-binding unit. In some embodiments, the nucleic acid molecule encodes i) a first polypeptide chain comprising a first single-stranded Fab (e.g., any one of B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23, and B7H6#24) and a first Fc domain, and / or ii) The second polypeptide chain comprises a second single-chain Fab (e.g., any one of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5 and CD3#6, preferably CD3#1) and a second Fc domain that are specific to CD3.

[0388] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding a first single-stranded Fab specific to B7H6 and / or a second single-stranded Fab of SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215 or SEQ ID NO:216. In a preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a first scFab specific to B7H6 of any one of SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208 or SEQ ID NO:215; and / or a nucleotide sequence encoding a second scFab specific to CD3 comprising the amino acid sequence of SEQ ID NO:305.

[0389] Another aspect of the invention provides an expression vector comprising a DNA molecule containing a nucleotide sequence encoding a first and / or a second antigen-binding domain (e.g., the first and / or second single-stranded Fab of the present invention). Preferably, in addition to the nucleic acid molecule and, preferably, the DNA molecule encoding the first and / or second Fc domain linked to the nucleic acid molecule, the expression vector also preferably comprises a DNA molecule encoding the first and / or the second antigen-binding domain (e.g., the first and / or the second single-stranded Fab). Thus, the expression vector comprises a nucleotide sequence encoding a polypeptide chain comprising a first single-stranded Fab linked to the first Fc domain and / or a nucleotide sequence encoding a polypeptide chain comprising a second single-stranded Fab linked to the second Fc domain.

[0390] In a preferred embodiment, the expression vector contains a DNA molecule comprising nucleotide sequences encoding a first polypeptide chain specific to B7H6 and / or a second polypeptide chain specific to CD3 of the present invention. In a preferred embodiment, the expression vector comprises a nucleotide sequence encoding a first polypeptide chain of any one of SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, or SEQ ID NO:240 and / or a nucleotide sequence encoding a second polypeptide chain comprising SEQ ID NO:311.

[0391] In other preferred embodiments, the expression vector comprises a nucleotide sequence encoding a first polypeptide chain of any one of SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232 and SEQ ID NO:239 and / or a nucleotide sequence encoding a second polypeptide chain comprising SEQ ID NO:311.

[0392] In a particularly preferred embodiment, two expression vectors can be used, one for expressing a first polypeptide chain specific to B7H6 and the other for expressing a second polypeptide chain specific to CD3, and then the two expression vectors can be transfected into host cells to express the recombinant protein.

[0393] Preferably, the expression vector will be a vector containing the nucleic acid molecule(s) operably linked to at least one regulatory sequence, wherein the regulatory sequence may be a promoter, enhancer, or terminator sequence, and most preferably a heterologous promoter, enhancer, or terminator sequence.

[0394] In another aspect, the present invention relates to a host cell having an expression vector encoding a first polypeptide chain of the present invention that is specific to B7H6 and an expression vector encoding a second polypeptide chain of the present invention that is specific to CD3.

[0395] In a particularly preferred embodiment, the host cell is a eukaryotic cell, such as a mammalian cell. In another embodiment, the host cell is a bacterial cell. Other useful cell lines include yeast cells or other fungal cells.

[0396] Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins may also be used.

[0397] Anti-B7H6 antibody

[0398] Another aspect of the present invention provides an anti-B7H6 antibody molecule comprising

[0399] i) The light chain CDR containing the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2), and SEQ ID NO:3 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2), and SEQ ID NO:6 (CDR3); or

[0400] ii) The light chain CDR containing the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3); or

[0401] iii) The light chain CDR containing the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:15 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:16 (CDR1), SEQ ID NO:17 (CDR2), and SEQ ID NO:18 (CDR3); or

[0402] iv) The light chain CDR containing the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2), and SEQ ID NO:21 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2), and SEQ ID NO:24 (CDR3); or

[0403] v) The light chain CDR containing the amino acid sequences of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2), and SEQ ID NO:30 (CDR3); or

[0404] vi) The light chain CDR containing the amino acid sequences of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2), and SEQ ID NO:33 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2), and SEQ ID NO:36 (CDR3); or

[0405] vii) The light chain CDR containing the amino acid sequences of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:39 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2), and SEQ ID NO:42 (CDR3); or

[0406] viii) The light chain CDR containing the amino acid sequences of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2), and SEQ ID NO:45 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2), and SEQ ID NO:48 (CDR3); or

[0407] ix) a light chain CDR containing the amino acid sequences of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3); or

[0408] x) The light chain CDR containing the amino acid sequences of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3); or

[0409] xi) The light chain CDR containing the amino acid sequences of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2), and SEQ ID NO:63 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2), and SEQ ID NO:66 (CDR3); or

[0410] xii) The light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2), and SEQ ID NO:69 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2), and SEQ ID NO:72 (CDR3); or

[0411] xiii) The light chain CDR containing the amino acid sequences of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2), and SEQ ID NO:75 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2), and SEQ ID NO:78 (CDR3); or

[0412] xiv) The light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2), and SEQ ID NO:81 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2), and SEQ ID NO:84 (CDR3); or

[0413] xv) light chain CDRs containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3); or

[0414] xvi) The light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2), and SEQ ID NO:93 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2), and SEQ ID NO:96 (CDR3); or

[0415] xvii) light chain CDRs containing the amino acid sequences of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2), and SEQ ID NO:99 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2), and SEQ ID NO:102 (CDR3); or

[0416] xviii) The light chain CDR containing the amino acid sequences of SEQ ID NO:103 (CDR1), SEQ ID NO:104 (CDR2), and SEQ ID NO:105 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2), and SEQ ID NO:108 (CDR3); or

[0417] xix) a light chain CDR containing the amino acid sequences of SEQ ID NO:109 (CDR1), SEQ ID NO:110 (CDR2), and SEQ ID NO:111 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:112 (CDR1), SEQ ID NO:113 (CDR2), and SEQ ID NO:114 (CDR3); or

[0418] xx) light chain CDRs containing the amino acid sequences of SEQ ID NO:115 (CDR1), SEQ ID NO:116 (CDR2), and SEQ ID NO:117 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:118 (CDR1), SEQ ID NO:119 (CDR2), and SEQ ID NO:120 (CDR3); or

[0419] xxi) light chain CDRs containing the amino acid sequences of SEQ ID NO:121 (CDR1), SEQ ID NO:122 (CDR2), and SEQ ID NO:123 (CDR3) and heavy chain CDRs containing the amino acid sequences of SEQ ID NO:124 (CDR1), SEQ ID NO:125 (CDR2), and SEQ ID NO:126 (CDR3); or

[0420] xxii) The light chain CDR containing the amino acid sequences of SEQ ID NO:127 (CDR1), SEQ ID NO:128 (CDR2), and SEQ ID NO:129 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:130 (CDR1), SEQ ID NO:131 (CDR2), and SEQ ID NO:132 (CDR3); or

[0421] xxiii) The light chain CDR containing the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and the heavy chain CDR containing the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3); or

[0422] xxiv) Light chain CDR containing the amino acid sequences of SEQ ID NO:139 (CDR1), SEQ ID NO:140 (CDR2) and SEQ ID NO:141 (CDR3) and heavy chain CDR containing the amino acid sequences of SEQ ID NO:142 (CDR1), SEQ ID NO:143 (CDR2) and SEQ ID NO:144 (CDR3).

[0423] The antibodies i) to xxiv) as summarized above are respectively designated as B7H6#1, B7H6#2, B7H6#3, B7H6#4, B7H6#5, B7H6#6, B7H6#7, B7H6#8, B7H6#9, B7H6#10, B7H6#11, B7H6#12, B7H6#13, B7H6#14, B7H6#15, B7H6#16, B7H6#17, B7H6#18, B7H6#19, B7H6#20, B7H6#21, B7H6#22, B7H6#23 and B7H6#24. A sequence listing is provided herein, which readily allows for the identification of individual amino acid sequences of the specific antibodies against this invention.

[0424] In some embodiments, the anti-B7H6 antibody of the present invention is a chimeric, humanized, human, or optimized antibody molecule. In some embodiments, the antibody molecule is a monoclonal antibody Fab, F(ab)2, Fv, or scFv. In some embodiments, the anti-B7H6 antibody molecule of the present invention comprises a heavy chain constant region selected from the group consisting of constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. In some embodiments, the light chain constant region of the anti-B7H6 antibody molecule of the present invention is κ or λ.

[0425] In some embodiments, the anti-B7H6 antibody of the present invention has a heavy chain variable domain comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, and 192. Preferably, the antibody molecule has a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192.

[0426] In some embodiments, the anti-B7H6 antibody molecule has a light chain variable domain comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, and 191. Preferably, the antibody molecule has a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 and 191.

[0427] The methods for calculating amino acid sequence identity are known in the art and have been further discussed in the definition section of this specification.

[0428] In some embodiments, the anti-B7H6 antibody molecule has i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:146 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:145 (B7H6#1), or ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:148 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:147 (B7H6#2); or iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:150 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:149 (B7H6#3), or iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:152 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:151 (B7H6#4); or v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:154 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:145. The light chain variable domain of the amino acid sequence of SEQ ID NO:153 (B7H6#5); or vi) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:156 and the light chain variable domain of the amino acid sequence of SEQ ID NO:155 (B7H6#6); or vii) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:158 and the light chain variable domain of the amino acid sequence of SEQ ID NO:157 (B7H6#7); or viiii) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:160 and the light chain variable domain of the amino acid sequence of SEQ ID NO:159 (B7H6#8); or ix) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:162 and the light chain variable domain of the amino acid sequence of SEQ ID NO:161 (B7H6#9); or x) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:164 and the light chain variable domain of SEQ ID NO:155 (B7H6#6). The light chain variable domain of the amino acid sequence NO:163 (B7H6#10); or xi) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:166 and the light chain variable domain of the amino acid sequence of SEQ ID NO:165 (B7H6#11); or xii) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:168 and the light chain variable domain of the amino acid sequence of SEQ ID NO:167 (B7H6#12); or xiii) the heavy chain variable domain of the amino acid sequence of SEQ ID NO:170 and the light chain variable domain of the amino acid sequence of SEQ ID NO:169 (B7H6#13);Or xiv) the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:172 and the light chain variable domain containing the amino acid sequence of SEQ ID NO:171 (B7H6#14); or xv) the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:174 and the light chain variable domain containing the amino acid sequence of SEQ ID NO:173 (B7H6#15); or xvi) the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:176 and the light chain variable domain containing the amino acid sequence of SEQ ID NO:175 (B7H6#16); or xvii) the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:178 and the light chain variable domain containing the amino acid sequence of SEQ ID NO:177 (B7H6#17); or xviii) the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:180 and the light chain variable domain containing the amino acid sequence of SEQ ID NO:172 and the light chain variable domain containing the amino acid sequence of SEQ ID NO:173 (B7H6#15); The light chain variable domain of the amino acid sequence NO:179 (B7H6#18); or xix) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:182 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:181 (B7H6#19); or xx) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:184 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:183 (B7H6#20); or xxi) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:186 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:185 (B7H6#21); or xxii) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:188 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:187 (B7H6#22); or xxiii) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:182 ...3 (B7H6#20); or xxi) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:182 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:183 (B7H6#21); or xxi) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:182 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:183 (B7H6#22); or xxi) the heavy chain variable domain of the amino acid sequence containing SEQ ID NO:182 and the light chain variable domain of the amino acid sequence containing SEQ ID NO:183 (B7H6#22); or xxi) the heavy chain variable domain of the amino acid sequence containing The heavy chain variable domain of the amino acid sequence of SEQ ID NO: 190 and the light chain variable domain of the amino acid sequence of SEQ ID NO: 189 (B7H6#23); or xxiv) the heavy chain variable domain of the amino acid sequence of SEQ ID NO: 192 and the light chain variable domain of the amino acid sequence of SEQ ID NO: 191 (B7H6#24).

[0429] In some embodiments, the anti-B7H6 antibody of the present invention is a mouse monoclonal antibody. In the context of the present invention, a mouse monoclonal antibody comprises antibodies with VH and VL obtained by: immunizing mice with human B7H6 protein, subsequently selecting suitable VH and VL sequences with a specific affinity for human B7H6 to bind, and then further conjugating the VH and VL sequences to constant domains derived from mice (e.g., mouse IgG2a) via recombinant technology; and it is produced by recombinant expression in host cells. The present invention further covers chimeric antibodies, for example, comprising variable and constant regions from different species. In some embodiments, the antibody molecule of the present invention is a chimeric antibody comprising mouse-derived VH and VL domains as described above and further comprising constant domains derived from another species (e.g., human, rabbit, rat, goat, donkey). In some embodiments, the chimeric antibody comprises mouse-derived VH and VL domains and is further humanized or sequence-optimized as defined above and further comprises constant domains derived from another species. In some embodiments, the chimeric antibody comprises VH and VL domains derived from a transgenic animal (e.g., a mouse) containing a human IgG sequence, and thus comprises human VH and VL sequences, and further comprises a constant domain derived from another species. In any of the embodiments of the chimeric antibody as outlined above, the heavy chain constant region is a mouse, human, rabbit, rat, goat, or donkey heavy chain region.

[0430] In some embodiments, the anti-B7H6 antibody molecule of the present invention has a constant domain selected from the group consisting of constant domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. In a preferred embodiment, the anti-B7H6 antibody has a constant domain of IgG2a. In some embodiments, the anti-B7H6 antibody molecule has a light chain constant domain of κ or λ, preferably a κ light chain constant domain, and preferably includes the sequence of SEQ ID NO:247.

[0431] The B7H6-specific antibodies provided herein can be used to label, locate, identify, or target cells expressing B7H6 by linking them to dyes, drugs, or other molecules that have binding specificity to different antigens (e.g., in ELISA, FACS, immunohistochemistry, or the like).

[0432] Another aspect of the invention provides isolated nucleic acid molecules that encode the heavy chain variable domain and / or light chain variable domain of the anti-B7H6 antibody molecule of the invention.

[0433] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable domain of any one of SEQ ID NO: 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, or 192. Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable domain of any one of SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or 191.

[0434] Another aspect of the invention provides an expression vector containing a DNA molecule comprising a nucleotide sequence encoding a heavy chain variable domain and / or a light chain variable domain of the anti-B7H6 antibody molecule of the present invention.

[0435] Preferably, in addition to the nucleic acid molecule, the expression vector also includes a DNA molecule preferably linked to the nucleic acid molecule that encodes a constant domain of the heavy chain and / or a constant domain of the light chain, and preferably a DNA molecule that encodes a variable domain of the heavy chain and / or a variable domain of the light chain, respectively.

[0436] In a particularly preferred embodiment, two expression vectors can be used, one for expressing the heavy chain and the other for expressing the light chain, and then the two expression vectors can be transfected into host cells to express the recombinant protein.

[0437] Preferably, the expression vector will be a vector containing the nucleic acid molecule(s) operably linked to at least one regulatory sequence, wherein the regulatory sequence may be a promoter, enhancer, or terminator sequence, and most preferably a heterologous promoter, enhancer, or terminator sequence.

[0438] In another aspect, the present invention relates to a host cell having an expression vector encoding the heavy chain of the anti-B7H6 antibody molecule of the present invention and an expression vector encoding the light chain of the anti-B7H6 antibody molecule of the present invention.

[0439] According to a particularly preferred embodiment, the host cell line is a eukaryotic cell, such as a mammalian cell. In another embodiment, the host cell line is a bacterial cell. Other useful cell lines include yeast cells or other fungal cells.

[0440] Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins may also be used.

[0441] Manufacturing and purification methods

[0442] The present invention further provides a method for manufacturing the multispecific binding protein of the present invention, the method generally comprising the following steps:

[0443] - Culture host cells containing an expression vector, which contains nucleic acid encoding the binding protein of the present invention, under conditions that allow for the formation of the binding protein of the present invention; and

[0444] - Self-cultured organisms recover binding proteins expressed by host cells; and

[0445] - Optional further purification and / or modification and / or formulation of the binding protein of the present invention.

[0446] The present invention further provides a method for manufacturing the anti-B7H6 antibody of the present invention, the method generally comprising the following steps:

[0447] - Culture host cells containing an expression vector, which contains nucleic acid encoding the antibody molecule of the present invention, under conditions that allow for the formation of antibody molecules; and

[0448] - Self-cultured antibody molecules expressed by host cells; and

[0449] - Optional further purification and / or modification and / or formulation of the antibody molecules of the present invention.

[0450] The nucleic acid of the present invention may be, for example, a DNA molecule comprising a coding sequence and a regulatory sequence and optionally natural or artificial introns, or may be a cDNA molecule. It may have its initial codon or may have optimized codon usage, which has been specifically modified for expression in the intended host cell or host organism. According to one embodiment of the invention, the nucleic acid of the present invention is in a substantially segregated form as defined above.

[0451] The nucleic acids of the present invention can be prepared or obtained in a manner known per se (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the proteins of the present invention as given herein.

[0452] The nucleic acids of this invention are typically incorporated into expression vectors, which provide vectors for protein expression when transfected into suitable host cells or other expression systems.

[0453] In order to manufacture the binding protein or antibody of the present invention, those skilled in the art may choose from a variety of expression systems well known in the art, such as those reviewed by Kipriyanow and Le Gall, 2004.

[0454] Expression vectors include plasmids, retroviruses, viscera, EBV-derived cell-free genomes, and the like. An expression vector and expression control sequence compatible with the host cell are selected. The nucleotide sequences encoding a first antigen-binding unit (e.g., a B7H6-specific single-stranded Fab or a full-length B7H6 chain of the binding protein of the present invention) and a second antigen-binding unit (e.g., a CD3-specific single-stranded Fab or a full-length CD3 chain of the binding protein of the present invention) can be inserted into separate vectors. In some embodiments, both DNA sequences are inserted into the same expression vector. The nucleotide sequences encoding the light chain of a B7H6 antibody and the heavy chain of a B7H6 antibody can be inserted into separate vectors. In some embodiments, both DNA sequences are inserted into the same expression vector.

[0455] Convenient vectors are those encoding functionally complete human CH (constant heavy chain) immunoglobulin sequences with engineered, appropriately restricted sites that allow for the easy insertion and expression of any antigen-binding unit (e.g., a single-chain Fab sequence) or any heavy / light chain variable domain, as described above. For antibody heavy chains, these can be, but are not limited to, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.

[0456] Recombinant expression vectors may also encode signal peptides that promote the secretion of full-length CD3 or B7H6 chains from host cells or the secretion of the light / heavy chain of anti-B7H6 antibodies. DNA encoding the protein chain can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of mature full-length DNA. The signal peptide may be an immunoglobulin signal peptide or a heterologous peptide derived from a non-immunoglobulin. Alternatively, the full-length DNA sequence encoding the protein of this invention may already contain the signal peptide sequence.

[0457] In addition to the DNA sequence encoding the B7H6 / CD3 chain or the DNA sequence encoding the heavy / light chain of the B7H6 antibody, recombinant expression vectors typically also carry regulatory sequences, optionally heterologous regulatory sequences, including promoters, enhancers, termination and polyadenylation signals, and other expression control components that control the expression of the protein chain in the host cell. Examples of promoter sequences (illustrated for expression in mammalian cells) are promoters and / or enhancers derived from CMV (e.g., the CMV simian virus 40 (SV40) promoter / enhancer), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomatous and strong mammalian promoters, such as innate immunoglobulin and actin promoters. Examples of polyadenylation signals are BGH polyA, SV40 late or early polyA; alternatively, 3'UTRs of immunoglobulin genes, etc., may be used.

[0458] Recombinant expression vectors may also carry sequences (e.g., origin of replication) regulating vector replication in host cells and selection marker genes. Nucleic acid molecules encoding a full-length chain having a first antigen-binding unit (single-stranded Fab and Fc domains) or its antigen-binding portion, and / or a full-length chain having a second antigen-binding unit (single-stranded Fab and Fc domains) or its antigen-binding portion, and vectors containing said DNA molecules, may be introduced into host cells (e.g., bacterial cells or higher eukaryotic cells, such as mammalian cells) using transfection methods well known in the art (including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer via viral vectors).

[0459] Preferably, the DNA molecules encoding the B7H6 and CD3 strands of the protein of the present invention are present on two expression vectors co-transfected into host cells (preferably mammalian cells).

[0460] Mammalian cell lines that can be used as hosts for expression are well known in the art, and particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human cancer cells (e.g., Hep G2 and A-549 cells), 3T3 cells, or any derivatives / progeny of such cell lines. Other mammalian cells (including, but not limited to, human, mouse, rat, monkey, and rodent cell lines), or other eukaryotic cells (including, but not limited to, yeast, insect, and plant cells), or prokaryotic cells (e.g., bacteria) may be used.

[0461] The proteins of this invention are produced by culturing host cells for a period sufficient to allow protein expression within the host cells. The protein molecules are preferably recovered from the culture medium as secreted polypeptides, or, if expressed, for example, in the absence of a secretion signal, from host cell lysates. The protein molecules need to be purified using standard protein purification methods for recombinant proteins and host cell proteins to obtain substantially homogeneous protein formulations. For example, state-of-the-art purification methods currently available for obtaining the protein molecules of this invention include the removal of cell and / or granular cell debris from the culture medium or lysates as a first step. The proteins are then purified from contamination with soluble proteins, polypeptides, and nucleic acids, for example, by fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed-phase HPLC, Sephadex chromatography, or chromatography on silica or cation exchange resins. As a final step in obtaining the protein molecule formulation, the purified protein molecules can be dried, for example, lyophilized, as described below for therapeutic applications.

[0462] This invention relates to binding proteins that have binding specificity to at least two different targets. In this invention, the binding molecules are derived from antibodies. Techniques for preparing the binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin chains with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168; Atwell et al., JMB, 1997, 270, 26-35). The binding protein of the present invention can also be prepared by: engineering an electrostatic steering effect to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (e.g., see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific proteins (e.g., see Kostelny et al., Immunol., 148(5): 1547-1553 (1992)); using “bivalent antibody” technology to prepare bispecific antibody fragments (e.g., see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (e.g., see Gruber et al., Immunol., 152:5368 (1994); and the preparation of trispecific antibodies, as described (for example) in Tutt et al. Immunol. 147: 60 (1991).

[0463] The compositions (e.g., multispecific binding proteins and anti-B7H6 antibodies) and methods disclosed herein cover polypeptides and nucleic acids having a specified sequence or a sequence substantially identical or similar to it (e.g., a sequence that is at least 85%, 90%, 95% identical or higher to a specified sequence). In the context of amino acid sequences, the term "substantially identical" is used herein to mean that the first amino acid contains a sufficient or minimum number of i) amino acid residues identical to the matched amino acid residues in the second amino acid sequence, or ii) conserved substitutions of the matched amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences may have a common structural domain and / or common functional activity. For example, an amino acid sequence containing a common structural domain that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence (e.g., the sequence provided herein). In the context of nucleotide sequences, the term “substantially identical” is used herein to refer to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to those aligned to a second nucleic acid sequence, such that the first and second nucleotide sequences encode a polypeptide with common functional activity, or a polypeptide encoding a common structural polypeptide domain or common functional polypeptide activity, for example, a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence.

[0464] The nucleic acid molecules of this invention include, but are not limited to, DNA molecules encoding polypeptide sequences shown in the sequence listing. Furthermore, this invention also relates to nucleic acid molecules that hybridize with DNA molecules encoding polypeptide sequences shown in the sequence listing under highly stringent binding and washing conditions, as defined in WO 2007 / 042309. Preferred molecules (from an mRNA perspective) are those having at least 75% or 80% (preferably at least 85%, more preferably at least 90%, and most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein. For example, the DNA sequences shown in the sequence listing are designed to match codon usage in eukaryotic cells for antibody expression. If antibody expression in *E. coli* is desired, the sequence can be modified to match *E. coli* codon usage. Variants of the DNA molecules of this invention can be constructed using several different methods, as described, for example, in WO 2007 / 042309.

[0465] The proteins of the present invention may have modified N-terminal sequences, such as the deletion of one or more N-terminal amino acids, or the exchange of, for example, the first N-terminal amino acid (e.g., glutamate to alanine), to optimize the molecule for expression using certain expression systems (e.g., specific vectors or host cells), or for expression in inclusion bodies or in soluble form, or for secretion into culture media or periplasmic space or inclusion within cells, or for producing more homogeneous products. The polypeptides of the present invention may have modified C-terminal sequences (e.g., additional alanine), and / or further amino acid exchanges at other defined positions in the C-terminal portion or within any frame region, as explained, for example, in WO2012 / 175741, WO2011 / 075861, or WO2013 / 024059, to, for example, further enhance the stability of said polypeptide or reduce its immunogenicity.

[0466] For the avoidance of doubt, all embodiments relating to the pharmaceutical compositions, kits, treatment methods, medical uses, combinations, administration methods and dosages described herein are intended for use with any of the multispecific binding proteins described herein, alone or in combination with other therapeutic agents (as described in more detail below).

[0467] Drug composition, method of administration, dosage

[0468] The present invention further relates to pharmaceutical compositions for treating diseases (as described in more detail below), wherein said compositions comprise at least one multispecific binding protein of the present invention. The present invention further covers methods for treating diseases using at least one multispecific binding protein of the present invention or the pharmaceutical compositions described below (as described in more detail below), and further covers the preparation of medicaments for treating the disease using the binding protein or pharmaceutical composition of the present invention.

[0469] The binding proteins of this invention (e.g., B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H...) Any one of B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, B7H6#24 / CD3#1 (as defined by the sequences shown in Table 1) and / or compositions containing thereof may be administered to patients in need in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Therefore, the binding protein and / or compositions comprising it of the present invention can be administered, for example, intravenously (iv), subcutaneously (sc), intramuscularly (im), intraperitoneally (ip), percutaneously, orally, sublingually (e.g., in the form of sublingual tablets, sprays or drops placed under the tongue and absorbed through the capillary network under the tongue via the mucosa), nasally (e.g., in the form of nasal sprays and / or as aerosols), topically, by means of suppositories, by inhalation, or by any other suitable manner in an effective amount or dose. The binding protein can be administered by infusion, concentration, or injection. In a preferred embodiment, administration is performed by intravenous infusion or subcutaneous injection.

[0470] The binding proteins and / or compositions comprising them of the present invention are administered according to a treatment regimen suitable for treating and / or alleviating a disease, symptom, or condition to be treated or alleviated. Clinicians are typically able to determine an appropriate treatment regimen based on factors such as the disease, symptom, or condition to be treated or alleviated, the severity of the disease, the severity of its symptoms, the specific binding protein of the present invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, age, sex, weight, diet, the patient's general condition, and similar factors known to the clinician. Typically, the treatment regimen will involve administering one or more binding proteins of the present invention, or one or more compositions comprising them, at a therapeutically effective amount or dose.

[0471] Typically, for the treatment and / or alleviation of the diseases, symptoms, and conditions mentioned herein, and depending on the specific disease, symptom, or condition to be treated, the potency of the specific binding protein of the present invention to be used, the specific route of administration, and the specific pharmaceutical formulation or composition, the binding protein of the present invention is usually administered in an amount between 0.005 and 20.0 mg / kg body weight, preferably between 0.05 and 10.0 mg / kg / dose, continuously (e.g., by infusion) or more preferably as a single dose (e.g., twice weekly, once weekly, once every two or three weeks, or once monthly; see below), but can vary significantly, particularly depending on the parameters mentioned above. Therefore, in some cases, using a dose lower than the minimum given above may be sufficient, while in other cases it may be necessary to exceed the upper limit. When administering in large doses, it is recommended to divide it into multiple smaller doses distributed over a period of time (e.g., two days or more).

[0472] According to the invention, the specific binding protein and its particular pharmacokinetics and other properties can be administered daily, every second day, every third day, every fourth day, every fifth day or every sixth day, weekly, every two or three weeks, monthly, and so on. Administration regimens may include long-term treatment. “Long-term” means a duration of at least two weeks, and preferably several months or years.

[0473] The efficacy of the multispecific binding proteins of the present invention and compositions comprising them can be tested, depending on the specific disease involved, using any suitable in vitro assay, cell-based assay, in vivo assay, and / or animal models known per se, or any combination thereof. Suitable assays and animal models will be apparent to those skilled in the art, and include, for example, the assays and animal models used in the following embodiments.

[0474] Mixtures

[0475] For pharmaceutical use, the binding protein of the present invention can be formulated into a pharmaceutical preparation comprising (i) at least one binding protein of the present invention (e.g., B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7 (i) any one of H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, B7H6#24 / CD3#1) and (ii) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and / or stabilizer, and (iii) optionally one or more other pharmacologically active peptides and / or compounds.

[0476] "Pharmaceutically acceptable" means that, when administered to an individual, the individual material does not exhibit any biological or other undesirable effects and does not interact in a harmful manner with any other component (e.g., the active pharmaceutical ingredient) of the pharmaceutical composition comprising it. Specific examples can be found in standard manuals, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, USA (1990). For example, the binding protein of the present invention can be formulated and administered in any manner known per se for use with conventional antibodies and antibody fragments and other pharmaceutically active proteins. Therefore, according to yet another embodiment, the present invention relates to pharmaceutical compositions or formulations containing at least one binding protein of the present invention and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and / or stabilizer, and optionally one or more other pharmacologically active substances, in lyophilized or other dried formulations or in aqueous or non-aqueous solutions or suspensions.

[0477] Pharmaceutical formulations intended for non-enteral administration (e.g., intravenous, intramuscular, subcutaneous, or intravenous infusion) may be, for example, sterile solutions, suspensions, dispersions, emulsions, or powders containing the active ingredient, and optionally, after further dissolution or dilution steps, suitable for infusion or injection. Suitable carriers or diluents for said formulations include, for example (but not limited to), sterile water and pharmaceutically acceptable aqueous buffers and solutions, such as physiological phosphate-buffered saline, Ringer's solutions, dextran solutions, and Hank's solution; water-oil; glycerol; ethanol; glycols, such as propylene glycol; and mineral oils, animal oils, and vegetable oils, such as peanut oil, soybean oil, and suitable mixtures thereof.

[0478] The protein-binding solution of the present invention may also contain preservatives to prevent microbial growth, such as antibacterial and antifungal agents, such as parabens, chlorobutanol, phenols, sorbic acid, thimerosal, ethylenediaminetetraacetic acid (an alkali metal salt), and the like. In many cases, isotonic agents, such as sugars, buffer solutions, or sodium chloride, are preferably included. Optionally, emulsifiers and / or dispersants may be used. Appropriate flowability may be maintained, for example, by forming liposomes, by maintaining the desired particle size (in the case of dispersions), or by using surfactants. Other agents that delay absorption, such as aluminum monostearate and gelatin, may also be added. The solution may be filled into vials, ampoules, infusion bottles, and the like.

[0479] In all cases, the final dosage form should be sterile, free-flowing, and stable under manufacturing and storage conditions. Sterile injectable solutions are prepared by adding the required amount of the active compound to a suitable solvent containing, if desired, other components listed above, followed by filtration and sterilization. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce powders containing the active ingredient present in the previously sterile filtered solution, plus any additional desired components.

[0480] Typically, aqueous solutions or suspensions are preferred. Suitable formulations of therapeutic proteins (e.g., the binding protein of the present invention) are typically buffer solutions, such as solutions comprising a protein at a suitable concentration (e.g., 0.001 to 400 mg / ml, preferably 0.005 to 200 mg / ml, more preferably 0.01 to 200 mg / ml, even more preferably 1.0 to 100 mg / ml, e.g., 1.0 mg / ml (IV administration) or 100 mg / ml (SC administration)) and an aqueous buffer, wherein the buffer is, for example:

[0481] - Phosphate-buffered saline, pH 7.4

[0482] - Other phosphate buffers, pH 6.2 to 8.2,

[0483] - Acetate buffer, pH 3.2 to 7.5, preferably pH 4.8 to 5.5.

[0484] - Histidine buffer, pH 5.5 to 7.0,

[0485] - Succinate buffer, pH 3.2 to 6.6, and

[0486] - Citrate buffer, pH 2.1 to 6.2,

[0487] Optionally, salts (e.g., NaCl) and / or sugars (e.g., sucrose and trehalose) and / or other polyols (e.g., mannitol and glycerol) are used to provide isotonicity of the solution.

[0488] Additionally, the solution may include other reagents (e.g., cleaning agents, such as 0.02% TWEEN™ 20 or TWEEN™-80). Formulations for subcutaneous application may include significantly higher concentrations of the antibodies of the present invention, such as up to 100 mg / ml or even higher. However, it will be apparent to those skilled in the art that the components and amounts given above represent only one preferred option. Alternatives and variations will be immediately apparent to those skilled in the art, or may readily conceive of from the foregoing disclosure. The above formulations may optionally be provided in the form of lyophilized formulations to be reconstituted in solution, for example, in water for injection (WFI).

[0489] According to another aspect of the invention, the binding protein of the invention can be used in combination with a device for protein administration (e.g., a syringe, injection pen, micropump or other device).

[0490] Treatment

[0491] Another aspect of the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the binding protein of the present invention to a patient in need.

[0492] Another aspect of the present invention provides the binding protein of the present invention for use in a method of treating cancer.

[0493] Another aspect of the invention is the use of the binding protein of the invention in the preparation of pharmaceutical compositions for treating cancer.

[0494] For the avoidance of doubt, the medical applications of this invention may include any of the specific binding proteins of this invention as described above (e.g., B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B...). (Any one of the following: 7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, B7H6#24 / CD3#1).

[0495] Regardless of the type or stage of the invading histopathology, the term “cancer” as used herein is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs.

[0496] Exemplary cancers that can use the multispecific binding protein described herein to inhibit their growth are any tumors expressing B7H6, preferably colorectal cancer (e.g., metastatic colorectal cancer, mCRC), non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).

[0497] Exemplary cancers whose growth can be inhibited using the multispecific binding protein described herein are any tumors expressing B7H6, including (but not limited to) T-cell lymphoma, myeloid leukemia, breast cancer; ovarian cancer, oral squamous cell carcinoma, and gastrointestinal cancer. Gastrointestinal cancers include (but are not limited to) esophageal cancer (e.g., gastroesophageal junction cancer), gastric cancer, hepatocellular carcinoma, biliary tract cancer (e.g., bile duct cancer), gallbladder cancer, pancreatic cancer, or colorectal cancer (CRC).

[0498] In some embodiments, the following cancers, tumors, and other proliferative diseases can be treated with the multispecific binding protein of the present invention: head and neck cancer, preferably HNSCC; lung cancer; preferably NSCLC; breast cancer; thyroid cancer; cervical cancer; ovarian cancer; endometrial cancer; liver cancer (hepatoblastoma or hepatocellular carcinoma); pancreatic cancer; prostate cancer; gastric sarcoma; gastrointestinal stromal tumor, esophageal cancer; colon cancer; colorectal cancer; kidney cancer; skin cancer; brain tumor; glioblastoma; non-Hodgkin lymphomas (T- or B-cell lymphomas); leukemia (chronic or acute myeloid leukemia, non-lymphocytic leukemia) or multiple myeloma.

[0499] In a preferred embodiment of the invention, the cancer is mCRC.

[0500] The above-mentioned cancers, tumors, cysts, etc., characterized by their specific location / origin in the body, are intended to include both primary tumors and metastatic tumors derived from them.

[0501] If a patient has a cancer characterized by high B7H6 expression, that patient is more likely to respond to treatment with the binding protein of the present invention (as described herein). Therefore, in some embodiments, the cancer to be treated with the binding protein of the present invention is a cancer with high B7H6 expression, for example, B7H6 expression higher than the average expression in cancer cells of a patient population with the same type of B7H6-expressing cancer.

[0502] The binding protein of the present invention can be used in treatment regimens in the context of first-line, second-line, or any other line of treatment and maintenance therapy.

[0503] The binding protein of the present invention can be used for the prevention, short-term or long-term treatment of the diseases mentioned above, optionally in combination with radiotherapy, one or more additional therapeutic agents and / or surgery.

[0504] In a preferred embodiment, the protein of the present invention is combined with a PD-1 antagonist (e.g., an anti-PD-1 antibody or an anti-PDL-1 antibody) for the treatment of cancer. Preferably, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5, as described herein (as defined in Table A below) and WO2017 / 198741 (incorporated herein by reference). Preferably, the anti-PDL-1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab. In a particularly preferred embodiment, the binding proteins of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B...) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) can be used in combination with PD1-1 for cancer treatment. In a particularly preferred embodiment, the binding proteins of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B...) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) can be used in combination with PD1-2 for cancer treatment.In a particularly preferred embodiment, the binding proteins of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B...) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) can be used in combination with PD1-3 for cancer treatment. In a particularly preferred embodiment, the binding proteins of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B...) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) can be used in combination with PD1-4 for cancer treatment. In a particularly preferred embodiment, the binding protein of the present invention is (preferably any one of B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, B7H6#24 / CD3#1).

[0505] Table A: Amino acid sequences and SEQ ID NO of the heavy and light chains of anti-PD1 antibodies PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5.

[0506]

[0507]

[0508]

[0509] According to the preferred embodiments described herein and any other aspect of the invention, antibodies PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 are antibody molecules as disclosed in WO2017 / 198741 and are defined by the sequences shown in Table A above.

[0510] Therefore, PD1-1 has a heavy chain containing the amino acid sequence of SEQ ID NO:331 and a light chain containing the amino acid sequence of SEQ ID NO:332;

[0511] PD1-2 has a heavy chain containing the amino acid sequence of SEQ ID NO:333 and a light chain containing the amino acid sequence of SEQ ID NO:334;

[0512] PD1-3 has a heavy chain containing the amino acid sequence of SEQ ID NO:335 and a light chain containing the amino acid sequence of SEQ ID NO:336;

[0513] PD1-4 has a heavy chain containing the amino acid sequence of SEQ ID NO:337 and a light chain containing the amino acid sequence of SEQ ID NO:338; and

[0514] PD1-5 has a heavy chain containing the amino acid sequence of SEQ ID NO:339 and a light chain containing the amino acid sequence of SEQ ID NO:340.

[0515] The foregoing also includes the use of the binding protein of the present invention in various methods of treating the above-mentioned diseases by administering a therapeutically effective dose to a patient in need, the use of the binding protein in the manufacture of a medicament for treating the above-mentioned diseases, pharmaceutical compositions including the binding protein of the present invention, and the preparation and / or manufacture of a medicament including the binding protein of the present invention, and the like.

[0516] Combination with other active substances or therapies

[0517] The binding proteins of this invention (e.g., B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1) Any one of B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, and B7H6#24 / CD3#1 can be used alone or in combination with other cancer therapies (e.g., surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, or combinations thereof). For example, the binding protein of the present invention can be combined with one or more additional therapeutic agents, specifically with cytotoxic or cell growth-inhibiting chemotherapeutic agents, therapeutically active compounds that inhibit angiogenesis, or signal transduction pathway inhibitors (e.g., EGFR inhibitors, immunomodulators, immune checkpoint inhibitors, mitotic checkpoint inhibitors, or hormone therapeutic agents) for the treatment of cancer.

[0518] Additional therapeutic agents may optionally be administered as a component of the same drug formulation concurrently with, before, or after the administration of the B7H6 / CD3 binding protein.

[0519] Cell growth inhibitory and / or cytotoxic active substances that can be combined with the molecules of the present invention include (but are not limited to) hormones, hormone analogs and anti-hormones, aromatase inhibitors, LHRH agonists and antagonists, growth factor inhibitors (such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, such as HER2, HER3, HER4) and hepatocyte growth factor (HGF), etc.), where the inhibitor is, for example, (anti) growth factor antibodies, (anti) growth factor antibodies, etc. Sub-receptor antibodies and tyrosine kinase inhibitors, such as cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib, and trastuzumab; antimetabolites (e.g., antifolate agents such as methotrexate and raltitrexed); pyrimidine analogues such as 5-fluorouracil (5-FU) and FOLFOX. (Combination regimens of folate, 5-FU, and oxaliplatin), FOLFIRI (combination regimens of folate, 5-FU, and irinotecan), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine, and gemcitabine; purine and adenosine analogues, such as mercaptopurine, thioguanine, cladribine, pentostatin, and cytarabine. C) Fludarabine; antitumor antibiotics (e.g., anthracyclines); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustin, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas (e.g., carmustin and lomustin), thiotepa);Antimitotic agents (e.g., vinca alkaloids, such as vinblastine, vindesin, vicorylbin, and vincristine; and taxanes, such as paclitaxel and docetaxel); angiogenesis inhibitors (including bevacizumab, ramucirumab, and aflibercept); and microtubule inhibitors.DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxin, etoposide, etopophos, teniposide, amsacrin, topotecan, irinotecan, mitoxantrone), serine / threonine kinase inhibitors (e.g., PDK1 inhibitors, Raf inhibitors, A-Raf inhibitors, B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (e.g., vorasetib), CDK inhibitors (including CDK9 inhibitors and aurora kinase inhibitors), tyrosine kinases. Inhibitors (e.g., PTK2 / FAK inhibitors), protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF-1R inhibitors, Bcl-xL inhibitors, Bcl-2 inhibitors, Bcl-2 / Bcl-xL inhibitors, ErbB receptor inhibitors, BCR-ABL inhibitors, ABL inhibitors, Src inhibitors, rapamycin analogs (e.g., everolimus, temsirolimus, ridaforolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, and immunotherapeutic agents, such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / Immunoglobulins, such as ipilimumab, nivorumab, and pembrolizumab; and various chemotherapeutic agents, such as amifostine, anagrelid, clodronat, filgrastin, interferon, interferon-alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer; proteasome inhibitors (e.g., bortezomib); Smac and BH3 mimics; and agents that restore p53 function, including MDM2-p53 antagonists.Inhibitors of the Wnt / β-catenin signaling pathway; and / or cyclin-dependent kinase 9 inhibitors.

[0520] Particularly preferred treatments are those using the combination of the conjugating molecule of the present invention with one or more immunotherapeutic agents, said immunotherapeutic agents including anti-PD-1 and anti-PD-L1 agents and anti-LAG3 agents: Exemplary anti-PD-1 agents include (but are not limited to) the anti-PD-1 antibody PDR-001, pembrolizumab, nivorumab, pilizumab, and PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5, as disclosed herein (Table A) and WO2017 / 198741. Exemplary anti-PD-L1 agents include (but are not limited to) atezolizumab, avelumab, and durvalumab. In a preferred embodiment, the binding molecules of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) combined with PD1-1. In a preferred embodiment, the binding molecules of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) combined with PD1-2.In a preferred embodiment, the binding molecules of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) combined with PD1-3. In a preferred embodiment, the binding molecules of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) combined with PD1-4. In a preferred embodiment, the binding molecules of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1) Any one of B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1) combined with PD1-5.

[0521] In some implementations, the additional therapeutic agent may be other immunotherapeutic agents, such as modulators of the following: TIM-1, TIM-3, TIM-4, PD-L2, LAG3, CTLA-4, galactoglobulin 9, galactoglobulin-1, CD69, CD113, GPR56, CD48, GARP, CAECAM-1, BTLA, TIGIT, CD160, LAIR1, 2B4, CEACAM, CD39, TGFβ, IL-10, Fas ligand, ICOS, B7 family (B7-1, B7-2, B7-H1 (PDL-1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5). (VISTA)), gp49B, PIR-B, KIR family receptor, SIRPα (CD47), ILT-2, ILT-4, IDO, CD39, arginase, CD73 HHLA2, lactolipoprotein or A2aR.

[0522] In some implementations, the additional immunotherapeutic agent is a member of the TNF family that binds to a member of the homologous TNF receptor family, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, CD137 / FAP, GITR, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, etc. RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LIGHT, DCR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR. Preferably, the additional immunotherapy agent is CD137 / FAP.

[0523] In some implementations, the additional immunotherapeutic agent is selected from (i) antagonists of cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-B, VEGF; "immunosuppressive cytokines") and / or (ii) agonists of cytokines that stimulate T cell activation and / or cytokines that stimulate immune responses (e.g., IL2), for example for the treatment of proliferative diseases, such as cancer.

[0524] In some implementations, the additional immunotherapeutic agent is an agonist of a protein that stimulates T cell activation, such as CD28, GITRL, OX40L, CD27, and CD28H or STING agonists.

[0525] In some implementations, the additional therapeutic agent is an oncolytic virus, including (but not limited to) oncolytic viruses derived from vaccinia virus, adenovirus (AdV), herpes simplex virus (HSV1 or HSV2), reovirus, myxoma virus (MYXV), poliovirus, vesicular stomatitis virus (VSV), Maraba virus, varicella-zoster virus, measles virus (MV), or Newcastle disease virus (NDV).

[0526] Reagent test kit

[0527] This invention also covers kits containing at least one of the multispecific binding proteins of this invention (e.g., B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#6 / CD3#1, B7H6#7 / CD3#1, B7H6#8 / CD3#1, B7H6#9 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / C). Any one of D3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, B7H6#24 / CD3#1) and other components of the group consisting of one or more other drugs used to treat the above-mentioned diseases and symptoms.

[0528] In one embodiment, the kit comprises a composition containing an effective amount of the binding protein of the present invention in unit dosage form.

[0529] The present invention also covers kits comprising at least one of the multispecific binding proteins of the present invention and one or more other components selected from the group consisting of other drugs for treating the above-mentioned diseases and conditions.

[0530] In one embodiment, the kit comprises, in unit dosage form, an effective amount of the multispecific binding protein of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B...). A composition of any one of 7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, or B7H6#24 / CD3#1. In another embodiment, the kit comprises, in unit dosage form, an effective amount of the multispecific binding protein of the present invention (preferably B7H6#1 / CD3#1, B7H6#2 / CD3#1, B7H6#3 / CD3#1, B7H6#4 / CD3#1, B7H6#5 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#10 / CD3#1, B7H6#11 / CD3#1, B7H6#12 / CD3#1, B7H6#13 / CD3#1, B7H6#14 / CD3#1, B7H6#15 / CD3#1, B7H6#16 / CD3#1, B7H6#17 / CD3#1, B7H6#18 / CD3#1, B7H6#19 / CD3#1, B7H6#10 ... Compositions of CD3#1, B7H6#19 / CD3#1, B7H6#20 / CD3#1, B7H6#21 / CD3#1, B7H6#22 / CD3#1, B7H6#23 / CD3#1, and B7H6#24 / CD3#1, and compositions in unit dosage form containing an effective amount of a PD-1 antagonist (e.g., an anti-PD-1 antibody, most preferably PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 as described herein (e.g., Table A) and WO2017 / 198741).

[0531] In some embodiments, the kit includes a sterile container containing the composition; said container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing pharmaceutical agents. Furthermore, the kit may contain the pharmaceutical composition in a first container having the binding protein of the invention in lyophilized form, and the second container having a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent may be used to reconstitute or dilute the binding protein.

[0532] If desired, the multispecific binding protein of the present invention is provided together with instructions for administering the multispecific binding protein to an individual suffering from cancer. The instructions typically include information on the use of the composition to treat or prevent cancer. In other embodiments, the instructions include at least one of the following: instructions for the therapeutic agent; a dosage schedule and administration for the treatment or prevention of cancer or its symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present) or applied to the container by marking, or may be in the form of a separate sheet, brochure, card, or binder supplied with or in connection with the container.

[0533] The proposed kit components have been proven to be packaged in a manner suitable for use by those skilled in the art. For example, the proposed kit components may be provided as a solution or as a liquid dispersion or similar.

[0534] Example

[0535] The following embodiments illustrate the present invention. These embodiments should not be construed as limiting the scope of the invention.

[0536] Example 1: Design and construction of B7H6 / CD3 binding protein

[0537] The inventors have developed a multispecific binding protein that binds to B7H6 and CD3 and induces T cell activation, thereby leading to the lysis of B7H6-expressing tumor cells. The molecular design incorporates an IgG antibody scaffold and an IgG-like structure. A key feature is the segment-entry technique in the Fc region for heterodimerization of the segment and acupoint arms. Furthermore, the binding protein has a flexible peptide sequence between the light chain and the corresponding heavy chain in each arm. Therefore, the binding protein comprises two arms, one binding to CD3 and the other to B7H6, each arm containing a single-chain Fab and an Fc region (see [link to Fc region]). Figure 1 ).

[0538] Preferably, the binding molecule is bispecific and divalent (monovalent for each of the two targets).

[0539] The binding domains recognizing B7H6 and CD3 were prepared using high-throughput V gene recovery from hybridomas and cultured single B cells.

[0540] To obtain the anti-B7H6 binder, hybridomas or single B cells derived from B7H6-immunized wild-type and ALIVAMAB™ humanized mice (Ablexis, San Francisco, CA, USA: ALIVAMAB MOUSE™ transgenic mouse platform with human immunoglobulin loci) were cultured in vitro. Supernatants binding to recombinant human B7H6 were screened using the ALPHALISA® immunoassay kit (PerkinElmer, Waltham, MA, USA), and supernatants binding to NCI-H716 cells (ATCC®, CCL-251TM) expressing human B7H6 were screened using flow cytometry. Supernatants binding to recombinant cynomolgus monkey B7-H6 expressed on CHO cells were also screened.

[0541] Then, the immunoglobulin (Ig) VH and VL genes were amplified from the self-identified positive pure lines. To isolate RNA from the hybridoma, approximately 2 × 10⁻⁶ cells from the single pure lines were used. 6 Cell pellets were collected and used as source material. For single B cells, 100 to 500 cells expanded from a single isolated B cell were used as source material. RNA was isolated using the RNeasy® plus mini RNA extraction kit (Qiagen, Hilden, Germany). cDNA was then synthesized using the SMARTer® cDNA synthesis kit (Clontech, MountainView, CA) according to the manufacturer's instructions. To facilitate cDNA synthesis, reverse transcription of all messenger RNA was initiated using oligodT, followed by "5' capping" with SMARTer IIA oligonucleotides. Amplification of the VH and VL fragments was then performed using a two-step PCR amplification with a 5' primer targeting the SMARTer IIA cap and a 3' primer targeting the common region in CH1. In short, each 50 μl PCR reaction consisted of a 20 μM mixture of forward and reverse primers, 25 μl of PrimeSTAR® Max DNA polymerase premix (Clontech), 2 μl of unpurified cDNA, and 21 μl of double-distilled H2O. The cyclic program started at 94°C for 3 min, followed by 35 cycles (30 sec at 94°C, 1 min at 50°C, and 1 min at 68°C), and ended at 72°C for 7 min. The second round of PCR was performed using the second-round primers for VL and VH, which contained a 15 bp complementary extension that overlapped with specific regions in their respective pTT5 parent vectors (VH and VL). The second round of PCR was performed using the same PCR cycling program.

[0542] The In-Fusion® HD Cloning Kit (Clontech, USA) is used for the directed cloning of the VL gene into the pTT5huIgK vector and the VH gene into the pTT5huIgG1KO vector. To facilitate In-Fusion® HD cloning, the PCR products are purified and treated with cloning enhancers prior to In-Fusion HD cloning. Cloning and transformation are performed according to the manufacturer's protocol (Clontech, USA). Small-scale DNA preparations are subjected to Sanger sequencing to confirm the acquisition of the complete V-gene fragment.

[0543] Using this method, IgVH and VL gene pairs encoding a binding domain specific to B7H6 were prepared. Recombinant antibodies were generated by transient transfection of CHO-E37 cells with plasmids encoding the corresponding heavy and light chains.

[0544] To obtain additional anti-CD3 binding agents, WT mice were immunized using the huCD3ε peptide 1-27 construct. Hybridoma supernatants binding to recombinant huCD3E+ G-Fc protein and recombinant cyCD3E+ G-Fc protein, as well as hybridoma supernatants binding to huCD3-positive and cyCD3-positive cells, were screened. Variable regions of positive homologous lines were recovered and cloned into IgG or IgG-like bispecific constructs for further evaluation.

[0545] Humanization / Optimization of B7H6 and CD3 Binders

[0546] The sequences of the aforementioned B7H6 or CD3 binding agents, as well as the CD3 binding agents described in the literature (Pessano et al., EMBO J. Feb. 1985; 4(2): 337-44; Salmerón A et al., J Immunol. Nov. 1, 1991; 147(9): 3047-52), were humanized and / or optimized. Antibody sequence optimization / humanization is a method for engineering antibodies (targeting specific antigens / epitopes) generated in non-human species to be used as therapeutic agents similar to those generated in humans, thereby eliminating potential adverse effects (e.g., immunogenicity) while retaining specificity. The sequence optimization / humanization method used in this paper is described by Singh et al., 2015 (Singh S et al., mAbs 2015: 7(4): 778-91). In short, closely matching human lineages were identified in a computer, and optimized / humanized variants were evaluated using phage screening methods. The final leader candidate sequence was selected based on binding, percentage human score, and EpiVax® (a computer-aided predictive tool for potential immunogenicity).

[0547] Construction of a dual-specific protein binding to B7H6 and CD3

[0548] The variable regions of the B7H6 and CD3 binding agents were cloned into the expression vector pTT5 (National Research Council, Canada) using conventional molecular biology techniques to form a bispecific binding protein with one B7H6-specific binding arm and one CD3-specific binding arm. The B7H6-specific binding arm comprises a single-stranded Fab and Fc region binding to B7H6 (this binding unit is also referred to herein as the "B7H6 arm" or "B7H6 chain"), and the CD3-specific binding arm comprises a single-stranded Fab and Fc region binding to CD3 (this binding unit is also referred to herein as the "CD3 arm" or "CD3 chain"). The Fc regions of both the B7H6 and CD3 arms include "W" or "SAV" mutations (Atwell et al., JMB, 1997, 270, 26-35), and the respective chains are referred to as the W or SAV chains. For multi-fragment DNA assembly, Gibson assembly and NEBuilder® HiFi DNA assembly methods were used, following the manufacturer's protocol (New England Biolabs, Ipswich, MA). (USA). Sequencing of small-scale DNA preparations.

[0549] Each expression vector contains a eukaryotic promoter assembly for the chain-coding gene (B7H6 or CD3 arm / chain), i.e., the gene encoding the signal sequence and the light and heavy chains, an expression cassette for the prokaryotic selection marker gene (e.g., ampicillin), and an origin of replication. The DNA plastids are propagated and purified in ampicillin-resistant E. coli colonies and cultures.

[0550] Example 2: Expression and purification of a bispecific binding protein that binds to B7H6 and CD3

[0551] CHO-E cells were transiently transfected with a pTT5 vector carrying the B7H6 / CD3 chain encoding gene (one chain being the W chain and the other the SAV chain) to generate a bispecific molecule binding to B7H6 and CD3. In short, transfected CHO-E cells, suspended in serum-free medium, were cultured in shake flasks at 140 rpm, 37°C, and 5% CO2, maintaining exponential growth conditions. On the day of transfection, cells were chemically transfected using Mirus Bio TransIT Pro® transfection reagent with W-chain and SAV-chain plasmids at a mass ratio of 1:3. Cells were then seeded at 1 to 2 × 10^6 cells / ml in 1 L Gibco® FreeStyle™ CHO expression medium (LifeTechnologies, NY, US). Then, on day 7, cells were cultured for 10 days with a single-use feed and 200 ml of commercial feed solution under orbital oscillation to optimize protein expression. Antibody titers in cell culture supernatants were determined using an Octet® instrument (Pall ForteBio, CA, US) and a ProtA biosensor tip, following the manufacturer’s instructions.

[0552] Recombinant B7H6 / CD3 binding protein was purified from culture supernatant using a two-step process with a GE Healthcare Life Sciences ÄKTA™ protein purification system. First, samples were captured from harvested cell culture medium using protein A affinity chromatography with a MabSelect™ column (GE Healthcare). The protein bound to protein A at neutral pH and was washed with high salt (1M NaCl) to remove cell culture medium components and any non-specifically bound proteins or components to protein A. Antibody or antibody-like construct samples were lysed in isocratic mode using 30 mM sodium acetate (pH 3.5). The lysed sample was neutralized to pH 5.0 using 1% 3M sodium acetate solution (pH 9.0). The neutralized protein was aseptically filtered using a 0.22 μm filtration system. Concentrations were measured at UV280 using a nanodrop 8000 spectrophotometer. In the second purification, cation exchange chromatography was performed using a POROS™ 50 HS cation exchange resin column (Applied Biosystems, Carlsbad, CA, USA) or size exclusion chromatography was performed using a HiLoad® 26 / 600 Superdex® 200 pg column (GE Healthcare). The two-step purified material was stored in a final buffer of 50 mM sodium acetate and 100 mM NaCl (pH 5.0). The purity and heterogeneity of the samples were evaluated by analytical size exclusion chromatography, mass spectrometry, and analytical ultracentrifugation. Advanced samples used for functional testing included two-step purified material with approximately 95% to 99% monomer content.

[0553] Table 1: Amino acid sequences and SEQ ID NO of the CDR, VH, VL, scFab, B7H6 arm, and CD3 arm sequences of the protein / antibody constructs described in this paper:

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623] Example 3: Generation of Recombinant Proteins

[0624] • Human B7H6-His

[0625] The complete extracellular domain of human-B7H6 was expressed using a pTT vector (encoding human B7H6-His, SEQ ID NO: 317) with a His 6-tag via transient transfection using the Lenti-X™ lentiviral system (Clontech). Molecular transfection was performed in Gibco™ Freestyle™ F17 expression medium (Thermo Fisher Scientific) at a concentration of 1.6 × 10⁻⁶. 6 HEK 293F cells / ml (Thermo Fisher). A 1:3 DNA:PEI complex and 1 mg / L DNA were pre-cultured for 5 minutes, filtered, and pre-cultured again at room temperature for 15 minutes before being added to the cells. Cells were cultured at 37°C, 5% CO2, and 140 rpm with shaking. 24 hours post-transfection, tryptone N1 was added to the cells to a final concentration of 0.5%. 48 hours post-transfection, cells were re-fed with 2 mM glutamine and 2 g / L glucose. Simultaneously, the temperature was lowered to 33°C. 120 hours post-transfection, 2 mM glutamine and 1 g / L glucose were added to the final feed. 144 hours post-transfection, cells were harvested by centrifugation at 6000 rpm for 15 minutes. The supernatant was clarified using a G4 filter.

[0626] Protein purification was performed in two steps. First, affinity chromatography was performed using a Ni-NTA column. The sample was precipitated for 10 CVs with 1 × PBS (pH 7.2) + 10 mM imidazole wash buffer, followed by 10 CVs with 1 × PBS (pH 7.2) + 20 mM imidazole wash buffer, with a precipitation gradient of 4-60% 1 × PBS (pH 7.2), supplemented with 0.5 M imidazole. The fractions were collected and analyzed by SDS-PAGE before pooling and concentration. Second, gel filtration chromatography (GE Healthcare Life Sciences) was performed using a Superdex® 200, 16 / 600, 120 ml column. 5 ml of the concentrated pooled product from affinity chromatography was loaded onto the column at a flow rate of 0.5 ml / min. The buffer was prepared with 20 mM HEPES, 100 mM NaCl, and 5% sucrose at pH 7.4. The fractions were collected and analyzed by SDS-PAGE before aggregation, and then sterilized using a 0.2 μm filter.

[0627] •Cyno B7H6-His

[0628] The complete extracellular domain of Cyno-B7H6 was expressed using a pTT vector (encoding cyno B7H6-His, SEQ ID NO: 320) with a His6-tag via transient transfection using the Lenti-X™ lentiviral system (Clontech). Molecular transfection was performed in Gibco™ Freestyle™ F17 expression medium (Thermo Fisher Scientific) at a concentration of 1.6 × 10⁻⁶. 6 HEK 293F cells / ml (Thermo Fisher). A 1:3 DNA:PEI complex and 1 mg / L DNA were pre-cultured for 5 minutes, filtered, and pre-cultured again at room temperature for 15 minutes before being added to the cells. Cells were cultured at 37°C, 5% CO2, and 140 rpm with shaking. 24 hours post-transfection, tryptone N1 was added to the cells to a final concentration of 0.5%. 48 hours post-transfection, cells were re-fed with 2 mM glutamine and 2 g / L glucose. Simultaneously, the temperature was lowered to 33°C. 120 hours post-transfection, 2 mM glutamine and 1 g / L glucose were added to the final feed. 144 hours post-transfection, cells were harvested by centrifugation at 6000 rpm for 15 minutes. The supernatant was clarified using a G4 filter.

[0629] Protein purification was performed in two steps. First, affinity chromatography was performed using a Ni-NTA column. The sample was precipitated for 10 CVs with wash buffer (1×PBS, 0.2 M sucrose, 0.01% CHAPS, 5% glycerol (pH 7.2) + 10 mM imidazole), followed by 10 CVs with wash buffer (1×PBS, pH 7.2) + 20 mM imidazole, using a 4-60% gradient of 1×PBS, 0.2 M sucrose, 0.01% CHAPS, 5% glycerol (pH 7.2), supplemented with 0.5 M imidazole. The fractions were collected and analyzed by SDS-PAGE before pooling and concentration. Second, gel filtration chromatography (GE Healthcare Life Sciences) was performed using a Superdex® 200, 16 / 600 column. 10 ml of the concentrated pooled product from affinity chromatography was loaded onto the column at a flow rate of 1.0 ml / min. The prepared buffer consisted of 1X PBS, 0.2M sucrose, 0.01% CHAPS, and 5% glycerol, at pH 7.2. Splits were collected and analyzed by SDS-PAGE prior to collection, and then sterilized using a 0.2 μm filter.

[0630] • Human CD3 E+G HuFc-6xHis (E+G indicates the εγ subunit)

[0631] A cell line for producing human CD3 E+G HuFc-6xHis was generated using HEK-293 cells (Thermo Fisher), the Lenti-X™ lentiviral system (Clontech), and plasmids encoding human CD3 E+G HuFc-6xHis (Human CD3E accession number: P07766; Human CD3E+G-HuFc-His: SEQ ID NO: 322). For expression, cells were cultured and expanded in Freestyle™ 293 medium (Thermo Fisher Scientific) at 37°C, humidified 8% CO2, and shaking at 135 rpm. The conditioned culture supernatant was harvested on day 6 by centrifugation at 9300 x g for 30 min. Expression was monitored by SDS-PAGE and Western blotting. The conditioned culture supernatant was adjusted with 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, and 10 mM imidazole. The pH was then adjusted to 7.2. Purification was performed in two steps: affinity purification using Ni / NTA resin (incubated overnight at 4°C followed by precipitation with 250 mM imidazole); then size exclusion chromatography was performed on a Superdex® 200 column (GE Healthcare Life Sciences) in a target buffer PBS containing 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, and 1 mM TCEP (pH 7.2). The collected material was concentrated using a 10K MWCO PES membrane Vivacell® 100 centrifuge before final analysis and storage. The purified material was qualitatively analyzed by mass spectrometry and analytical ultracentrifugation.

[0632] •Cyno CD3 E+G HuFc-6xHis (E+G indicates the εγ subunit)

[0633] A cell line for producing Cyno CD3 E+G HuFc-6xHis was generated using HEK-293 cells (Thermo Fisher), the Lenti-X™ lentiviral system (Clontech), and plasmids encoding Cyno CD3 E+G HuFc-6xHis (cynomolgus monkey CD3E accession number: Q95LI5<, cyno CD3 E+G huFc-His: SEQ ID NO:323). For expression, cells were cultured and expanded in Freestyle™ 293 medium (Thermo Fisher Scientific) at 37°C, humidified with 8% CO2, and shaking at 135 rpm. The conditioned culture supernatant was harvested on day 6 by centrifugation at 9300 x g for 30 min. Expression was monitored by SDS-PAGE and Western blotting. The conditioned culture supernatant was adjusted with 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, and 10 mM imidazole. The pH was then adjusted to 7.2. Purification was performed in two steps: affinity purification using Ni / NTA resin (incubated overnight at 4°C followed by precipitation with 250 mM imidazole); then size exclusion chromatography was performed on a Superdex® 200 column (GE HealthcareLife Sciences) in a target buffer PBS containing 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, and 1 mM TCEP (pH 7.2). The collected material was concentrated using a 10K MWCO PES membrane Vivacell® 100 centrifuge before final analysis and storage. The purified material was qualitatively identified by mass spectrometry and analytical ultracentrifugation.

[0634] • Human B7H6 ECD marked with Fc-His

[0635] In this construct, the huB7H6 ECD is followed by a GS adapter, then a huIgG1-Fc domain and a C-terminal His6 tag (SEQ ID NO:318). The construct was expressed by transient transfection of HEK293-6E cells with a DNA:PEI ratio of 1:3 and 1 mg DNA / L culture medium. The PEI reagent was linear PEI MAX [Mw 40,000] (Polysciences: catalog number 24765-2). Transfected cells were incubated at 37°C, 5% CO2, and 130 rpm. 24 hours after transfection, tryptone N1 (Organotechnie; catalog number 19553) and glucose were added to final concentrations of 0.5% and 1 g / L, respectively. Cells were harvested after 5 days. After centrifugation, the supernatant was filtered through a 0.2 μm membrane filter. The huB7H6-ECD-Fc-His protein was purified using a two-step purification process: first, affinity chromatography on a Ni NTA agarose matrix, followed by gel filtration using a Superdex® 200, 26 / 600 column (GE Healthcare Life Sciences). The collected fractions were filtered and stored in a buffer solution of 1 x PBS, 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, pH 7.2.

[0636] • Fc-His-tagged human Ala-mutated B7H6 ECD (NKp30 interaction sites aa35-38 and aa102-105 are replaced by Ala).

[0637] This construct (SEQ ID NO:319) possesses huB7H6 with Ala substitutions at positions 35-38 and 102-105, thus it does not bind NKp30. The huB7H6-Ala-ECD is followed by a GS adapter, then a huIgG1 Fc domain and a C-terminal His6 tag. This construct was expressed in HEK293-6E cells via transient transfection, purified using a two-step purification method, and stored as described above for the Fc-His-tagged huB7H6-ECD construct.

[0638] •Human B7H1-Fc

[0639] This construct (SEQ ID NO:324) contains huB7H1, which has a cMyc tag, a thrombin cleavage site, and a huFc domain. The construct was expressed using HEK293f cells, a DNA:PEI ratio of 1:1.5, and transient transfection with 1 mg DNA / L culture. Flasks were incubated at 37°C in a humidified 8% CO2 environment with shaking at 135 rpm. Cells were harvested after 3 days. After centrifugation, the protein was purified from the supernatant. First, affinity purification was performed using nProtein A Sepharose® 4 rapid flow medium (GE Healthcare, #17-5280-03), and the elution was dialyzed in 20 mM Tris, 100 mM NaCl, 10% glycerol, 1 mM TCEP, and 3 mM CaCl2 (pH 8.0). Next, the sample was incubated with thrombin CleanCleave™ resin (1 mL, Sigma). Third, the aggregate from the previous step was reconjugated with nProtein A Sepharose® 4 rapid flow medium. Unbound material was preserved, polished, and concentrated by further gel filtration on a Superdex® 75 (GE Healthcare) column equilibrated in PBS, 1 mM TCEP (pH 7.2) buffer.

[0640] Example 4: Determination of SPR-based affinity and interspecies cross-reactivity for recombinant B7H6 and CD3 εγ subunits

[0641] To determine the affinity of human and cynomolgus monkey B7H6 and human B7H1 for the B7H6 / CD3 binding protein, experiments were performed on a Biacore™ 8K instrument (GE Healthcare Life Sciences). In short, the B7H6 / CD3 binding protein was captured via protein A / G. Run buffers and all serial dilutions for this experiment were prepared in HBS-EP+. The CM5 sensor chip was activated by passing an equal volume mixture of EDC / NHS through two flow cells at a flow rate of 10 μL / min for 420 s, and immobilized with recombinant protein A / G (50 μG / ml, in 10 mM NaOAc, pH 4.5) through all flow cells at a flow rate of 10 μL / min for 420 s, resulting in approximately 2500 RU of protein A / G on the surface. The sensor chip was inactivated by passing 1M ethanolamine-HCl through all flow cells at a flow rate of 10 μL / min for 420 s.

[0642] Approximately 700 RU of B7H6 / CD3-binding protein was captured in flow cell 2 on the protein A / G surface at a flow rate of 10 μL / min for 60 s. Analytes HuB7H6, CyB7H6, and HuB7H1 were injected into the captured B7H6 / CD3-binding protein through both flow cells at a flow rate of 30 µL / min for 300 s, followed by dissociation at 1200 s. The concentrations of HuB7H6 and CyB7H6 were 0 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. The concentrations of HuB7H1 were 0 nM and 1 μM. The surface was regenerated by injecting 10 mM glycine-HCl (pH 1.5) through both flow cells at a flow rate of 30 µL / min for 20 s.

[0643] The reference flow cell 1 (interacting with the sensor surface) and the blank (HBS-EP+ or 0nM analyte) were subtracted from the raw data. The sensor plot was then fitted to a 1:1 Langmuir matrix using Biacore™ 8K evaluation software to provide the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) values.

[0644] To determine the affinity of the B7H6 / CD3 binding protein for human and cyno CD3E+G-hFc, experiments were performed on a Bio-Rad ProteOn™ XPR36 instrument. Briefly, HuCD3E+G-hFc and CyCD3E+G were amine-coupled onto the ProteOn™ GLM sensor chip (Bio-Rad), and the B7H6 / CD3 binding protein was allowed to flow over the immobilized surface. Run buffer and all serial dilutions for this experiment were prepared in HBS-EP+. The GLM sensor chip was normalized according to Bio-Rad's recommendations. The sensor chip was activated for 300 s in a horizontal direction with an equal volume mixture of EDC / s-NHS at a flow rate of 30 μL / min. HuCD3E+G-hFc was fixed in the vertical direction at a flow rate of 30 µL / min at 0.4 µg / mL, 0.2 µg / mL and 0.1 µg / mL in 10 mM acetate (pH 4.5) for 300 s at L1, L2 and L3 respectively. This resulted in the production of approximately 100 RU of HuCD3E+G-hFc at L1, approximately 40 RU of HuCD3E+G-hFc at L2 and approximately 0 RU of HuCD3E+G-hFc at L3. CyCD3E+G-hFc was immobilized vertically at a flow rate of 30 µL / min in 10 mM acetate (pH 4.5) at concentrations of 0.4 µg / mL, 0.2 µg / mL, and 0.1 µg / mL for 300 s at L4, L5, and L6, respectively. This resulted in approximately 385 RU of CyCD3E+G-hFc at L4, approximately 170 RU at L5, and approximately 50 RU at L6. The sensor chip was then deactivated horizontally at a flow rate of 30 µL / min using 1 M ethanolamine-HCl for 300 s. The sensor chip was then regenerated twice horizontally and vertically using 0.85% phosphate at a flow rate of 100 µL / min for 18 s.

[0645] The B7H6 / CD3 binding protein analyte was horizontally injected onto a fixed surface at a flow rate of 30 µL / min for 300 s, followed by dissociation for 600 s. The concentrations of the B7H6 / CD3 binding protein used were 0 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM. The surface was regenerated twice by horizontally injecting 0.85% phosphate at a flow rate of 100 µL / min for 18 s.

[0646] Midpoints (interactions with the sensor surface) and blanks (HBS-EP+ or 0 nM analytes) were subtracted from the raw data. The sensor map was then fitted to a 1:1 Langmuir matrix using Bio-Rad ProteOn™ management software to provide the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) values.

[0647] Table 2 shows the affinity of exemplary B7H6 / CD3 binding proteins (B7H6 / CD3 binding proteins comprising the B7H6 chain of SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239 or SEQ ID NO: 240 and the CD3 chain of SEQ ID NO: 311, respectively, and B7H6 binding proteins comprising the B7H6 chain of SEQ ID NO: 230 or SEQ ID NO: 239 and the CD3 chain of SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315 or SEQ ID NO: 316) as described above.

[0648] Table 2: Affinities (KD) of B7H6 / CD3 binding proteins with human B7H6, cynoB7H6, human CD3 Ɛγ subunit, cynoCD3 Ɛγ subunit, and human B7H1, as determined by SPR analysis. No detectable binding is indicated by “nb”.

[0649]

[0650]

[0651] Example 5: Generation of recombinant CHO-K1 cell lines expressing the extracellular domain of cynomolgus monkey B7H6 on the cell surface

[0652] To generate stable CHO-K1 cells (NCBI: XP_005578557) expressing the extracellular domain of cynomolgus monkey B7H6 on the cell surface, the individual coding sequences (aa 25-262 of XP_005578557.1) were cloned into pcDNA3.1 (Thermo Fisher Scientific). The construct contained an N-terminal mouse IgG Vk leader sequence, followed by a 6-His-myc-tag and the cynomolgus monkey B7H6 extracellular domain (aa 25-262 of NCBI XP_005578557.1). To ensure cell surface localization of the B7H6 extracellular domain, the construct was followed by an adapter and the transmembrane and intracellular domains of EpCAM (Uniprot P16422). The expression of the B7H6 domain on the cell surface was verified by flow cytometry using a mouse monoclonal anti-myc antibody (AbD Serotec). The sequences used are listed in Table 3, and a schematic diagram of the construct is shown in [the table]. Figure 2 middle.

[0653] Table 3: Amino acid sequence of the B7H6 subdomain expressed on CHO-K1 cells

[0654]

[0655] Example 6: Binding of an exemplary B7H6 binding protein to a recombinant human B7H6 extracellular domain protein

[0656] To evaluate the binding of the B7H6 / CD3 binding protein to recombinant human Fc-His-labeled B7H6 ECD and human Fc-His-labeled Ala-mutant B7H6 extracellular protein, as described in Example 3, MediSorp was used at 4°C. TM Plates (Nunc, 467320) were coated overnight with 2 µl / ml of recombinant protein. The following day, the plates were blocked at room temperature (RT) for 1 hour with 0.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The plates were then washed with OBS containing 0.05% TWEEN® 20 viscous liquid, and B7H6 / CD3 binding protein was incubated at concentrations ranging from 0.00001 to 10 μg / ml. After an additional washing step, the bound B7H6 / CD3 binding protein was detected using a peroxidase-conjugated goat anti-human IgG F(ab')2 specific secondary antibody (Jackson Immunoresearch) and visualized using TMB substrate solution (Bender MedSystems). Figure 3 Small image A + Small image B and Figure 4Small images A and B show exemplary B7H6 / CD3 binding proteins (containing the B7H6 chain and the CD3 chain of SEQ ID NO:311 of SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 or SEQ ID NO:240, respectively), and B7H6 / CD3 binding proteins containing SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 or SEQ ID NO:240, respectively). B7H6 binding protein of the B7H6 chain of SEQ ID NO:230 or SEQ ID NO:239 and the CD3 chain of SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315 or SEQ ID NO:316 and recombinant human B7H6 ECD ( Figure 3 Small image A + small image B) and human Ala mutation B7H6 ECD ( Figure 4 (Small image A + Small image B) Protein binding.

[0657] All exemplary B7H6 / CD3 binding proteins tested showed comparable binding to recombinant human B7H6 ECD. Figure 3Small image A + Small image B), and only containing the B7H6 chain of SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 or SEQ ID NO:240 and the CD3 chain of SEQ ID NO:311, as well as B7H6 / CD3 binding proteins containing the B7H6 chain of SEQ ID NO:230 or SEQ ID NO:239 and SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315 or SEQ ID NO:240. The B7H6-binding protein of the CD3 chain in NO:316 showed strong binding to the human Fc-His-tagged Ala-mutated B7H6 extracellular protein, with the NKp30 binding site mutated to alanine. The B7H6 / CD3-binding proteins of the B7H6 chains (SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227) and the CD3 chain (SEQ ID NO:311) showed only weak binding or no binding to the Fc-His-tagged Ala-mutated B7H6 extracellular protein at high concentrations.

[0658] Example 7: Binding with B7H6-positive HCT cells

[0659] The binding of B7H6 / CD3 binding protein to HCT-15 (a human (colorectal cancer) CRC cell line) was tested by flow cytometry. In previous experiments, HCT-15 cells were confirmed to express B7-H6 at both the RNA and protein levels, with approximately 8,000 B7-H6 receptors expressed on the cell surface (data not shown). The B7H6 / CD3 binding protein was generated as described in Example 2. HCT-15 cells were stained with a two-step purified B7H6 / CD3 binding protein in increased concentrations in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). The bound molecules were detected using a PE-conjugated anti-human secondary antibody (Sigma-Aldrich, #P8047). Figure 5Small images A and B show exemplary B7H6 / CD3 binding proteins (containing the B7H6 chain and the CD3 chain of SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239 or SEQ ID NO:240, respectively) and B7H6 / CD3 binding proteins containing the B7H6 chain and the CD3 chain of SEQ ID NO:311, respectively. The binding of the B7H6 chain of SEQ ID NO:230 or SEQ ID NO:239 and the B7H6 binding protein of the CD3 chain of SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315 or SEQ ID NO:316 to human HCT-15 cells.

[0660] Example 8: Cross-reaction with cynomolgus monkey B7H6

[0661] The binding of the B7H6 / CD3 binding protein to recombinant CHO-K1 cells expressing cynomolgus monkey B7H6 was tested by flow cytometry. The B7H6 / CD3 binding protein was generated as described in Example 2. A recombinant cell line expressing cynomolgus monkey B7H6 was generated as described in Example 5. Cells were stained with an increased concentration of the two-step purified B7H6 / CD3 binding protein in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). The binding molecules were detected using a PE-conjugated anti-human secondary antibody (Sigma-Aldrich, #P8047). Figure 6This demonstrates the binding of exemplary B7H6 / CD3 binding proteins (B7H6 / CD3 binding proteins comprising the B7H6 chain of SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239 or SEQ ID NO: 240 and the CD3 chain of SEQ ID NO: 311, respectively, and B7H6 binding proteins comprising the B7H6 chain of SEQ ID NO: 230 or SEQ ID NO: 239 and the CD3 chain of SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315 or SEQ ID NO: 316) to recombinant cells expressing cynomolg...

Claims

1. A protein comprising a first antigen-binding unit specifically binding to B7H6 and a second antigen-binding unit specifically binding to CD3, wherein the first antigen-binding unit specifically binding to B7H6 is selected from the group consisting of i) to xxiv): i) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:1 (CDR1), SEQ ID NO:2 (CDR2) and SEQ ID NO:3 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:4 (CDR1), SEQ ID NO:5 (CDR2) and SEQ ID NO:6 (CDR3); ii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2) and SEQ ID NO:9 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2) and SEQ ID NO:12 (CDR3); iii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2) and SEQ ID NO:15 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:16 (CDR1), SEQ ID NO:17 (CDR2) and SEQ ID NO:18 (CDR3); iv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:19 (CDR1), SEQ ID NO:20 (CDR2) and SEQ ID NO:21 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:24 (CDR3); v) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2) and SEQ ID NO:27 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2) and SEQ ID NO:30 (CDR3); vi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2) and SEQ ID NO:33 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2) and SEQ ID NO:36 (CDR3); vii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2) and SEQ ID NO:39 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2) and SEQ ID NO:42 (CDR3); viii) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2) and SEQ ID NO:45 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2) and SEQ ID NO:48 (CDR3); ix) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2) and SEQ ID NO:51 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2) and SEQ ID NO:54 (CDR3); x) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2) and SEQ ID NO:57 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2) and SEQ ID NO:60 (CDR3); xi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2) and SEQ ID NO:63 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2) and SEQ ID NO:66 (CDR3); xii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2) and SEQ ID NO:69 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2) and SEQ ID NO:72 (CDR3); xiii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2) and SEQ ID NO:75 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2) and SEQ ID NO:78 (CDR3); xiv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2) and SEQ ID NO:81 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2) and SEQ ID NO:84 (CDR3); xv) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2) and SEQ ID NO:87 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2) and SEQ ID NO:90 (CDR3); xvi) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2) and SEQ ID NO:93 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2) and SEQ ID NO:96 (CDR3); xvii) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2) and SEQ ID NO:99 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2) and SEQ ID NO:102 (CDR3); xviii) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:103 (CDR1), SEQ ID NO:104 (CDR2) and SEQ ID NO:105 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2) and SEQ ID NO:108 (CDR3); xix) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:109 (CDR1), SEQ ID NO:110 (CDR2) and SEQ ID NO:111 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:112 (CDR1), SEQ ID NO:113 (CDR2) and SEQ ID NO:114 (CDR3); xx) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:115 (CDR1), SEQ ID NO:116 (CDR2) and SEQ ID NO:117 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:118 (CDR1), SEQ ID NO:119 (CDR2) and SEQ ID NO:120 (CDR3); xxi) antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:121 (CDR1), SEQ ID NO:122 (CDR2) and SEQ ID NO:123 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:124 (CDR1), SEQ ID NO:125 (CDR2) and SEQ ID NO:126 (CDR3); xxii) Antigen-binding unit, comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:127 (CDR1), SEQ ID NO:128 (CDR2) and SEQ ID NO:129 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:130 (CDR1), SEQ ID NO:131 (CDR2) and SEQ ID NO:132 (CDR3); xxiii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:133 (CDR1), SEQ ID NO:134 (CDR2), and SEQ ID NO:135 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:136 (CDR1), SEQ ID NO:137 (CDR2), and SEQ ID NO:138 (CDR3); and xxiv) Antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:139 (CDR1), SEQ ID NO:140 (CDR2) and SEQ ID NO:141 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:142 (CDR1), SEQ ID NO:143 (CDR2) and SEQ ID NO:144 (CDR3).

2. The protein of claim 1, wherein the first antigen-binding unit that specifically binds to B7H6 is selected from the group consisting of i) to xxiv): i) A light chain variable domain containing the amino acid sequence of SEQ ID NO:145 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

146. ii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:147 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:148; iii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:149 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:150; iv) A light chain variable domain containing the amino acid sequence of SEQ ID NO:151 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:152; v) The light chain variable domain containing the amino acid sequence of SEQ ID NO:153 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

154. vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:155 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

156. vii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:157 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

158. viii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:159 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

160. ix) The light chain variable domain containing the amino acid sequence of SEQ ID NO:161 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:162; x) The light chain variable domain containing the amino acid sequence of SEQ ID NO:163 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

164. xi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:165 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:166; xii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:167 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

168. xiii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:169 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

170. xiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:171 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

172. xv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:173 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:174; xvi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:175 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

176. xvii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:177 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:178; xviii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:179 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:180; xix) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:181 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:

182. xx) The light chain variable domain containing the amino acid sequence of SEQ ID NO:183 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:184; xxi) contains a light chain variable domain comprising the amino acid sequence of SEQ ID NO:185 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:

186. xxii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:187 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:188; xxiii) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:189 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:190; and xxiv) The light chain variable domain containing the amino acid sequence of SEQ ID NO:191 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

192.

3. The protein according to claim 1 or 2, wherein the second antigen-binding unit that specifically binds to CD3 is selected from the group consisting of i) to vi): i) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2) and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2) and SEQ ID NO:262 (CDR3); ii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:263 (CDR1), SEQ ID NO:264 (CDR2) and SEQ ID NO:265 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:266 (CDR1), SEQ ID NO:267 (CDR2) and SEQ ID NO:268 (CDR3); iii) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:269 (CDR1), SEQ ID NO:270 (CDR2) and SEQ ID NO:271 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:272 (CDR1), SEQ ID NO:273 (CDR2) and SEQ ID NO:274 (CDR3); iv) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:275 (CDR1), SEQ ID NO:276 (CDR2) and SEQ ID NO:277 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:278 (CDR1), SEQ ID NO:279 (CDR2) and SEQ ID NO:280 (CDR3); v) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:281 (CDR1), SEQ ID NO:282 (CDR2), and SEQ ID NO:283 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:284 (CDR1), SEQ ID NO:285 (CDR2), and SEQ ID NO:286 (CDR3); and vi) An antigen-binding unit comprising a light chain CDR containing the amino acid sequences of SEQ ID NO:287 (CDR1), SEQ ID NO:288 (CDR2) and SEQ ID NO:289 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:290 (CDR1), SEQ ID NO:291 (CDR2) and SEQ ID NO:292 (CDR3).

4. The protein according to any one of claims 1 to 3, wherein the second antigen-binding unit that specifically binds to CD3 is selected from the group consisting of i) to vi): i) The light chain variable domain containing the amino acid sequence of SEQ ID NO:293 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:294; ii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:295 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:296; iii) The light chain variable domain containing the amino acid sequence of SEQ ID NO:297 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:298; iv) A light chain variable domain containing the amino acid sequence of SEQ ID NO:299 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:300; v) A light chain variable domain comprising the amino acid sequence of SEQ ID NO:301 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:302; and vi) The light chain variable domain containing the amino acid sequence of SEQ ID NO:303 and the heavy chain variable domain containing the amino acid sequence of SEQ ID NO:

304.

5. The protein according to any one of claims 1 to 4, wherein i) The first antigen-binding unit that specifically binds to B7H6 comprises, from its N-terminus to its C-terminus, a first light chain variable domain, a first light chain constant domain, a first peptide linker, a first heavy chain variable domain, and a first heavy chain constant CH1 domain; and ii) The second antigen-binding unit that specifically binds to CD3 includes, from its N-terminus to its C-terminus, a second light chain variable domain, a second light chain constant domain, a second peptide linker, a second heavy chain variable domain, and a second heavy chain constant CH1 domain.

6. The protein according to claim 5, wherein... i) The first antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2), and SEQ ID NO:69 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2), and SEQ ID NO:72 (CDR3), and the second antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3); or ii) The first antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2), and SEQ ID NO:81 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2), and SEQ ID NO:84 (CDR3), and the second antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3); or iii) The first antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3), and the second antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2), and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2), and SEQ ID NO:262 (CDR3); or iv) The first antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2) and SEQ ID NO:93 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2) and SEQ ID NO:96 (CDR3), and the second antigen-binding unit comprises a light chain CDR containing the amino acid sequences of SEQ ID NO:257 (CDR1), SEQ ID NO:258 (CDR2) and SEQ ID NO:259 (CDR3) and a heavy chain CDR containing the amino acid sequences of SEQ ID NO:260 (CDR1), SEQ ID NO:261 (CDR2) and SEQ ID NO:262 (CDR3).

7. The protein according to claim 5 or 6, wherein i) The first antigen-binding unit includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO:167 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:168, and the second antigen-binding unit includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294; or ii) The first antigen-binding unit includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO:171 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:172, and the second antigen-binding unit includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:294; or iii) The first antigen-binding unit comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO:173 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:174, and the second antigen-binding unit comprises a light chain variable domain containing the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:294; or iv) The first antigen-binding unit includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO:175 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:176, and the second antigen-binding unit includes a light chain variable domain comprising the amino acid sequence of SEQ ID NO:293 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:

294.

8. The protein according to any one of claims 5 to 7, wherein the first and / or second peptide linker comprises 26 to 42 amino acids, preferably any one of 30 to 40 amino acids, 34 to 40 amino acids, or 36 to 39 amino acids, more preferably 38 amino acids.

9. The protein according to any one of claims 5 to 8, wherein the first linker and / or the second linker is a Gly-Ser linker, preferably comprising the amino acid sequence of SEQ ID NO:250, more preferably the first and second peptide linkers comprising the same sequence (e.g., SEQ ID NO:250).

10. The protein according to any one of claims 5 to 9, wherein the first light chain constant domain and the second light chain constant domain comprise a human κ or λ domain.

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