Multispecific binding proteins for cancer therapy

Abstract

The present invention relates to novel B7H6 / CD3 binding proteins. The present invention also relates to nucleic acids encoding said proteins; methods for preparing 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

TECHNICAL FIELD

[0001] The present invention relates to multispecific binding proteins comprising a first antigen binding unit specific for B7H6 (also referred to herein as "B7-H6") and a second antigen binding unit specific for CD3. The invention further relates to nucleic acids encoding said binding proteins, methods for the production of said binding proteins; host cells expressing or capable of expressing said binding proteins, compositions comprising said binding proteins, and uses of said binding proteins or said compositions, in particular in the field of cancer diseases for therapeutic purposes. BACKGROUND

[0002] B7H6 is a tumor-selective B7 family member that has been described to attract innate immunity to target cells and shares similar functions with other B7 family members, having two Ig-like domains in the extracellular domain, an N-terminal IgV-like domain and a C-terminal IgC1 -like domain. B7H6 triggers activation of human natural killer (NK) cells mediated by NKp30, leading to degranulation and IFNy secretion. (Brandt et al., J. Exp. Med. 2009 206(7); 1495-1503). Currently available data suggest a role of B7H6 in the inflammatory response to infectious conditions and in solid tumors.

[0003] It has been shown that B7H6 is expressed on CD14 + CD16 + cells isolated from peripheral blood of septic patients due to the inflammatory process in this acute disease state. The finding has been confirmed by an upregulation of B7H6 on the cell surface of CD14 + CD16 + proinflammatory monocytes and neutrophils upon stimulation with IL-1 β and TNFα in vitro. (Matta et al., Blood 2013 122(3)), suggesting a role of B7H6 in the inflammatory response to septic conditions.

[0004] In addition to the sepsis condition mentioned above, B7H6 is selectively expressed in other ways in tumor cells and cannot be detected in normal human tissues at steady state. For example, the expression of B7H6 has been described for 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 tissue (Zhang et al., Int J clin Exp Pathol 2014; 7(10): 6936-6942), gastrointestinal tumor tissue (Chen et al., Pathol. Oncol. Res. 2014 20:203-207; Zhao et al., Cell Proliferation 2018; e12468), ovarian cancer tissue (Zhou et al., Int clin Exp Pathol 2015 8(8), oral squamous cell carcinoma tissue (Wang et al., J Oral Pathol Med. 2017; 46:766-772) and hepatocellular carcinoma tissue (Li et al., Int. J. Mol. Sci. 2019, 20, 156), however, the function of B7H6 in tumors is not yet fully understood.

[0005] Therapeutic applications are described in WO 2009 / 046407 A2 and WO 2011 / 07044 A2, including the treatment of cancer using anti-B7H6 antibodies or anti-B7H6 antibody-drug conjugates that engage the ADCC / CDC pathway.

[0006] However, B7H6 targeting therapy based on ADCC / CDC activity is not the most preferred mode of action, because the cell surface expression of B7H6 is low and the success rate of using conventional antibodies with ADCC / CDC activity in solid tumors is low.

[0007] Targeting therapy based on B7H6-specific antibody drug conjugates (ADCs) can also have limitations, because most patients relapse after chemotherapy treatment and due to the low expression of B7H6 on the cell surface. In addition, the ADC approach usually has off-target toxicity caused by free drug, which is a result of linker instability or degradation.

[0008] CAR-T cells and T cell engager antibodies are other approaches to targeted therapy of 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 Jun 1; 194(11):5305-11) describe preclinical data for a B7H6-specific BiTE, BiTE standing for bispecific T cell engager, which is a fusion protein of approximately 55 Kda consisting of two single-chain variable fragments (scFv). In this case, a B7H6-specific BiTE was engineered based on the OKT3-CD3- binder and a 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 CD3 and thus does not allow preclinical toxicology testing in cynomolgus monkeys, which is the preferred test species for preparing clinical trials (Chatenoud et al., The Rev Diabet Stud 2012; 9(4):372-381). An additional challenge is the short half-life of the relatively small, easily degradable BiTE molecule, which requires continuous intravenous administration in the clinic. Therefore, it is not proven whether this approach is successful. To date, there is no targeted therapy for tumors expressing B7-H6, and there remains an unmet need that is not addressed by current approaches.

[0009] 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 present with overt metastasis at initial presentation, and metastatic disease develops in 40-50% of newly diagnosed patients. Despite recent improvements in chemotherapy and targeted therapies, the survival duration of metastatic CRC is prolonged, but most patients will die from their disease.

[0010] In view of the poor prognosis of cancer patients with advanced disease, there is a need to identify more effective therapies, in particular effective therapies with improved tolerability.

[0011] It is therefore an object of the present application to provide pharmacologically active agents, compositions and / or methods of treatment that offer certain advantages over the agents, compositions and / or methods currently used and / or known in the art. Such advantages include, inter alia, improved therapeutic and pharmacological properties, such as in vivo potency, fewer side effects, reduced immunogenicity, improved therapeutic window, reduced number of administrations (e.g. infusions), lower dosages, prolonged half-life to allow less frequent dosing, and other advantageous properties, such as improved ease of manufacture, stability, compatibility with conventional antibody methods, or reduced cost of goods, compared to candidate drugs known in the art. SUMMARY

[0012] The present application is based on a bispecific T cell engager approach that employs a multispecific binding protein having a binding arm to CD3 expressed on T cells and a binding arm to B7H6 expressed on the cell surface of tumor cells. By simultaneously binding to T cells and tumor cells, the T cell engager of the present application forces the formation of a cytolytic synapse between the two cells and thereby selectively redirects T cell activity towards the targeted tumor cells.

[0013] In one aspect, the present application provides a multispecific binding protein comprising a first antigen binding unit that specifically binds to B7H6 and a second antigen binding unit that specifically binds to CD3, wherein the first antigen binding unit that specifically binds to B7H6 is selected from the group consisting of i) to xxiv):

[0014] i) an antigen binding unit that comprises light chain CDRs comprising the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3);

[0015] ii) an antigen binding unit that comprises light chain CDRs comprising 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 comprising the amino acid sequences of SEQ ID NO: 10 (CDR1), SEQ ID NO: 11 (CDR2), and SEQ ID NO: 12 (CDR3);

[0016] iii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3);

[0017] iv) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 19 (CDR1), SEQ ID NO: 20 (CDR2), and SEQ ID NO: 21 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 24 (CDR3);

[0018] v) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 25 (CDR1), SEQ ID NO: 26 (CDR2), and SEQ ID NO: 27 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 28 (CDR1), SEQ ID NO: 29 (CDR2), and SEQ ID NO: 30 (CDR3);

[0019] vi) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 31 (CDR1), SEQ ID NO: 32 (CDR2), and SEQ ID NO: 33 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 34 (CDR1), SEQ ID NO: 35 (CDR2), and SEQ ID NO: 36 (CDR3);

[0020] vii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 37 (CDR1), SEQ ID NO: 38 (CDR2), and SEQ ID NO: 39 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 40 (CDR1), SEQ ID NO: 41 (CDR2), and SEQ ID NO: 42 (CDR3);

[0021] viii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2), and SEQ ID NO:45 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2), and SEQ ID NO:48 (CDR3);

[0022] ix) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3);

[0023] x) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3);

[0024] xi) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2), and SEQ ID NO:63 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2), and SEQ ID NO:66 (CDR3);

[0025] xii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2), and SEQ ID NO:69 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2), and SEQ ID NO:72 (CDR3);

[0026] xiii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2), and SEQ ID NO:75 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2), and SEQ ID NO:78 (CDR3);

[0027] xiv) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2), and SEQ ID NO:81 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2), and SEQ ID NO:84 (CDR3);

[0028] xv) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3);

[0029] xvi) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2), and SEQ ID NO:93 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2), and SEQ ID NO:96 (CDR3);

[0030] xvii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2), and SEQ ID NO:99 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2), and SEQ ID NO:102 (CDR3);

[0031] xviii) an antigen binding unit which comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 103 (CDR1), SEQ ID NO: 104 (CDR2), and SEQ ID NO: 105 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 106 (CDR1), SEQ ID NO: 107 (CDR2), and SEQ ID NO: 108 (CDR3);

[0032] xix) an antigen binding unit which comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 109 (CDR1), SEQ ID NO: 110 (CDR2), and SEQ ID NO: 111 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 112 (CDR1), SEQ ID NO: 113 (CDR2), and SEQ ID NO: 114 (CDR3);

[0033] xx) an antigen binding unit which comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 115 (CDR1), SEQ ID NO: 116 (CDR2), and SEQ ID NO: 117 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 118 (CDR1), SEQ ID NO: 119 (CDR2), and SEQ ID NO: 120 (CDR3);

[0034] xxi) an antigen binding unit which comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 121 (CDR1), SEQ ID NO: 122 (CDR2), and SEQ ID NO: 123 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 124 (CDR1), SEQ ID NO: 125 (CDR2), and SEQ ID NO: 126 (CDR3);

[0035] xxii) an antigen binding unit which comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 127 (CDR1), SEQ ID NO: 128 (CDR2), and SEQ ID NO: 129 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 130 (CDR1), SEQ ID NO: 131 (CDR2), and SEQ ID NO: 132 (CDR3);

[0036] xxiii) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 133 (CDR1), SEQ ID NO: 134 (CDR2), and SEQ ID NO: 135 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 136 (CDR1), SEQ ID NO: 137 (CDR2), and SEQ ID NO: 138 (CDR3); and

[0037] xxiv) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 139 (CDR1), SEQ ID NO: 140 (CDR2), and SEQ ID NO: 141 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 142 (CDR1), SEQ ID NO: 143 (CDR2), and SEQ ID NO: 144 (CDR3).

[0038] In some embodiments of the binding proteins of the application, the first antigen binding unit that specifically binds to B7H6 is selected from the group consisting of i) to xxiv):

[0039] i) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 145 and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 146;

[0040] ii) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 147 and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 148;

[0041] iii) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 149 and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 150;

[0042] iv) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 151 and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 152;

[0043] v) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 153 and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 154;

[0044] vi) a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 155 and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 156;

[0045] vii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158;

[0046] viii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160;

[0047] ix) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162;

[0048] x) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164;

[0049] 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;

[0050] xii) 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;

[0051] xiii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170;

[0052] xiv) 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;

[0053] xv) 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;

[0054] 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;

[0055] xvii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178;

[0056] 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;

[0057] xix) 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;

[0058] 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;

[0059] xxi) 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;

[0060] xxii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188;

[0061] 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

[0062] xxiv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 191 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 192.

[0063] In some embodiments of the binding proteins of the application, the second antigen binding unit that specifically binds to CD3 is selected from the group consisting of i) - vi):

[0064] i) an antigen binding unit comprising light chain CDRs comprising the amino acid sequences of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3);

[0065] ii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:263 (CDR1), SEQ ID NO:264 (CDR2), and SEQ ID NO:265 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:266 (CDR1), SEQ ID NO:267 (CDR2), and SEQ ID NO:268 (CDR3);

[0066] iii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:269 (CDR1), SEQ ID NO:270 (CDR2), and SEQ ID NO:271 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:272 (CDR1), SEQ ID NO:273 (CDR2), and SEQ ID NO:274 (CDR3);

[0067] iv) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:275 (CDR1), SEQ ID NO:276 (CDR2), and SEQ ID NO:277 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:278 (CDR1), SEQ ID NO:279 (CDR2), and SEQ ID NO:280 (CDR3);

[0068] v) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:281 (CDR1), SEQ ID NO:282 (CDR2), and SEQ ID NO:283 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:284 (CDR1), SEQ ID NO:285 (CDR2), and SEQ ID NO:286 (CDR3); and

[0069] vi) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:287 (CDR1), SEQ ID NO:288 (CDR2), and SEQ ID NO:289 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:290 (CDR1), SEQ ID NO:291 (CDR2), and SEQ ID NO:292 (CDR3).

[0070] In some embodiments of the binding proteins of the application, the second antigen binding unit that specifically binds to CD3 is selected from the group consisting of i) to vi):

[0071] i) 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;

[0072] ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 295 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 296;

[0073] iii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 297 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 298;

[0074] iv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 299 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 300;

[0075] 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

[0076] vi) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 303 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 304.

[0077] In some embodiments of the binding proteins of the application, the first antigen binding unit that specifically binds to B7H6 comprises, from its N-terminus to 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 CHI domain; and the second antigen binding unit that specifically binds to CD3 comprises, from its N-terminus to 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 CHI domain. In some embodiments of the binding proteins of the application, the first and / or second peptide linker comprises any of 26 to 42 amino acids, preferably 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 application, 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 linker comprise the same sequence (e.g. SEQ ID NO: 250). In some embodiments of the application, the first light chain constant domain and the second light chain constant domain independently comprise a human kappa or lambda domain.

[0078] In some embodiments, the first antigen binding unit specific for B7H6 of the binding proteins of the application 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 the second antigen binding unit specific for CD3 comprises an amino acid sequence selected from the group consisting of 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, preferably SEQ ID NO: 305.

[0079] In some embodiments, the binding proteins of the application further comprise a first and a second Fc domain, wherein the first Fc domain is covalently linked to the first antigen binding unit, preferably to the C-terminus of the first antigen binding unit, and the second Fc domain is covalently linked to the second antigen binding unit, preferably to the C-terminus of the second antigen binding unit.

[0080] In some embodiments of the application,

[0081] i) the first Fc domain comprises a tyrosine (Y) at position 366 [T366Y] and the second Fc domain comprises a threonine (T) at position 407 [Y407T], or

[0082] ii) the first Fc domain comprises a tryptophan (W) at position 366 [T366W] and the second Fc domain comprises a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A] and a valine (V) at position 407 [Y407V], or

[0083] iii) the second Fc domain comprises a tyrosine (Y) at position 366 [T366Y] and the first Fc domain comprises a threonine (T) at position 407 [Y407T], or

[0084] iv) the second Fc domain comprises a tryptophan (W) at position 366 [T366W] and the first Fc domain comprises a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A] and a valine (V) at position 407 [Y407V],

[0085] Preferably, wherein the first or second Fc domain further comprises an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F]. In some embodiments, the first and / or second Fc domain comprises an alanine at position 234 [L234A] and an alanine at position 235 [L235A].

[0086] In some embodiments, the binding proteins of the present application comprise 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: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, and SEQ ID NO: 316, preferably SEQ ID NO: 311.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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

[0092] Figure 1Schematic of a bispecific binding protein of the present invention.

[0093] Figure 2 Schematic of an extracellular B7H6 protein expressed on the cell surface of CHO-K1 cells.

[0094] Figure 3 Binding of 34 exemplary B7H6 / CD3 binding proteins to recombinant human B7H6 extracellular protein.

[0095] Figure 4 Binding of 34 exemplary B7H6 / CD3 binding proteins to recombinant human alanine mutated B7H6 extracellular protein.

[0096] Figure 5 Binding of 34 exemplary B7H6 / CD3 binding proteins to HCT-15 cells expressing endogenous human B7H6.

[0097] Figure 6 Binding of 23 exemplary B7H6 / CD3 binding proteins to recombinant CHO-K1 cells expressing cynomolgus monkey B7H6.

[0098] Figure 7 Binding of 23 exemplary B7H6 / CD3 binding proteins to human T cells expressing CD3.

[0099] Figure 8 Binding of 23 exemplary B7H6 / CD3 binding proteins to B7H6 negative CHO-K1 cells.

[0100] Figure 9 Inhibitory activity of 17 exemplary B7H6 / CD3 binding proteins on B7H6-dependent IFNy secretion by NK-92MI cells.

[0101] Figure 10 Efficiency of 11 exemplary B7H6 / CD3 binding proteins in lysing target cells in redirecting unstimulated T cells to human HCT-15 cells.

[0102] Figure 11 Efficiency of 23 exemplary B7H6 / CD3 binding proteins in lysing target cells in redirecting unstimulated T cells to human HCT-15 cells.

[0103] Figure 12 Efficiency of exemplary B7H6 / CD3 binding proteins in lysing target cells at various effector to target (E:T) cell ratios.

[0104] Figure 13: Potency of 23 exemplary B7H6 / CD3 binding proteins in redirecting unstimulated T cells to recombinant CHO cells transfected with B7-H6 and Cho wt cells in lysed cells.

[0105] Figure 14 : Potency of 6 exemplary B7H6 / CD3 binding proteins in CD25 upregulation on T cells in the presence of HCT-15 cells.

[0106] Figure 15 : Potency of 6 exemplary B7H6 / CD3 binding proteins in perforin expression upregulation in T cells in the presence of HCT-15 cells.

[0107] Figure 16 : Potency of 6 exemplary B7H6 / CD3 binding proteins in granzyme B expression upregulation in T cells in the presence of HCT-15 cells.

[0108] Figure 17 : Potency of 6 exemplary B7H6 / CD3 binding proteins in T cell proliferation in the presence of HCT-15 cells.

[0109] Figure 18 : Potency of 5 exemplary B7H6 / CD3 binding proteins in T cell secretion of IFNy in the presence of HCT-15 cells.

[0110] Figure 19 : Pharmacokinetic profile of an exemplary B7H6 / CD3 binding protein.

[0111] Figure 20 : Anti-tumor activity of an exemplary B7H6 / CD3 binding protein in a T cell engrafted mouse xenograft model.

[0112] Figure 21 : T cell infiltration in NCI-H716 xenograft tumor tissue with an exemplary B7H6 / CD3 binding protein.

[0113] Figure 22 : Pharmacokinetic profile of four exemplary B7H6 binding proteins.

[0114] Figure 23 : Anti-tumor activity of four exemplary B7H6 / CD3 binding proteins in a T cell engrafted mouse xenograft model.

[0115] Figure 24 : Anti-tumor activity of an exemplary B7H6 / CD3 binding protein in a T cell engrafted mouse xenograft model at q7d or as a single dose. DETAILED DESCRIPTION

[0116] Terms and definitions used

[0117] The above and other aspects and embodiments of the present application will become apparent from the further description herein, wherein:

[0118] Unless otherwise indicated or defined, all terms used have their common general meaning as is understood by one of ordinary skill in the art. Reference is made, for example, to standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), as well as the general background art cited herein. Furthermore, all methods, steps, techniques and manipulations which are not explicitly described in detail can be performed and carried out in a manner known per se, unless otherwise indicated, as will be apparent to the skilled person. Reference is again made, for example, to the standard handbooks, the general background art cited above and other references cited therein.

[0119] The term "comprising", and variations thereof such as "comprises" and "comprise", as used herein, can be substituted with the term "containing" or "including" or "having".

[0120] Unless the context requires otherwise, the term "sequence" (e.g. in terms such as "heavy / light chain sequence", "antibody sequence", "variable domain sequence", "constant domain sequence" or "protein sequence") as used herein is generally to be understood as including the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding the same.

[0121] The term "antigen binding unit" as used herein comprises the minimum structural requirements from an antibody which allow binding to its specific target or antigen (i.e. the minimum structural requirements normally present in an antibody). Thus, 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.

[0122] The general structure of antibodies or immunoglobulins is well known to those skilled in the art. The molecules are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains that are usually referred to as full-length antibodies. Each light chain is covalently linked to a heavy chain by one disulfide bond, to form a heterodimer, and the heterotetrameric molecule is formed by the covalent disulfide bonds between the two identical heavy chains. Although the light and heavy chains are connected by one 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 intrachain disulfide bonds of regular spacing. Each heavy chain has at its N-terminus a variable domain (VH) followed by three or four constant domains (CHI, CH2, CH3, and CH4 in the case of IgE) and a hinge region between CHI and CH2. Each light chain has two domains, a variable domain (VL) at the N-terminus, and a constant domain (CL) at the C-terminus. The VL domain associates with the VH domain to form a single variable region, while the CL domain is covalently linked to the CHI domain via a disulfide bond. It is believed that specific amino acid residues form an interface between the light and heavy chain variable domains (Chothia et al., 1985, J. Mol. Biol. 186:651-663). The variable domains are also referred to herein as variable regions or Fv and represent the portions of the antibody that confer specificity to the antigen.

[0123] "Light chain variable domain" (or "light chain variable region") and "heavy chain variable domain" (or "heavy chain variable region") as used herein have the same general structure and each domain essentially consists of four framework (FR) regions of generally conserved sequence, referred to herein and in the art below as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively; interrupted by three hypervariable regions HVRs (or CDRs), referred to herein and in the art below as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The framework regions adopt a beta-sheet conformation and the CDRs can form loops connecting the beta-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.

[0124] Various definitions of CDRs are known in the art, e.g., based on the CCG, also known as IMGT, definition (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. 2003 Jan;27(l):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 the Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196:901-917) and Kabat (E.A. Kabat, T.T. Wu, H. Bilofsky, M. Reid-Miller, and H. Perry, Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983)) definitions. Within the context of the present application, the reference to CDRs is based on the CCG (IMGT) definition.

[0125] The term "constant domain" or "constant region" as used herein in the context of the present application denotes the domains and regions of an antibody other than the variable regions. Said constant domains and regions are well known in the state of the art and are described, for example, by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication no. 91-3242 (1991)). Depending on the amino acid sequence of the constant region of the heavy chain, antibodies or immunoglobulins are assigned to the following classes: IgA, IgD, IgE, IgG, and IgM. Depending on the heavy chain constant region, the different classes of immunoglobulins are designated a, δ, ε, γ, and μ, respectively. Several of said classes can be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2.

[0126] The "Fc portion" or "Fc domain" of an antibody is not directly involved in binding of the antibody to antigen, but exhibits various effector functions. The "Fc portion / domain" of an antibody is well known to the person skilled in the art and is a term defined based on papain cleavage of antibodies. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, Clq binding and Fc receptor binding. Complement activation (CDC) is initiated by the binding of the complement factor Clq to the Fc portion of most IgG antibody subclasses. While the influence of an antibody on the complement system depends on certain conditions, binding to Clq is caused by defined binding sites in the Fc portion. Said binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to Eu numbering (Edelman et al., Proc Natl Acad Sci U S A. 1969 May; 63(1): 78-85)). The most critical of said residues mediating Clq and Fc gamma receptor binding in IgGl are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168, Shields et al. (2001) JBC, 276(9): 6591-6604). Antibodies of the subclasses IgGl and IgG3 usually show complement activation as well as Clq and C3 binding, while IgG2 and IgG4 do not activate the complement system and do not bind Clq and C3.

[0127] 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 (in particular Fv, Fab, Fab' or F(ab')2 fragments), single-chain antibodies (in particular single-chain variable fragments (scFv), single-chain Fab fragments (scFab)), small modular immunopharmaceuticals (SMIPs), domain antibodies, Bispecific antibodies. Antibodies can have an effector function, e.g., ADCC or CDC, which is typically mediated by the Fc portion of the antibody, or they can lack an effector function, e.g., lack an Fc portion or have a blocked, masked Fc portion, an Fc portion that is not recognized, or not sufficiently recognized, by immune cells or components of the immune system, such as the complement system, in nature.

[0128] Monoclonal antibodies (mAbs) are monospecific antibodies of uniform amino acid sequence. They can be produced by the hybridoma technique from a hybrid cell line (called a hybridoma) representing a clonal line of a fusion between a B cell producing a specific antibody and a myeloma (B cell cancer) cell (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 by 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." J Immunol Methods 204 (1): 77-87; see also below). A "recombinant antibody" or "recombinant binding protein" is an antibody or binding protein that has been produced by a recombinantly engineered host cell. It is optionally isolated or purified.

[0129] 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.

[0130] Numerous techniques have been developed to place variable domains of immunoglobulins, or molecules derived from said variable domains, into different molecular environments. These should also be considered "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 immunoglobulins 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). "Single-domain antibody" or " "Single-domain antibodies" possess an antigen binding site in a single Ig-like domain (WO 94 / 04678; WO03 / 050531, Ward et al., Nature. 1989 Oct 12; 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 for the same or different antigens can be linked together. "Diabodies" are bivalent antibody molecules consisting of two amino acid chains comprising two variable domains (WO94 / 13804, Holliger et al., Proc Natl Acad Sci U S A. 1993 Jul 15; 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). A different concept leads to so-called "small modular immunopharmaceuticals (SMIPs)", which comprise a Fv domain linked to a single chain hinge and an effector domain lacking the constant domain CH1 (WO 02 / 056910). "Single-chain Fabs" or "scFabs" are fusions of a light chain Fab domain (i.e. a light chain variable domain (VL) linked to one light chain constant domain (CL)) and a heavy chain Fab domain (i.e. a heavy chain variable domain (VH) linked to one heavy chain constant domain (CH1)). Single-chain Fabs are capable of recognizing and binding antigens. scFabs can also optionally contain a linker (e.g. a peptide linker) between the CL and VH domains (Hust et al. BMC Biotechnology 2007, 7: 14).

[0131] For use in humans, it is often desirable to reduce the immunogenicity of therapeutic molecules, such as antibodies or binding proteins comprising an antigen binding unit 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 methods known as "humanization". In this context, a "chimeric antibody" or "chimeric antigen binding unit" is understood to be an antibody or antigen binding unit comprising a sequence portion (e.g. variable domain) derived from one species (e.g. mouse) fused to a sequence portion (e.g. constant domain) derived from a different species (e.g. human). In this context, a "humanized antibody", "humanized antigen binding unit" or "humanized VL / VH domain" comprises an antibody, antigen binding unit or VH / VL domain whose variable domain is originally derived from a non-human species, wherein certain amino acids have been mutated so that the overall sequence of the variable domain more closely resembles the sequence of a human variable domain. Methods for humanization of antibodies are well known in the art (Billetta R, Lobuglio AF. "Chimeric antibodies". Int Rev Immunol. 1993; 10(2-3): 165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature: 332:323).

[0132] The term "human antibody", "human antigen binding unit" or "human VH / VL domain" as used herein includes an antibody, antigen binding unit or VH / VL domain having variable (and, if applicable, constant) regions derived from human germline immunoglobulin sequences. The term "human antibody", "human antigen binding unit" or "human VH / VL domain" as used herein is not intended to include antibodies in which CDR sequences derived from another (mammalian) species germline have been grafted onto human framework sequences. Thus, the term "human antibody", "human antigen binding unit" or "human VH / VL domain" as used herein refers to an antibody, antigen binding unit or VH / VL domain in which each portion of the protein (e.g. CDR, framework, CL, CH domain (e.g. CH1, CH2, CH3), hinge, VL, VH) is substantially non-immunogenic in humans, with only minimal sequence alterations or changes as further described herein below.

[0133] Techniques for producing the "human antibody", "human antigen binding unit" or "human VH / VL domain" have been described and include, but are not limited to, phage display or the use of transgenic animals (WWW.Ablexis.com / technology-alivamab.php; WO 90 / 05144; D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. 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.; Bruggemann M, Taussig MJ. "Production of human antibody repertoires in transgenic mice". Curr Opin Biotechnol. 1997 Aug; 8(4): 455-8.).

[0134] Thus, a human antibody, human antigen binding unit or human VH / VL domain is distinct from, for example, a chimeric or humanized antibody. It is noted that a human antibody, human antigen binding unit or human VH / VL domain can be produced by a non-human animal or prokaryotic or eukaryotic cell capable of expressing a functionally rearranged human immunoglobulin (e.g. heavy and / or light chain) gene.

[0135] The chimeric, humanized or human antibodies, antigen binding units or VH / VL domains of the application can be further optimized; also referred to herein as "optimized" or "sequence-optimized" antibodies, antigen binding units or VH / VL domains. The optimization includes, but is not limited to, removal or exchange of undesired amino acids, e.g. to reduce immunogenicity in humans, or to avoid deamidation, undesired charge or lipophilicity or non-specific binding. The removal or exchange of undesired amino acids can be introduced, e.g., by in vitro random or site-specific mutagenesis or by in vivo somatic mutation. Furthermore, with regard to chimeric or humanized antibodies, antigen binding units or VH / VL domains, it is understood that certain mouse FR residues can be important for the function of the optimized antibodies, antigen binding units and VH / VL domains. Thus, the important amino acid residues can be retained in the optimized antibodies, antigen binding units and VH / VL domains.

[0136] The term "monomer" refers to a homomer 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 the present application, a monomer means a monomeric protein of the application as described herein having a single antigen binding unit specific for B7H6 and a single antigen binding unit specific for CD3. For example, a monomer of a binding protein described herein can have two polypeptide chains, i.e. a first polypeptide chain comprising a single chain Fab specific for B7H6 and a first Fc domain and a second polypeptide chain comprising a single chain Fab specific for CD3 and a second Fc domain.

[0137] An epitope is the region of an antigen that is bound by an antibody or antigen binding moiety, e.g. an antigen binding unit of a protein described herein. The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody or antigen binding moiety. In certain embodiments, epitope determinants include chemical groups on a molecule such as amino acids, glycan side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics. Conformational and linear epitopes are distinguished in that the former, but not the latter, are dependent upon the conformation of the protein for their identification.

[0138] An antigen binding molecule / protein (e.g., an immunoglobulin, an antibody, an antigen binding unit, or a fragment of the antigen binding molecule / protein) that can "bind", "bind to", "bind specifically to", or "bind specifically to", be "a binding (to)" or "a specific binding to", have "affinity for", "specificity for", and / or "specificity to" a particular epitope, antigen, or protein (or at least a portion, fragment, or epitope thereof) is referred to as a "binding" molecule / protein "against" or "directed against" the epitope, antigen, or protein, or involving the epitope, antigen, or protein. The terms are used interchangeably herein.

[0139] As used herein, the terms "bind" and "bind specifically" refer to the binding of an antigen binding molecule / protein (e.g., an immunoglobulin, an antibody, an antigen binding unit, or a fragment of the antigen binding molecule / protein) to an antigenic epitope with purified wild-type antigen in an in vitro assay, preferably in a surface plasmon resonance assay (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 Apr; 5(2): 282-6.). Antibody affinity can also be measured using Kinetic Exclusion Assay (KinExA) technology (Darling, R.J. and Brault P-A., "Kinetic exclusion assay technology: Characterization of Molecular Interactions." ASSAY and Drug Development Technologies. 2004 Dec 2(6): 647-657). For example, a binding protein or protein of the present application binds to an epitope of B7H6 with its first antigen binding unit / first polypeptide chain and to an epitope of CD3 with its second antigen binding unit / second polypeptide chain.

[0140] 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.

[0141] 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.

[0142] The term "isolated" as used herein refers to material that is removed from its original or natural environment (e.g., the natural environment if it is natural). For example, a native polynucleotide or polypeptide that is present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials of the natural system by the intervention of man, is isolated. The polynucleotide can be part of a vector and / or the polynucleotide or polypeptide can be part of a composition, and still be isolated in that the vector or composition is not part of a natural environment found in nature. For example, a nucleic acid, protein / polypeptide molecule is considered to be in a "substantially isolated" form when compared to its natural biological source and / or reaction or culture medium - when the nucleic acid, protein / polypeptide molecule is 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) with which it is ordinarily associated in the source or medium. In particular, a nucleic acid or protein / polypeptide is considered to be "substantially isolated" if it is purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. A nucleic acid or protein / polypeptide molecule in "substantially isolated" form is preferably substantially uniform, as determined using appropriate techniques (e.g., an appropriate chromatographic technique, such as polyacrylamide gel electrophoresis).

[0143] As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity," means that sequences are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence. To determine identity, sequences are compared to determine the best overall match between the sequences. For example, gaps can be introduced in a sequence for optimal alignment. Then the amino acid residues or nucleotides at the corresponding positions are 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 then the molecules are identical at that position. The percent of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences being compared are the same length, after introduction of gaps if appropriate (e.g., to exclude additional sequence that extends beyond the sequence being compared). For example, when comparing variable region sequences, leader (signal peptide) and / or constant domain sequences are not considered. For sequence comparison of two sequences, a "corresponding" CDR refers to a CDR in the same position in both sequences (e.g., CDR-H1 of each sequence).

[0144] Determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm that is used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm that is used to compare sequences is the algorithm of Myers and Miller, CABIOS (1989). This algorithm is incorporated in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap 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 Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup = 2, similar regions in the two sequences being compared are found by observing pairs of aligned residues; 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 CLUSTALW algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0145] The term "covalently linked" or "covalently bound" as used herein means a direct covalent bond between residues, or an indirect linkage / bond, wherein two residues are not directly bonded, but are both covalently bonded to an intermediate molecule or domain, such as an intermediate domain of an immunoglobulin or a linker.

[0146] Multispecific binding proteins of the application

[0147] The present application provides multispecific binding proteins comprising at least one antigen binding unit that specifically binds to B7H6 (first antigen binding unit) and at least one antigen binding unit that specifically binds to CD3 (second antigen binding unit). By simultaneously binding to a tumor cell antigen and CD3 on T cells, the binding proteins act as T cell activating proteins and are also referred to herein as T cell engagers. The term "(multispecific) binding protein" is used herein interchangeably with the term "(multispecific) binding molecule". Other terms used herein in relation to the multispecific binding proteins of the present application are "proteins of the application", "binding proteins of the application", "antigen binding proteins" and "multispecific proteins".

[0148] The present inventors surprisingly found that the multispecific binding proteins of the present application induce potent and selective lysis of B7H6 positive colorectal cancer cell lines in the presence of T cells and are already active at low effector to target cell ratios. Importantly, the binding proteins of the present application do not lyse B7H6 negative cells and do not cause T cell activation, T cell proliferation and cytokine secretion in the absence of B7H6 positive cells. Notably, the proteins of the present application that do not inhibit B7H6 dependent NK cell activation via NKp30 in vitro are more potent in lysing B7H6 positive tumor cells. This activity is described in the in vitro assays of e.g. Example 11.

[0149] For the avoidance of doubt, B7H6 as used herein refers to human B7H6 of UniProt Q68D85 and to nucleic acid sequences encoding this protein. CD3 as used herein refers to the human CD3 epsilon (UniProt P07766) and CD3 gamma (UniProt: P09693) complex (human CD3 epsilon gamma complex). The skilled person will understand that the terms B7H6 and B7-H6 are used interchangeably herein.

[0150] In one aspect, the multispecific binding proteins of the present application comprise a first antigen binding unit that specifically binds to B7H6 and a second antigen binding unit that specifically binds to CD3, wherein the first binding unit is selected from the group consisting of i) to xxiv):

[0151] i) an antigen binding unit comprising light chain CDRs comprising the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3), and heavy chain CDRs comprising 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);

[0152] ii) an antigen binding unit comprising light chain CDRs comprising 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 comprising 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);

[0153] iii) an antigen binding unit comprising light chain CDRs comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and heavy chain CDRs comprising 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);

[0154] iv) an antigen binding unit comprising light chain CDRs comprising the amino acid sequences of SEQ ID NO: 19 (CDR1), SEQ ID NO: 20 (CDR2), and SEQ ID NO: 21 (CDR3), and heavy chain CDRs comprising 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);

[0155] v) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2), and SEQ ID NO:30 (CDR3) (antigen binding unit B7H6#5);

[0156] vi) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2), and SEQ ID NO:33 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2), and SEQ ID NO:36 (CDR3) (antigen binding unit B7H6#6);

[0157] vii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:39 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2), and SEQ ID NO:42 (CDR3) (antigen binding unit B7H6#7);

[0158] viii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2), and SEQ ID NO:45 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2), and SEQ ID NO:48 (CDR3) (antigen binding unit B7H6#8);

[0159] ix) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3) (antigen binding unit B7H6#9);

[0160] x) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 55 (CDR1), SEQ ID NO: 56 (CDR2), and SEQ ID NO: 57 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 58 (CDR1), SEQ ID NO: 59 (CDR2), and SEQ ID NO: 60 (CDR3) (antigen binding unit B7H6#10);

[0161] xi) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 61 (CDR1), SEQ ID NO: 62 (CDR2), and SEQ ID NO: 63 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 64 (CDR1), SEQ ID NO: 65 (CDR2), and SEQ ID NO: 66 (CDR3) (antigen binding unit B7H6#11);

[0162] xii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 70 (CDR1), SEQ ID NO: 71 (CDR2), and SEQ ID NO: 72 (CDR3) (antigen binding unit B7H6#12);

[0163] xiii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 76 (CDR1), SEQ ID NO: 77 (CDR2), and SEQ ID NO: 78 (CDR3) (antigen binding unit B7H6#13);

[0164] xiv) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 79 (CDR1), SEQ ID NO: 80 (CDR2), and SEQ ID NO: 81 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 82 (CDR1), SEQ ID NO: 83 (CDR2), and SEQ ID NO: 84 (CDR3) (antigen binding unit B7H6#14);

[0165] xv) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO:85 (CDR1 ), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:88 (CDR1 ), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3) (antigen binding unit B7H6#15);

[0166] xvi) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO:91 (CDR1 ), SEQ ID NO:92 (CDR2), and SEQ ID NO:93 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:94 (CDR1 ), SEQ ID NO:95 (CDR2), and SEQ ID NO:96 (CDR3) (antigen binding unit B7H6#16);

[0167] xvii) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO:97 (CDR1 ), SEQ ID NO:98 (CDR2), and SEQ ID NO:99 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:100 (CDR1 ), SEQ ID NO:101 (CDR2), and SEQ ID NO:102 (CDR3) (antigen binding unit B7H6#17);

[0168] xviii) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO:103 (CDR1 ), SEQ ID NO:104 (CDR2), and SEQ ID NO:105 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:106 (CDR1 ), SEQ ID NO:107 (CDR2), and SEQ ID NO:108 (CDR3) (antigen binding unit B7H6#18);

[0169] xix) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO:109 (CDR1 ), SEQ ID NO:110 (CDR2), and SEQ ID NO:111 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:112 (CDR1 ), SEQ ID NO:113 (CDR2), and SEQ ID NO:114 (CDR3) (antigen binding unit B7H6#19);

[0170] xx) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 115 (CDR1 ), SEQ ID NO: 116 (CDR2), and SEQ ID NO: 117 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 118 (CDR1 ), SEQ ID NO: 119 (CDR2), and SEQ ID NO: 120 (CDR3) (antigen binding unit B7H6#20);

[0171] xxi) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 121 (CDR1 ), SEQ ID NO: 122 (CDR2), and SEQ ID NO: 123 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 124 (CDR1 ), SEQ ID NO: 125 (CDR2), and SEQ ID NO: 126 (CDR3) (antigen binding unit B7H6#21 );

[0172] xxii) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 127 (CDR1 ), SEQ ID NO: 128 (CDR2), and SEQ ID NO: 129 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 130 (CDR1 ), SEQ ID NO: 131 (CDR2), and SEQ ID NO: 132 (CDR3) (antigen binding unit B7H6#22);

[0173] xxiii) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 133 (CDR1 ), SEQ ID NO: 134 (CDR2), and SEQ ID NO: 135 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 136 (CDR1 ), SEQ ID NO: 137 (CDR2), and SEQ ID NO: 138 (CDR3) (antigen binding unit B7H6#23); and

[0174] xxiv) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 139 (CDR1 ), SEQ ID NO: 140 (CDR2), and SEQ ID NO: 141 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 142 (CDR1 ), SEQ ID NO: 143 (CDR2), and SEQ ID NO: 144 (CDR3) (antigen binding unit B7H6#24).

[0175] In some embodiments of the binding proteins of the application, the second antigen binding unit that specifically binds to CD3 is selected from the group consisting of i) - vi):

[0176] i) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3) (antigen binding unit CD3#1);

[0177] ii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 263 (CDR1), SEQ ID NO: 264 (CDR2), and SEQ ID NO: 265 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 266 (CDR1), SEQ ID NO: 267 (CDR2), and SEQ ID NO: 268 (CDR3) (antigen binding unit CD3#2);

[0178] iii) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 269 (CDR1), SEQ ID NO: 270 (CDR2), and SEQ ID NO: 271 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 272 (CDR1), SEQ ID NO: 273 (CDR2), and SEQ ID NO: 274 (CDR3) (antigen binding unit CD3#3);

[0179] iv) an antigen binding unit comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 275 (CDR1), SEQ ID NO: 276 (CDR2), and SEQ ID NO: 277 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 278 (CDR1), SEQ ID NO: 279 (CDR2), and SEQ ID NO: 280 (CDR3) (antigen binding unit CD3#4);

[0180] v) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 281 (CDR1), SEQ ID NO: 282 (CDR2), and SEQ ID NO: 283 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 284 (CDR1), SEQ ID NO: 285 (CDR2), and SEQ ID NO: 286 (CDR3) (antigen binding unit CD3#5); and

[0181] vi) an antigen binding unit that comprises light chain CDRs comprising an amino acid sequence of SEQ ID NO: 287 (CDR1), SEQ ID NO: 288 (CDR2), and SEQ ID NO: 289 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 290 (CDR1), SEQ ID NO: 291 (CDR2), and SEQ ID NO: 292 (CDR3) (antigen binding unit CD3#6).

[0182] The first antigen binding units i) to xxiv) as outlined 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 outlined above are referred to as CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, and CD3#6, respectively. A sequence listing is provided herein that readily allows the identification of the individual amino acid sequences for the specific antigen binding units and full length binding proteins of the present application. An overview is provided in Table 1 in Example 2.

[0183] As used herein, the terms "first" and "second" in general in relation to the antigen binding units are merely intended to indicate that the units are two different units (as they bind to different target antigens). Thus, the terms are not to be understood as referring to the exact order or sequence of the units within the binding proteins of the present application.

[0184] In some embodiments, the binding proteins of the application comprise 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 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.

[0185] In some embodiments, the binding proteins of the application comprise 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 of CD3#1 as defined by the respective CDR sequences shown in Table 1. In preferred embodiments, the binding proteins of the application comprise 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 of CD3#1 as defined by the respective CDR sequences shown in Table 1. In preferred embodiments, the binding proteins of the application comprise 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 of CD3#1 as defined by the respective CDR sequences shown in Table 1. In preferred embodiments, the binding proteins of the application comprise 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 of CD3#1 as defined by the respective CDR sequences shown in Table 1.

[0186] In addition to the CDR sequences as described herein, the antigen binding unit of the binding proteins of the application also comprises immunoglobulin framework region (FR) sequences. The sequences are preferably non-immunogenic in humans and are thus preferably human, humanized or optimized FR sequences. Suitable human, humanized or optimized FR sequences are known in the art. Particularly preferred FR sequences can be taken from the embodiments shown herein, which disclose complete antigen binding units and thus disclose both CDR sequences as well as FR sequences. In a preferred embodiment, the binding protein of the application comprises a first antigen binding unit specifically binding to B7H6 comprising light chain CDRs comprising the amino acid sequences of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2) and SEQ ID NO: 69 (CDR3) and heavy chain CDRs 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 light chain CDRs comprising the amino acid sequences of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2) and SEQ ID NO: 259 (CDR3) and heavy chain CDRs 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 specifically binding to B7H6 and CD3, respectively, each comprise CDRs as defined above within a VL / VH domain, e.g. a sequence optimized VL / VH domain (B7H6#12 / CD3#1), respectively. In a particularly preferred embodiment, the antigen binding units specifically binding to B7H6 and CD3, respectively (B7H6#12 / CD3#1), are each formed by a scFab and are each optionally linked to a Fc domain.

[0187] In a preferred embodiment, the binding protein of the application comprises a first antigen binding unit that specifically binds to B7H6 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 79 (CDR1), SEQ ID NO: 80 (CDR2), and SEQ ID NO: 81 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 82 (CDR1), SEQ ID NO: 83 (CDR2), and SEQ ID NO: 84 (CDR3), and a second antigen binding unit that specifically binds to CD3 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising an amino acid sequence 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, each comprise the CDRs as defined above within a VL / VH domain, e.g., a sequence optimized VL / VH domain, respectively (B7H6#14 / CD3#1). In particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, are each formed by a scFab and are each, optionally, linked to a Fc domain (B7H6#14 / CD3#1).

[0188] In a preferred embodiment, the binding protein of the application comprises a first antigen binding unit that specifically binds to B7H6 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 85 (CDR1), SEQ ID NO: 86 (CDR2), and SEQ ID NO: 87 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 88 (CDR1), SEQ ID NO: 89 (CDR2), and SEQ ID NO: 90 (CDR3), and a second antigen binding unit that specifically binds to CD3 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising an amino acid sequence 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, each comprise the CDRs as defined above within a VL / VH domain, e.g. a sequence optimized VL / VH domain, respectively (B7H6#15 / CD3#1). In particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, (B7H6#15 / CD3#1) are each formed by a scFab and are each, optionally, linked to a Fc domain.

[0189] In a preferred embodiment, the binding protein of the application comprises a first antigen binding unit that specifically binds to B7H6 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 91 (CDR1), SEQ ID NO: 92 (CDR2), and SEQ ID NO: 93 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 94 (CDR1), SEQ ID NO: 95 (CDR2), and SEQ ID NO: 96 (CDR3), and a second antigen binding unit that specifically binds to CD3 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising an amino acid sequence 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, each comprise the CDRs as defined above within a VL / VH domain, e.g. a sequence optimized VL / VH domain, respectively (B7H6#16 / CD3#1). In particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, (B7H6#16 / CD3#1) are each formed by a scFab and are each, optionally, linked to a Fc domain.

[0190] In a preferred embodiment, the binding protein of the application comprises a first antigen binding unit which specifically binds to B7H6 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 133 (CDR1), SEQ ID NO: 134 (CDR2), and SEQ ID NO: 135 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 136 (CDR1), SEQ ID NO: 137 (CDR2), and SEQ ID NO: 138 (CDR3), and a second antigen binding unit which specifically binds to CD3 comprising light chain CDRs comprising an amino acid sequence of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising an amino acid sequence 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, each comprise the CDRs as defined above within a VL / VH domain, e.g. a sequence optimized VL / VH domain, respectively (B7H6#23 / CD3#1). In particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively (B7H6#23 / CD3#1), are each formed by a scFab and are each, optionally, linked to a Fc domain.

[0191] In preferred embodiments of the binding proteins of the application, the first and second binding units each comprise a light chain variable domain and a heavy chain variable domain, which for the first antigen binding unit are defined by the CDR sequences 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, or B7H6#24, and for the second antigen binding unit are defined by the CDR sequences of any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6. In some embodiments of the binding proteins of the application, the VH and / or VL domains of the antigen binding unit 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, CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 are human, humanized or optimized VH and / or VL domains.

[0192] In preferred embodiments of the binding proteins of the application, the light / heavy chain variable domains of the first antigen binding unit that specifically bind to B7H6 are further defined as follows

[0193] 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

[0194] ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 147 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 148 (antigen binding unit B7H6#2); or

[0195] iii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 149 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 (antigen binding unit B7H6#3); or

[0196] 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

[0197] 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

[0198] vi) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 (antigen binding unit B7H6#6); or

[0199] vii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 (antigen binding unit B7H6#7); or

[0200] viii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 (antigen binding unit B7H6#8); or

[0201] ix) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 (antigen binding unit B7H6#9); or

[0202] x) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 (antigen binding unit B7H6#10); or

[0203] 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

[0204] xii) 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 (antigen binding unit B7H6#12); or

[0205] xiii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 (antigen binding unit B7H6#13); or

[0206] xiv) 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 (antigen binding unit B7H6#14); or

[0207] xv) 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 (antigen binding unit B7H6#15); or

[0208] 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

[0209] xvii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178 (antigen binding unit B7H6#17); or

[0210] 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

[0211] xix) 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 (antigen binding unit B7H6#19); or

[0212] 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

[0213] xxi) 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 (antigen binding unit B7H6#21 ); or

[0214] xxii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188 (antigen binding unit B7H6#22); or

[0215] 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

[0216] xxiv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 191 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 192 (antigen binding unit B7H6#24).

[0217] In a preferred embodiment of the binding proteins of the application, the light / heavy chain variable domains of the second antigen binding unit specifically binding to CD3 are further defined as follows

[0218] i) 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 (antigen binding unit CD3#1); or

[0219] ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 295 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 296 (antigen binding unit CD3#2); or

[0220] iii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 297 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 298 (antigen binding unit CD3#3); or

[0221] iv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 299 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 300 (antigen binding unit CD3#4); or

[0222] 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

[0223] vi) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 303 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 304 (antigen binding unit CD3#6).

[0224] In preferred embodiments, the binding protein of the present application 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#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, said first and second antigen binding units being defined by the CDR and / or VH and VL sequences of the antigen binding units as indicated in Table 1.

[0225] In preferred embodiments, the binding protein of the present application 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, said first and second antigen binding units being defined by the CDR and / or VH and VL sequences of the antigen binding units as indicated in Table 1.

[0226] In a preferred embodiment, the binding protein of the application 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, said first and second antigen binding units being 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 application 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, said first and second antigen binding units being 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 application comprises (i) a first antigen binding unit that specifically binds 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 that specifically binds 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 specifically binding to B7H6 and CD3, respectively (B7H6#12 / CD3#1) are each formed by a scFab, which is optionally covalently linked to a Fc domain, as defined above.

[0229] In a preferred embodiment, the binding protein of the application comprises (i) a first antigen binding unit that specifically binds to B7H6, which comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 171 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 172, and (ii) a second antigen binding unit that specifically binds to CD3, which comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 293 and a light 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, as defined above (B7H6#14 / CD3#1) are each formed by a scFab, which is optionally covalently linked to a Fc domain.

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

[0231] In a preferred embodiment, the binding protein of the application comprises (i) a first antigen binding unit that specifically binds to B7H6, which comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 171 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 172, and (ii) a second antigen binding unit that specifically binds to CD3, which comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 293 and a light 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, as defined above (B7H6#14 / CD3#1) are each formed by a scFab, which is optionally covalently linked to a Fc domain.

[0232] In a preferred embodiment, the binding protein of the application comprises (i) a first antigen binding unit that specifically binds 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 that specifically binds 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 particularly preferred embodiments, the antigen binding units specifically binding to B7H6 and CD3, respectively, as defined above (B7H6#23 / CD3#1) are each formed by a scFab, which is optionally covalently linked to a Fc domain.

[0233] In some embodiments, the binding protein of the application comprises i) a first antigen binding unit that specifically binds to B7H6 (e.g., 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 defined by the respective CDR or VH / VL sequences shown in Table 1), comprising a first light chain variable domain covalently linked to a first heavy chain variable domain using a first peptide linker, and / or ii) a second antigen binding unit that specifically binds to CD3 (e.g., any 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 using a second peptide linker. Optionally, the first and second antigen binding units are covalently linked to each other using a peptide linker.

[0234] In some embodiments of the binding proteins of the application, the first and / or second antigen binding unit further comprises a CL and a CH1 domain as in a conventional antibody molecule light / heavy Fab fragment, thus the first binding unit comprises a) a VL domain covalently attached (preferably directly bound) to a first CL domain (e.g. as defined by the light chain CDRs (LCCDRs) or VL 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, or B7H6#24), and b) a VH domain covalently attached (preferably directly bound) to a first CH1 domain (e.g. as defined by the heavy chain CDRs (HCCDRs) 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, or B7H6#24), and / or the second antigen binding unit comprises a) a VL domain covalently attached (preferably directly bound) to a second CL domain (e.g. as defined by the LCCDRs or VL sequence of any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6), and b) a VH domain covalently attached (preferably directly bound) to a second CH1 domain (e.g. as defined by the HCCDRs or VH sequence of any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6).

[0235] In the context of the present application, a CL domain is the constant domain of an antibody light chain, e.g., kappa (K) or lambda (l) light chain. An example of a constant region of a K light chain is shown in SEQ ID NO: 247. An example of a constant region of a l light chain is shown in SEQ ID NO: 248. In some embodiments, the first and second CL domains are identical, e.g., both the first and second CL domains are K light chain constant domains, or both the first and second CL domains are l light chain constant domains. In preferred embodiments, the first and second CL domains are different, e.g., the first CL domain is a constant K domain and the second CL domain is a constant l domain, or vice versa.

[0236] In the context of the present application, a CH1 domain is the first constant domain of an antibody heavy chain. An example of a constant CH1 domain is shown in SEQ ID NO: 249.

[0237] In preferred embodiments of the binding proteins of the application, the first antigen binding unit specific for B7H6 (e.g., 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, or B7H6#24 as defined by the CDRs and / or VH / VL sequences shown in Table 1) comprises, from N- to C-terminus: a first light chain variable domain, a first CL domain, a first linker peptide, a first VH domain, and a first CH1 domain, and / or the second binding unit of the binding proteins of the application (e.g., CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as defined by the CDRs and / or VH / VL sequences shown in Table 1) comprises, from N- to C-terminus: a second light chain variable domain, a second CL domain, a second linker peptide, a second VH domain, and a second CH1 domain. In said embodiments, the first and / or second binding unit has the structure of a single chain Fab. For both the first and / or second antigen binding unit, the order can be reversed when forming a single chain Fab, such that the antigen binding unit comprises, from N- to C-terminus: VH-CH1-[linker peptide]-VL-CL. In some embodiments of the proteins of the application, when the first and / or second antigen binding unit comprises a Fab or a single chain Fab, the constant domains can be of the same type (e.g., both CL domains are kappa or lambda light chain constant domains) or of different types (the first CL domain is kappa and the second CL domain is lambda light chain constant domain, or vice versa), preferably, the first and second CL domains are of different types. In preferred embodiments, the first antigen binding unit consists of a first single chain Fab specific for B7H6 (preferably any of B7H6#12, B7H6#14, B7H6#15, B7H6#16, or B7H6#23 as defined by the CDRs and / or VH / VL sequences shown in Table 1), and the second antigen binding unit consists of a second single chain Fab specific for CD3 (e.g., CD3#1 as defined by the CDRs and / or VH / VL sequences shown in Table 1).

[0238] The linker sequence of a B7H6 / CD3 binding protein, e.g. a B7H6 / CD3 scFab as described above, can be a natural sequence or a non-natural sequence. If used for therapeutic purposes, the linker is preferably not immunogenic in the individual to which the binding protein of the application is administered. Preferably, the linker comprises 26 to 42 amino acids, e.g. 30 to 40 amino acids. In another aspect, the linker used in the proteins of the application comprises 34 to 40 amino acids, e.g. 36 to 39 amino acids, e.g. 38 amino acids.

[0239] One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 1996 / 34103 and WO 1994 / 04678. Other examples are poly-alanine linker sequences, e.g. Ala-Ala-Ala.

[0240] Other preferred examples of linker sequences are Gly / Ser linkers of different lengths, e.g. (glyxsery)zlinkers, including, e.g. (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 NOs: 250, 251, 252, 253, 254, 255, or 256, preferably the linker of SEQ ID NO: 250.

[0241] In some embodiments of the binding proteins of the application, the VL domain of the first antigen binding unit (e.g., as defined by the light chain CDRs (LCCDRs) or VL 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) is covalently linked to the VH domain of the first antigen binding unit (e.g., as defined by the heavy chain CDRs (HCCDRs) 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) via a first Gly / Ser linker (e.g., any 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 of a Gly / Ser linker); and the VL domain of the second antigen binding unit (e.g., as defined by the light chain CDRs (LCCDRs) or VL sequence of any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as shown in Table 1) is covalently linked to the VH domain of the second antigen binding unit (e.g., as defined by the heavy chain CDRs (HCCDRs) or VH sequence of any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as shown in Table 1) via a second Gly / Ser linker (e.g., any 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 of a Gly / Ser linker). More preferably, the first and second linkers are the same. Even more preferably, the first and second linkers each comprise the amino acid sequence of SEQ ID NO: 250.

[0242] In a preferred embodiment of the binding proteins of the application, the first antigen binding unit specifically binding to B7H6 comprises from N-terminus to C-terminus i) a VL domain (e.g. as defined by the light chain CDRs (LCCDRs) 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) a first CL domain, iii) 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), iv) a VH domain (e.g. as defined by the heavy chain CDRs (HCCDRs) or VH 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 ), and v) a first CH1 domain, and / or the second antigen binding unit specifically binding to CD3 comprises from N-terminus to C-terminus i) a VL domain (e.g. as defined by the light chain CDRs (LCCDRs) 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. as defined by the heavy chain CDRs (HCCDRs) 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 each linked in the order i) to v) from the N-terminus to the C-terminus of the antigen binding unit via a direct covalent bond (each antigen binding unit thus has the structure of a scFab).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 preferred embodiments, the binding protein of the application comprises a first single chain Fab forming a first antigen binding unit specific for 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 for CD3 and comprising the sequence of SEQ ID NO: 305.

[0244] In preferred embodiments, the binding protein of the application comprises a first single chain Fab forming a first antigen binding unit specific for 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 for CD3 and comprising the sequence of SEQ ID NO: 305.

[0245] In a preferred embodiment, the binding protein of the application comprises a first single chain Fab forming a first antigen binding unit specific for 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 for CD3 and comprising the sequence of SEQ ID NO: 305.

[0246] In a preferred embodiment, the binding protein of the application 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, optionally each single chain Fab is further linked to an Fc domain and thereby forms a first polypeptide chain ("B7H6 chain") and a second polypeptide chain ("CD3 chain"). In a preferred embodiment, the binding protein of the application 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, optionally each single chain Fab is further linked to an Fc domain and thereby forms a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain). In a preferred embodiment, the binding protein of the application 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, optionally each single chain Fab is further linked to an Fc domain and thereby forms a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain). In a preferred embodiment, the binding protein of the application 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, optionally each single chain Fab is further linked to an Fc domain and thereby forms a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain). In a preferred embodiment, the binding protein of the application 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, optionally each single chain Fab is further linked to an Fc domain and thereby forms a first polypeptide chain (B7H6 chain) and a second polypeptide chain (CD3 chain).

[0247] In some embodiments, the first antigen binding unit (e.g., 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, or B7H6#24 as defined by the CDRs and / or VH / VL sequences shown in Table 1) and / or the second antigen binding unit (e.g., any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6 as defined by the CDRs and / or VH / VL sequences shown in Table 1) comprises a VL domain covalently (preferably directly) bound to a CL domain and a VH domain covalently (e.g., directly) bound to a CH1 domain (together forming a Fab fragment), and the CH1 domain is further covalently (e.g., directly) bound to an 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 a Fab fragment, i.e., a first and a second Fab fragment, which are each covalently (preferably directly) bound to a first and a second Fc domain, respectively, thereby forming a conventional heterotetrameric bispecific and bivalent (monovalent for B7H6 and CD3, respectively) antibody molecule.

[0248] In preferred embodiments, the binding protein of the application comprises (i) a first antigen binding unit comprising a first single chain Fab specifically binding to B7H6, i.e. an antibody light chain (VL-CL) covalently linked, e.g. directly bound, to a VH-CH1 domain of a heavy chain (VL and VH domains 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 or B7H6#24 as defined by the CDRs and / or VH / VL sequences shown in Table 1) covalently linked to the heavy chain via a peptide linker (e.g. a Gly / Ser linker of any 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, even more preferably the linker of SEQ ID NO: 250), which first antigen binding unit is covalently linked, e.g. directly bound, to a first Fc domain, and (ii) a second antigen binding unit comprising a second single chain Fab specifically binding to CD3, i.e. an antibody light chain (VL-CL) covalently linked to a VH-CH1 domain of a heavy chain (VL and VH domains of any 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), which second antigen binding unit is covalently linked, e.g. directly bound, to a second Fc domain. Thus, in preferred embodiments, the binding protein of the application comprises (i) a first polypeptide chain comprising (a) a first antigen binding unit specific for B7H6 comprising a first single chain Fab specific for B7H6 (preferably any of B7H6#12, B7H6#14, B7H6#15, B7H6#16 or B7H6#23 as defined by the CDRs and / or VL / VH sequences shown in Table 1), and (b) a first Fc domain (this first polypeptide chain is also referred to herein as “B7H6 chain”), and (ii) a second polypeptide chain specific for CD3 comprising (a) a second antigen binding unit comprising a second single chain Fab specific for CD3 (preferably CD3#1 as defined by the CDRs 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 “CD3 chain”). Thus, as used herein the term “polypeptide chain” comprises at least a scFab and a Fc domain. In some embodiments, the first and second Fc domains are identical. In preferred embodiments, the first and second Fc domains are different.The resulting binding proteins of the present application comprise two different polypeptide chains, which carry an intact Fc and have two independent binding sites, i.e. a first antigen binding unit formed by a first scFab specific for B7H6 and a second binding unit formed by a second scFab specific for CD3.

[0249] In preferred embodiments, the binding proteins of the application comprise two different polypeptide chains each comprising an antigen binding unit formed by a scFab, the two different polypeptide chains having different specificities, each covalently linked to an Fc domain, the polypeptide chains being covalently linked to each other via disulfide bonds or potentially via a peptide linker. In preferred embodiments, the binding proteins of the application are bispecific, bivalent (monovalent for B7H6 and CD3, respectively) heterodimeric proteins comprising two polypeptide chains, one polypeptide chain (first polypeptide chain or B7H6 chain) comprising an antigen binding unit formed by a scFab specifically binding to B7H6 (e.g. 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, or B7H6#24 as defined by the CDR and / or VH / VL sequences shown in Table 1) and an Fc domain (preferably the Fc domain of SEQ ID NO: 242), and the other polypeptide chain (second polypeptide chain or CD3 chain) comprising an antigen binding unit formed by a scFab specifically binding to CD3 (e.g. any of CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, or CD3#6) and an Fc domain (preferably the Fc domain of SEQ ID NO: 243). In some embodiments, the first antigen binding unit consists of a first single chain Fab and the second antigen binding unit consists of a second single chain Fab. In some embodiments of the binding proteins, the first polypeptide chain (B7H6 chain) specific for B7H6 consists of a) a first antigen binding unit consisting of a scFab (preferably any 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, and the second polypeptide chain (CD3 chain) specific for CD3 consists of a) a second antigen binding unit consisting of a 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 the scFab is linked to the N-terminus of the Fc domain via a direct covalent bond. Preferably, the first and second polypeptide chains are covalently linked to each other via disulfide bonds, and form an antibody-like structure similar to a conventional Y-shaped antibody molecule Figure 1

[0250] ​In the context of the present application, the Fc domain is for example derived from the heavy chain of an IgG, for example IgG1, IgG2 or IgG4. For example, the Fc domain of the present application is the Fc domain of the heavy chain of IgG1 or IgG4 and comprises the hinge region and two constant domains (C H2 and C H3 ). Examples of Fc domains (including the hinge region) are shown in SEQ ID NO: 241 and 244.

[0251] Unless otherwise specified, the numbering of the amino acids in the amino acid chain of the proteins of the present application is herein according to the Eu numbering system (Edelman et al., PNAS USA 1969 May, 63(1 ): 78-85; Cunningham et al. PNAS USA 1969 Nov, 64(3): 997-1003). This means that unless otherwise specified, the amino acid numbering indicated herein corresponds to the position in the heavy chain of the respective subtype (e.g. IgG1 or IgG4) according to the Eu numbering system.

[0252] In some embodiments, the first and second Fc domains in the proteins of the application each comprise one or more amino acid changes that reduce the formation of homodimers of the first or second polypeptide chain over the formation of heterodimers of the first and second polypeptide chains. Through the changes, a "protuberance" is created in one of the Fc domains by replacing one or more small amino acid side chains at the interface of one heavy chain with a larger side chain, such as 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 one, such as alanine or threonine. This provides a mechanism for increasing the yield of heterodimers over other undesired end products, such as homodimers, particularly of the Fc domain with the "protuberance" (see, e.g., 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 changes are tyrosine (Y) at position 366 of the first Fc domain [T366Y] and threonine (T) at position 407 of the second Fc domain [Y407T]. In some embodiments, the first Fc domain comprises serine (S) at position 366 [T366S] and the second Fc domain comprises tryptophan (W) at position 366 [T366W], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V]. In preferred embodiments, the first Fc domain comprises tryptophan (W) at position 366 [T366W] and the second Fc domain comprises serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V]. For example, position 366 of the Fc domain corresponding to amino acid position 146 in the human IgGl Fc sequence of SEQ ID NO: 241 changes from T at position 146 in SEQ ID NO: 241 to W at position 146 in SEQ ID NO: 242 according to Eu numbering; and positions 366, 368, and 407 corresponding to amino acid positions 146, 148, and 187 in SEQ ID NO: 241, respectively, change from T, L, and Y at the positions in SEQ ID NO: 241 to S, A, and V at the positions in SEQ ID NO: 243, respectively. In any of the embodiments, the amino acid changes described for the first Fc domain can be in the second Fc domain and the respective amino acid changes of the second Fc domain can be 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 IgGl or IgG4.

[0253] In some embodiments, the first Fc domain comprises a cysteine (C) at position 354 [S354C] in addition to the tryptophan (W) at position 366 [T366W], and the second Fc domain comprises a cysteine (C) at position 349 [Y349C] in addition to the serine (S) at position 366 [T366S], the alanine (A) at position 368 [L368A], and the valine (V) at position 407 [Y407V]. In one aspect, the Fc domain is an Fc domain derived from a heavy chain of an IgG4.

[0254] In some embodiments, the first or second Fc domain in the binding proteins of the application further comprises one or more amino acid changes that reduce binding of the Fc domain to Protein A. In some embodiments, the amino acid changes are an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F] of one of the Fc domains. Both changes are derived from the sequence of human IgG3 (IgG3 does not bind to Protein A). The two mutations are in the CH3 domain and are incorporated into one of the Fc domains to reduce binding to Protein A (see, e.g., Jendeberg et al. J Immunol Methods, 1997. 201(1): p. 25-34). The two changes favor the removal of homodimers of the heavy chain comprising the changes during protein purification.

[0255] In some embodiments, in the binding proteins of the application, the Fc domain comprising the threonine (T) at position 407 [Y407T] further comprises an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F]. In this case, the other heavy chain comprises a tyrosine (Y) at position 366 [T366Y] but does not include the two changes at positions 435 and 436. Alternatively, in some embodiments, in the proteins of the application, the Fc domain comprising the serine (S) at position 366 [T366S], the alanine (A) at position 368 [L368A], and the valine (V) at position 407 [Y407V] further comprises an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F]. In this case, the other Fc domain comprises a tryptophan (W) at position 366 [T366W] but does not include the two changes at positions 435 and 436. Thus, the Fc domain comprising the amino acid changes that create a "cavity" as described above also comprises amino acid changes that reduce binding to Protein A. By reducing binding to Protein A, homodimers comprising this Fc domain are removed. The production of homodimers of the other Fc domain comprising the "protrusion" is reduced by the presence of the "protrusion".

[0256] In some embodiments, the Fc domain of the present proteins can or can not further comprise the YTE mutation (M252Y / S254T / T256E, Eu numbering (Dall'Acqua et al. J. Biol. Chem. 2006, 281(33):23514-24). The mutation has been shown to improve the pharmacokinetic properties of the Fc domain by preferentially enhancing the binding affinity to the neonatal FcRn receptor at pH 6.0.

[0257] In some embodiments, the first and / or second Fc domain of the present application derived from IgGl also comprises the "KO" mutation (L234A, L235A) (Xu et al., Cellular Immunology 2000 Feb 25, 200(1): 16-26). In another aspect, the first and / or second Fc domain of the present application derived from IgG4 also comprises the Pro hinge mutation (S228P) (Angal et al., Molecular Immunology 1993, 30(1): 105-108; Labrijn et al., Nature Biotechnology 2009, 27:767-771).

[0258] In a preferred embodiment of the binding proteins of the present application, 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.

[0259] In preferred embodiments of the application, 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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 222 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#6 / CD3#1); or vii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 223 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#7 / CD3#1); or viii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 224 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#8 / CD3#1); or ix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 225 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#9 / CD3#1); or x) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#10 / CD3#1); or xi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 227 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#11 / CD3#1); or xii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 228 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#12 / CD3#1); or xiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 229 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#13 / CD3#1).or xiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 230 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#14 / CD3#1); or xv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 231 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#15 / CD3#1), or xvi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 232 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#16 / CD3#1); or xvii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 233 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#17 / CD3#1); or xviii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 234 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#18 / CD3#1); or xix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 235 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#19 / CD3#1); or xx) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 236 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#20 / CD3#1); or xxi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 237 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#21 / CD3#1); or xxii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 238 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#22 / CD3#1); or xxiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 239 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#23 / CD3#1); or xxiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 240 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#24 / CD3#1). Preferably, the first and second polypeptide chains are connected via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 ).

[0260] In a preferred embodiment, the first polypeptide chain comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 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 the amino acid sequence of any one of SEQ ID NOs: 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 are connected via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1

[0261] In one preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 comprising the amino acid sequence of SEQ ID NO: 228 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311.

[0262] In one preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 comprising the amino acid sequence of SEQ ID NO: 230 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311.

[0263] In one preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 comprising the amino acid sequence of SEQ ID NO: 231 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311.

[0264] In one preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 comprising the amino acid sequence of SEQ ID NO: 232 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311.

[0265] In one preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 comprising the amino acid sequence of SEQ ID NO: 239 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311.

[0266] ​For all embodiments described herein, it is understood that by using the term "comprising", it is intended to also include embodiments wherein the respective protein, molecule, antigen binding unit or polypeptide chain "consists of the amino acid sequence as indicated:

[0267] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 consisting of the amino acid sequence of SEQ ID NO: 228 and a second polypeptide chain specific for CD3 consisting of the amino acid sequence of SEQ ID NO: 311. Preferably, the first and second polypeptide chains are connected via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule Figure 1 ).

[0268] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 consisting of the amino acid sequence of SEQ ID NO: 230 and a second polypeptide chain specific for CD3 consisting of the amino acid sequence of SEQ ID NO: 311. Preferably, the first and second polypeptide chains are connected 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 for B7H6 consisting of the amino acid sequence of SEQ ID NO: 231 and a second polypeptide chain specific for CD3 consisting of the amino acid sequence of SEQ ID NO: 311. Preferably, the first and second polypeptide chains are connected via 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 for B7H6 consisting of the amino acid sequence of SEQ ID NO: 232 and a second polypeptide chain specific for CD3 consisting of the amino acid sequence of SEQ ID NO: 311. Preferably, the first and second polypeptide chains are connected 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 for B7H6 consisting of the amino acid sequence of SEQ ID NO: 239 and a second polypeptide chain specific for CD3 consisting of the amino acid sequence of SEQ ID NO: 311. Preferably, the first and second polypeptide chains are connected via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule Figure 1 ).

[0272] In another aspect, the present application provides a binding protein comprising a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 and a second polypeptide chain (CD3 chain) that specifically binds to CD3, wherein the first polypeptide chain that specifically binds to B7H6 comprises a first light chain covalently linked (preferably directly bound) to a first linker, which first linker is itself covalently linked (e.g., directly bound) to a first heavy chain, and wherein the second polypeptide chain that specifically binds to CD3 comprises a second light chain covalently linked (preferably directly bound) to a second linker, which second linker is itself covalently linked (e.g., directly bound) to a second heavy chain.

[0273] Unless otherwise defined herein, all definitions and preferred embodiments provided herein above with respect to the binding proteins of the present application having specifically recited antigen-binding units also apply mutatis mutandis to such binding proteins of the present application comprising first and second polypeptide chains.

[0274] In some embodiments, the first polypeptide chain (also referred to herein as B7H6 chain) comprises, starting from its N-terminus, a first light chain variable domain that specifically binds to B7H6, a first light chain constant domain, a first linker, a first heavy chain variable domain specific for B7H6, and a first heavy chain constant region. In some embodiments, the second polypeptide chain (also referred to herein as CD3 chain) comprises, starting from its N-terminus, a second light chain variable domain that specifically binds to CD3, a second light chain constant domain, a second linker, a second heavy chain variable domain specific for CD3, and a second heavy chain constant domain.

[0275] The resulting protein carries a complete Fc, is larger than IgG (due to the presence of a linker between the light and heavy chains) and has two independent binding sites (e.g., each binding site is monovalent for the respective antigen), a first binding site for B7H6 and a second binding site for CD3. Preferably, the first and second polypeptide chains are linked via one or more disulfide bonds. Thus, the protein of the present application is an antibody-like structure, having the Y-shaped structure of a conventional full-length antibody (see Figure 1 ), comprising two polypeptide chains each comprising a scFab and an Fc domain. In preferred embodiments, the protein of the present application comprises (i) a first polypeptide chain specific for B7H6 (B7H6 chain) consisting of a first scFab specific for B7H6 and a first Fc domain, and (ii) a second polypeptide chain specific for CD3 (CD3 chain) consisting of a second single chain Fab specific for CD3 and a second Fc domain.

[0276] Preferably, the first scFab is linked to the first Fc domain via a direct covalent bond and the second scFab is linked to the second Fc domain via a direct covalent bond. This bispecific format generally reduces heterogeneity upon expression and purification (e.g. by avoiding mispairing of light and medium variable domains with different binding specificities), while maintaining the functional property that binding moieties within the structure are less likely to generate unwanted immunogenic reactions. This also allows for good expression of the heterodimeric protein in e.g. mammalian cells.

[0277] In a preferred embodiment of the protein of the application, 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 comprising CDR sequences selected from the group consisting of i) to xxiv):

[0278] i) light chain CDRs comprising the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3);

[0279] ii) light chain CDRs comprising 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 comprising the amino acid sequences of SEQ ID NO: 10 (CDR1), SEQ ID NO: 11 (CDR2), and SEQ ID NO: 12 (CDR3);

[0280] iii) light chain CDRs comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3);

[0281] iv) light chain CDRs comprising the amino acid sequences of SEQ ID NO: 19 (CDR1), SEQ ID NO: 20 (CDR2), and SEQ ID NO: 21 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 24 (CDR3);

[0282] v) light chain CDRs comprising an amino acid sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2), and SEQ ID NO:30 (CDR3);

[0283] vi) light chain CDRs comprising an amino acid sequence of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2), and SEQ ID NO:33 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2), and SEQ ID NO:36 (CDR3);

[0284] vii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:39 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2), and SEQ ID NO:42 (CDR3);

[0285] viii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2), and SEQ ID NO:45 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2), and SEQ ID NO:48 (CDR3);

[0286] ix) light chain CDRs comprising an amino acid sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3);

[0287] x) light chain CDRs comprising an amino acid sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3);

[0288] xi) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 61 (CDR1), SEQ ID NO: 62 (CDR2), and SEQ ID NO: 63 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 64 (CDR1), SEQ ID NO: 65 (CDR2), and SEQ ID NO: 66 (CDR3);

[0289] xii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 67 (CDR1), SEQ ID NO: 68 (CDR2), and SEQ ID NO: 69 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 70 (CDR1), SEQ ID NO: 71 (CDR2), and SEQ ID NO: 72 (CDR3);

[0290] xiii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 73 (CDR1), SEQ ID NO: 74 (CDR2), and SEQ ID NO: 75 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 76 (CDR1), SEQ ID NO: 77 (CDR2), and SEQ ID NO: 78 (CDR3);

[0291] xiv) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 79 (CDR1), SEQ ID NO: 80 (CDR2), and SEQ ID NO: 81 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 82 (CDR1), SEQ ID NO: 83 (CDR2), and SEQ ID NO: 84 (CDR3);

[0292] xv) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 85 (CDR1), SEQ ID NO: 86 (CDR2), and SEQ ID NO: 87 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 88 (CDR1), SEQ ID NO: 89 (CDR2), and SEQ ID NO: 90 (CDR3);

[0293] xvi) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 91 (CDR1), SEQ ID NO: 92 (CDR2), and SEQ ID NO: 93 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 94 (CDR1), SEQ ID NO: 95 (CDR2), and SEQ ID NO: 96 (CDR3);

[0294] xvii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 97 (CDR1), SEQ ID NO: 98 (CDR2), and SEQ ID NO: 99 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 100 (CDR1), SEQ ID NO: 101 (CDR2), and SEQ ID NO: 102 (CDR3);

[0295] xviii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 103 (CDR1), SEQ ID NO: 104 (CDR2), and SEQ ID NO: 105 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 106 (CDR1), SEQ ID NO: 107 (CDR2), and SEQ ID NO: 108 (CDR3);

[0296] xix) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 109 (CDR1), SEQ ID NO: 110 (CDR2), and SEQ ID NO: 111 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 112 (CDR1), SEQ ID NO: 113 (CDR2), and SEQ ID NO: 114 (CDR3);

[0297] xx) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 115 (CDR1), SEQ ID NO: 116 (CDR2), and SEQ ID NO: 117 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 118 (CDR1), SEQ ID NO: 119 (CDR2), and SEQ ID NO: 120 (CDR3);

[0298] xxi) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 121 (CDR1), SEQ ID NO: 122 (CDR2), and SEQ ID NO: 123 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 124 (CDR1), SEQ ID NO: 125 (CDR2), and SEQ ID NO: 126 (CDR3);

[0299] xxii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 127 (CDR1), SEQ ID NO: 128 (CDR2), and SEQ ID NO: 129 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 130 (CDR1), SEQ ID NO: 131 (CDR2), and SEQ ID NO: 132 (CDR3);

[0300] xxiii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 133 (CDR1), SEQ ID NO: 134 (CDR2), and SEQ ID NO: 135 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 136 (CDR1), SEQ ID NO: 137 (CDR2), and SEQ ID NO: 138 (CDR3); and

[0301] xxiv) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 139 (CDR1), SEQ ID NO: 140 (CDR2), and SEQ ID NO: 141 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 142 (CDR1), SEQ ID NO: 143 (CDR2), and SEQ ID NO: 144 (CDR3).

[0302] The respective light / heavy chain variable domains defined by the CDR sequences are designated 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.

[0303] In preferred embodiments of the binding proteins of the application, the second polypeptide chain that specifically binds to CD3 (CD3 chain) comprises a second light chain variable domain and a second heavy chain variable domain comprising CDR sequences selected from the group consisting of:

[0304] i) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 257 (CDR1), SEQ ID NO: 258 (CDR2), and SEQ ID NO: 259 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3);

[0305] ii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 263 (CDR1), SEQ ID NO: 264 (CDR2), and SEQ ID NO: 265 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 266 (CDR1), SEQ ID NO: 267 (CDR2), and SEQ ID NO: 268 (CDR3);

[0306] iii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 269 (CDR1), SEQ ID NO: 270 (CDR2), and SEQ ID NO: 271 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 272 (CDR1), SEQ ID NO: 273 (CDR2), and SEQ ID NO: 274 (CDR3);

[0307] iv) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 275 (CDR1), SEQ ID NO: 276 (CDR2), and SEQ ID NO: 277 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 278 (CDR1), SEQ ID NO: 279 (CDR2), and SEQ ID NO: 280 (CDR3);

[0308] v) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 281 (CDR1), SEQ ID NO: 282 (CDR2), and SEQ ID NO: 283 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 284 (CDR1), SEQ ID NO: 285 (CDR2), and SEQ ID NO: 286 (CDR3); and

[0309] vi) a light chain CDR comprising the amino acid sequence of SEQ ID NO: 287 (CDR1), SEQ ID NO: 288 (CDR2), and SEQ ID NO: 289 (CDR3), and a heavy chain CDR comprising the amino acid sequence of SEQ ID NO: 290 (CDR1), SEQ ID NO: 291 (CDR2), and SEQ ID NO: 292 (CDR3).

[0310] The respective light / heavy chain variable domains defined by the CDR sequences are designated CD3#1, CD3#2, CD3#3, CD3#4, CD3#5, and CD3#6, respectively.

[0311] Preferably, the light and heavy chain CDR sequences are 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 above.

[0312] In one preferred embodiment, the binding protein of the application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6, comprising a first light chain variable domain having light chain CDRs comprising the amino acid sequence 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 heavy chain CDRs comprising the amino acid sequence of SEQ ID NO: 70 (CDR1), SEQ ID NO: 71 (CDR2), and SEQ ID NO: 72 (CDR3); and (ii) a second polypeptide chain that specifically binds to CD3, comprising a second light chain variable domain having light chain CDRs comprising the amino acid sequence 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 heavy chain CDRs comprising the amino acid sequence of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3).

[0313] In a preferred embodiment, the binding protein of the present application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprising a first light chain variable domain having light chain CDRs comprising an amino acid sequence 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 heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 82 (CDR1), SEQ ID NO: 83 (CDR2), and SEQ ID NO: 84 (CDR3); and (ii) a second polypeptide chain that specifically binds to CD3 comprising a second light chain variable domain having light chain CDRs comprising an amino acid sequence 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 heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3).

[0314] In a preferred embodiment, the binding protein of the present application comprises (i) a first polypeptide chain that specifically binds to B7H6 comprising a first light chain variable domain having light chain CDRs comprising an amino acid sequence 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 heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 88 (CDR1), SEQ ID NO: 89 (CDR2), and SEQ ID NO: 90 (CDR3); and (ii) a second polypeptide chain that specifically binds to CD3 comprising a second light chain variable domain having light chain CDRs comprising an amino acid sequence 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 heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3).

[0315] In a preferred embodiment, the binding protein of the application comprises (i) a first polypeptide chain that specifically binds to B7H6 comprising a first light chain variable domain having light chain CDRs comprising 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 heavy chain CDRs comprising 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 that specifically binds to CD3 comprising a second light chain variable domain having light chain CDRs comprising 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 heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3).

[0316] In a preferred embodiment, the binding protein of the application comprises (i) a first polypeptide chain that specifically binds to B7H6 comprising a first light chain variable domain having light chain CDRs comprising 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 heavy chain CDRs comprising 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 that specifically binds to CD3 comprising a second light chain variable domain having light chain CDRs comprising 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 heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 260 (CDR1), SEQ ID NO: 261 (CDR2), and SEQ ID NO: 262 (CDR3).

[0317] In a preferred embodiment of the protein of the application, the first polypeptide chain that specifically binds to B7H6 (B7H6 chain) 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):

[0318] 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 (B7H6#1);

[0319] ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 147 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 148 (B7H6#2);

[0320] iii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 149 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 (B7H6#3);

[0321] 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 (B7H6#4);

[0322] 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 (B7H6#5);

[0323] vi) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 (B7H6#6);

[0324] vii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 (B7H6#7);

[0325] viii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 (B7H6#8);

[0326] ix) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 (B7H6#9);

[0327] x) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 (B7H6#10);

[0328] 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 (B7H6#11);

[0329] xii) 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 (B7H6#12);

[0330] xiii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 (B7H6#13);

[0331] xiv) 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 (B7H6#14);

[0332] xv) 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 (B7H6#15);

[0333] 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 (B7H6#16);

[0334] xvii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178 (B7H6#17);

[0335] 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 (B7H6#18);

[0336] xix) 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 (B7H6#19);

[0337] 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 (B7H6#20);

[0338] xxi) 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 (B7H6#21);

[0339] xxii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188 (B7H6#22);

[0340] 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 (B7H6#23); and

[0341] xxiv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 191 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 192 (B7H6#24).

[0342] Preferably, the light chain variable and heavy chain variable 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.

[0343] In a preferred embodiment of the protein of the application, the second polypeptide chain (CD3 chain) that specifically binds to CD3 comprises 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:

[0344] i) 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 (CD3#1 );

[0345] ii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 295 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 296 (CD3#2);

[0346] iii) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 297 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 298 (CD3#3); iv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 299 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 300 (CD3#4);

[0347] iv) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 299 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 300 (CD3#4);

[0348] 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 (CD3#5);

[0349] vi) a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 303 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 304 (CD3#6).

[0350] In some embodiments, the binding proteins of the application comprise a first and a second polypeptide chain comprising CDRs and / or VH and VL sequences of light / heavy chain variable domains selected from the list 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#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 preferred embodiments, the binding proteins of the application comprise a first and a second polypeptide chain comprising CDRs and / or VH and VL sequences of light / heavy chain variable domains selected from the list consisting of: 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 comprises a Fc domain comprising the amino acid sequence of SEQ ID NO: 242 and the second polypeptide chain comprises a Fc domain comprising the amino acid sequence of SEQ ID NO: 243.

[0351] In a preferred embodiment, the binding protein of the present application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprising a light chain variable domain of SEQ ID NO: 167 and a heavy chain variable domain of SEQ ID NO: 168; and (ii) a second polypeptide chain (CD3 chain) that specifically binds to CD3 comprising a light chain variable domain of SEQ ID NO: 293 and a heavy chain variable domain of SEQ ID NO: 294.

[0352] In a preferred embodiment, the binding protein of the present application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprising 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) that specifically binds to CD3 comprising a light chain variable domain of SEQ ID NO: 293 and a heavy chain variable domain of SEQ ID NO: 294.

[0353] In a preferred embodiment, the binding protein of the present application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprising a light chain variable domain of SEQ ID NO: 173 and a heavy chain variable domain of SEQ ID NO: 174; and (ii) a second polypeptide chain (CD3 chain) that specifically binds to CD3 comprising 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 application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprising a light chain variable domain of SEQ ID NO: 175 and a heavy chain variable domain of SEQ ID NO: 176; and (ii) a second polypeptide chain (CD3 chain) that specifically binds to CD3 comprising a light chain variable domain of SEQ ID NO: 293 and a heavy chain variable domain of SEQ ID NO: 294.

[0355] In a preferred embodiment, the binding protein of the present application comprises (i) a first polypeptide chain (B7H6 chain) that specifically binds to B7H6 comprising a light chain variable domain of SEQ ID NO: 189 and a heavy chain variable domain of SEQ ID NO: 190; and (ii) a second polypeptide chain (CD3 chain) that specifically binds to CD3 comprising a light chain variable domain of SEQ ID NO: 293 and a heavy chain variable domain of SEQ ID NO: 294.

[0356] In preferred embodiments, the first polypeptide chain specific for B7H6 comprises a single chain Fab having the 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 for CD3 comprises a single chain Fab having the amino acid sequence of SEQ ID NO: 305.

[0357] Preferably, the first polypeptide chain comprises a single chain Fab comprising 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: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, and SEQ ID NO: 216, and the second polypeptide chain comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 305.

[0358] In a preferred embodiment, the first polypeptide chain specific for B7H6 (B7H6 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 204 and the second polypeptide chain specific for CD3 (CD3 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 305.

[0359] In a preferred embodiment, the first polypeptide chain specific for B7H6 (B7H6 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 206 and the second polypeptide chain specific for CD3 (CD3 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 305.

[0360] In a preferred embodiment, the first polypeptide chain specific for B7H6 (B7H6 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 207 and the second polypeptide chain specific for CD3 (CD3 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 305.

[0361] In a preferred embodiment, the first polypeptide chain specific for B7H6 (B7H6 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 208 and the second polypeptide chain specific for CD3 (CD3 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 305.

[0362] In a preferred embodiment, the first polypeptide chain specific for B7H6 (B7H6 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 215 and the second polypeptide chain specific for CD3 (CD3 chain) comprises a single chain Fab comprising the amino acid sequence of SEQ ID NO: 305.

[0363] Further, with respect to this specific embodiment for scFabs, the term comprising is also intended to include "consisting of the amino acid sequences defined above", which is a more general meaning.

[0364] In some embodiments of the binding proteins of the application, the first and second polypeptide chains comprise an Fc domain derived from a heavy chain of an IgG, e.g. IgGl, IgG2 or IgG4. For example, the Fc domain of the application is an Fc domain of a heavy chain of IgGl or IgG4 and comprises a hinge region and two constant domains (CH2 and CH3). Examples of Fc domains of human IgG are shown in SEQ ID NO: 241 and SEQ ID NO: 244.

[0365] In some embodiments of the binding proteins of the application, the heavy chain comprises one or more amino acid changes. For example, the amino acid changes are a tyrosine (Y) at position 366 of the first heavy chain [T366Y] and a threonine (T) at position 407 of the second heavy chain [Y407T]. In some embodiments, the first heavy chain comprises a serine (S) at position 366 [T366S] and the second heavy chain comprises a tryptophan (W) at position 366 [T366W], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V]. In preferred embodiments, the first heavy chain comprises a tryptophan (W) at position 366 [T366W] and the second heavy chain comprises a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V]. For example, position 366 of the Fc domain corresponding to amino acid position 146 in the human IgGl Fc sequence of SEQ ID NO: 241 changes from T at position 146 in SEQ ID NO: 241 to W at position 146 in SEQ ID NO: 242; and positions 366, 368, and 407 corresponding to amino acid positions 146, 148, and 187, respectively, in SEQ ID NO: 241 change from T, L, and Y at those positions in SEQ ID NO: 241 to S, A, and V at those positions in SEQ ID NO: 243, according to Eu numbering. In any of the embodiments described, the amino acid changes described for the first heavy chain can be in the second heavy chain and the respective amino acid changes of the second heavy chain can be in the first heavy chain. In other words, the terms "first" and "second" are interchangeable in the described embodiments. In some embodiments, the heavy chain is derived from a heavy chain of IgGl or IgG4.

[0366] In some embodiments, the first or second heavy chain in the binding proteins of the application further comprises one or more amino acid changes that reduce binding of the heavy chain to Protein A. In some embodiments, the amino acid changes are an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F] of one of the heavy chains.

[0367] In some embodiments, in the proteins of the application, the heavy chain comprising threonine (T) at position 407 [Y407T] further comprises arginine at position 435 [H435R] and phenylalanine at position 436 [Y436F]. In this case, the other heavy chain comprises tyrosine (Y) at position 366 [T366Y] but does not include the two changes at positions 435 and 436. Alternatively, in some embodiments, in the proteins of the application, the heavy chain comprising serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A] and valine (V) at position 407 [Y407V] further comprises arginine at position 435 [H435R] and phenylalanine at position 436 [Y436F]. In this case, the other heavy chain comprises tryptophan (W) at position 366 [T366W] but does not include the two changes at positions 435 and 436. Thus, the heavy chain comprising the amino acid changes that create a "cavity" as described above also comprises amino acid changes that reduce binding to Protein A. By reducing binding to Protein A, homodimers comprising the heavy chain are removed. Production of homodimers of the other heavy chain comprising the "protrusion" is reduced by the presence of the "protrusion".

[0368] In some embodiments, the heavy chain of the proteins of the application can or can not further comprise the YTE mutation (M252Y / S254T / T256E, Eu numbering (Dall'Acqua et al. J. Biol. Chem. 2006, 281(33):23514-24). The mutation has been shown to improve the pharmacokinetic properties of the heavy chain by preferentially enhancing the binding affinity to the neonatal FcRn receptor at pH 6.0.

[0369] In some embodiments, the first and / or second heavy chain of the application derived from IgGl also includes the "KO" mutation (L234A, L235A) (Xu et al., Cellular Immunology 2000 Feb 25, 200(1): 16-26). In another aspect, the first and / or second heavy chain of the application derived from IgG4 also includes the Pro hinge mutation (S228P) (Angal et al., Molecular Immunology 1993, 30(1): 105-108; Labrijn et al., Nature Biotechnology 2009, 27:767-771).

[0370] In a preferred embodiment of the binding proteins of the application, the first polypeptide chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 242 and the second polypeptide chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 243.

[0371] In preferred embodiments of the application, 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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 222 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#6 / CD3#1); or vii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 223 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#7 / CD3#1); or viii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 224 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#8 / CD3#1); or ix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 225 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#9 / CD3#1); or x) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#10 / CD3#1); or xi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 227 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#11 / CD3#1); or xii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 228 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#12 / CD3#1); or xiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 229 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#13 / CD3#1).or xiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 230 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#14 / CD3#1); or xv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 231 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#15 / CD3#1), or xvi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 232 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#16 / CD3#1); or xvii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 233 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#17 / CD3#1); or xviii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 234 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#18 / CD3#1); or xix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 235 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#19 / CD3#1); or xx) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 236 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#20 / CD3#1); or xxi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 237 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#21 / CD3#1); or xxii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 238 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#22 / CD3#1); or xxiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 239 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#23 / CD3#1); or xxiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 240 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 311 (B7H6#24 / CD3#1). Preferably, the first and second polypeptide chains are connected via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule. Figure 1 .

[0372] In a preferred embodiment, the binding protein comprises a first polypeptide chain specific for B7H6 comprising the amino acid sequence of SEQ ID NO: 228 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311. Preferably, the first and the second polypeptide chain are connected via 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 for B7H6 comprising the amino acid sequence of SEQ ID NO: 230 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311. Preferably, the first and the second polypeptide chain are connected 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 for B7H6 comprising the amino acid sequence of SEQ ID NO: 231 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311. Preferably, the first and the second polypeptide chain are connected 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 for B7H6 comprising the amino acid sequence of SEQ ID NO: 232 and a second polypeptide chain specific for CD3 comprising the amino acid sequence of SEQ ID NO: 311. Preferably, the first and the second polypeptide chain are connected via one or more disulfide bonds and form an antibody-like structure similar to a conventional Y-shaped antibody molecule Figure 1 ).

[0376] In another aspect, the protein of the present application comprises a first antigen binding unit or polypeptide chain specific for B7H6, wherein the affinity to human and cynomolgus B7H6 is preferably < 10 nM, more preferably < 1 nM, even more preferably < 0.1 nM. The affinity can be measured in a SPR SPR system (GE Healthcare Life Sciences) analysis using recombinant B7H6-protein as described e.g. in the examples or other methods well known to the person skilled in the art. The protein comprises a second antigen binding unit or polypeptide chain with an affinity to the human and cynomolgus CD3 epsilon gamma complex of preferably < 500 nM, more preferably < 100 nM, even more preferably < 10 nM.

[0377] In another aspect, the B7H6 / CD3 binding proteins of the application do not bind to B7H6 negative cells and do not cross-react with B7H1 (see, e.g., Example 10 and Example 4, respectively).

[0378] In preferred embodiments, the B7H6 / CD3 binding proteins of the application (e.g., any 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) do not inhibit the activation of natural killer cells. Notably, the B7H6 / CD3 binding proteins of the application that do not inhibit the activation of natural killer cells in vitro bind to B7H6 with an NKp30 interaction site that is substituted with alanine.

[0379] B7H6 on the cell surface binds to NKp30 on the cell surface of NK cells, which triggers NKp30-mediated activation of NK cells, NK cell cytotoxicity, and cytokine secretion (Brandt et al., J. Exp. Med. 2009, 206(7): 1495-503). This can be mimicked in vitro by culturing NK cell lines (e.g., NK92MI) or primary NK cells on plates coated with recombinant B7H6 extracellular domain protein, followed by analysis of upregulation of activation markers (e.g., CD25 or CD69) or cytokine secretion by NK cells. This assay setup is used to assess whether our B7H6 / CD3 binding proteins inhibit the interaction of B7H6 and NKp30, resulting in inhibition of IFNy secretion (Example 11).

[0380] Using recombinant Ala mutated B7H6 extracellular proteins in which the NKp30 interaction site was substituted by an alanine, it was observed that there were two groups of binding proteins: 1) binding proteins that found strong binding to wild-type B7H6 but no or only weak binding to recombinant Ala mutated B7H6 extracellular proteins inhibited B7H6-dependent IFNy secretion by NK cells in vitro ("inhibitors of B7H6-dependent NK cell activation"), and 2) binding proteins that found strong binding to wild-type B7H6 and maintained the ability to also bind to recombinant Ala mutated B7H6 extracellular proteins did not inhibit B7H6-dependent activation and associated IFNy secretion by NK cells in vitro ("non-inhibitors of B7H6-dependent NK cell activation") (see Examples 6 and 11, Figure 4 and 9 ). Surprisingly, the binding proteins of the application that were non-inhibitors of B7H6-dependent NK cell activation were more potent in T cell redirected lysis of B7H6 expressing tumor cells (see Examples 12, Figure 10 and 11 ). Without being bound by theory, non-inhibitors of B7H6-dependent NK cell activation can allow B7H6 NKp30 interaction without affecting the natural role of B7H6 in mediating innate immunity.

[0381] In another aspect, the B7H6 / CD3 binding proteins of the application were able to mediate T cell redirected cytotoxicity against tumor cells independently of NK cell activity (as shown in a mouse xenograft model in the absence of NK cells, see Example 19, Figure 20 and 23 , and in a cell lysis assay in the absence of NK cells, see Example 12, Figure 10 and 11 ).

[0382] Cytotoxicity mediated by the B7H6 / CD3 binding proteins of the application can be measured using a variety of methods. 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) T cells or subsets thereof (e.g. CD4, CD8) or unstimulated (human or cynomolgus) peripheral blood mononuclear cells (PBMC). Target cells should express at least the extracellular domain of (human or cynomolgus) B7H6 and can be cells with endogenous (native) B7H6 expression, such as the human small cell lung carcinoma cell line SHP77, NCI-H82, or can also 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. Cytotoxic activity of the B7H6 / CD3 binding molecules can be determined, for example, in an LDH release assay after 48 or 72 hours of incubation. Modifications of the incubation time and readout for the determination of cytotoxicity are possible and known to the person skilled in the art. Readout systems for cytotoxicity can include MTT / MTS assays, ATP-based assays, FACS-based assays, 51-chromium release assays, sulforhodamine B (SRB) assays, colorimetric (WST) assays, clonogenic assays, ECIS technology and bioluminescence assays.

[0383] Cytotoxic activity mediated by the B7H6 / CD3 binding proteins of the application is preferably measured in a cell-based cytotoxicity assay. Cytotoxicity is indicated by the EC 90 values measured in the cytotoxicity assay. The person skilled in the art is aware that, when purified T cells are used as effector cells, the EC 90 values are expected to be lower compared to PBMC, and the person skilled in the art is also aware that, when stimulated T cells are used, the EC 90 values are even lower. Furthermore, it can be expected that the EC 90 values are lower when the target cells express high numbers of B7H6 on the cell surface compared to cells expressing low numbers of B7H6 molecules on the cell surface. The EC 90 values of the B7H6 / CD3 binding proteins are preferably < 10 nM, more preferably < 5 nM and even more preferably < 1 nM.

[0384] Preferably, the multispecific binding proteins of the application do not induce / mmediate lysis of B7H6-negative cells. The term "do not induce / mmediate lysis of B7H6-negative cells" means that the B7H6 / CD3 binding molecules do not induce or mediate lysis of more than 30%, preferably not more than 20%, more preferably not more than 10% and especially not more than 5% of B7H6-negative cells, whereas lysis of B7H6-positive colorectal cell lines is set to 100%. This is generally true for concentrations of the binding proteins of up to 1000 nM.

[0385] Furthermore, the B7H6 / CD3 binding proteins of the present application show monomer content of more than 95% in a two-step purification process (see Example 20), have favorable pharmacokinetic properties and good downstream manufacturability, and are further expected to have a good biodistribution (e.g., see Example 18). The proteins of the present application further have favorable immunogenicity properties (see Example 22) and have good in vitro and in vivo stability (e.g., see Examples 21 and 18). Moreover, the B7H6 / CD3 binding proteins of the present application show favorable efficacy in a humanized in vivo xenograft mouse model. The B7H6 / CD3 binding proteins induce strong tumor regression, which has already started after the first administration of the B7H6 / CD3 binding proteins (e.g., see Example 19). Furthermore, the B7H6 / CD3 binding proteins of the present application induce tumor regression at an extremely low dose of 0.05 mg / kg administered once a week (q7d), further supporting their therapeutic applicability. In particular, the B7H6 / CD3 binding proteins of the present application induce 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, 17, respectively), and further significantly increase T cell infiltration into tumor tissue (see Example 24).

[0386] In another aspect, the present application provides an isolated nucleic acid molecule encoding any one of the first and / or second antigen binding units of the multispecific binding proteins of the present application (any one of 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, 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 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 second Fc domain as described herein, the first and / or second Fc domain is linked to the 3’ end of the nucleic acid molecule encoding the first and / or second antigen binding unit, respectively. In some embodiments, the nucleic acid molecule encodes i) a first polypeptide chain comprising a first single chain Fab specific for B7H6 (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) a second polypeptide chain comprising a second single chain Fab specific for CD3 (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.

[0387] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding a first single chain Fab specific for B7H6 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 / or a second single chain Fab of SEQ ID NO: 305. In a preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a first scFab specific for 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 for CD3 comprising the amino acid sequence of SEQ ID NO: 305.

[0388] A further aspect of the application provides an expression vector containing a DNA molecule comprising a nucleotide sequence encoding the first and / or second antigen binding domain (e.g. the first and / or second single chain Fab of the application). Preferably, the expression vector comprises in addition to the nucleic acid molecule, preferably a DNA molecule encoding the first and / or second Fc domain linked to the nucleic acid molecule, preferably also a DNA molecule encoding the first and / or second antigen binding domain (e.g. the first and / or second single chain Fab), respectively. Thus, the expression vector comprises a nucleotide sequence encoding a polypeptide chain comprising the first single chain Fab linked to the first Fc domain and / or a nucleotide sequence encoding a polypeptide chain comprising the second single chain Fab linked to the second Fc domain.

[0389] In preferred embodiments, the expression vector contains a DNA molecule comprising a nucleotide sequence encoding the first polypeptide chain specific for B7H6 and / or the second polypeptide chain specific for CD3 of the application. In preferred embodiments, the expression vector comprises a nucleotide sequence encoding the 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 the second polypeptide chain comprising SEQ ID NO: 311.

[0390] In other preferred embodiments, the expression vector comprises a nucleotide sequence encoding the 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 the second polypeptide chain comprising SEQ ID NO: 311.

[0391] In particularly preferred embodiments, two expression vectors can be used, one for expressing the first polypeptide chain specific for B7H6 and the other for expressing the second polypeptide chain specific for CD3, which can then be transfected into a host cell for expression of the recombinant protein.

[0392] Preferably, the expression vector will be a vector comprising the nucleic acid molecule(s) operably linked to at least one regulatory sequence, wherein the regulatory sequence can be a promoter, enhancer, or terminator sequence, and most preferably a heterologous promoter, enhancer, or terminator sequence.

[0393] In another aspect, the application relates to a host cell having an expression vector encoding the first polypeptide chain specific for B7H6 of the application and an expression vector encoding the second polypeptide chain specific for CD3 of the application.

[0394] According to 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 are yeast cells or other fungal cells.

[0395] 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 the expression of heterologous proteins can also be used.

[0396] Anti-B7H6 antibodies

[0397] Yet another aspect of the application provides an anti-B7H6 antibody molecule comprising

[0398] i) light chain CDRs comprising the amino acid sequences of SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3); or

[0399] ii) light chain CDRs comprising 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 comprising the amino acid sequences of SEQ ID NO: 10 (CDR1), SEQ ID NO: 11 (CDR2), and SEQ ID NO: 12 (CDR3); or

[0400] iii) light chain CDRs comprising the amino acid sequences of SEQ ID NO: 13 (CDR1), SEQ ID NO: 14 (CDR2), and SEQ ID NO: 15 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 16 (CDR1), SEQ ID NO: 17 (CDR2), and SEQ ID NO: 18 (CDR3); or

[0401] iv) light chain CDRs comprising the amino acid sequences of SEQ ID NO: 19 (CDR1), SEQ ID NO: 20 (CDR2), and SEQ ID NO: 21 (CDR3), and heavy chain CDRs comprising the amino acid sequences of SEQ ID NO: 22 (CDR1), SEQ ID NO: 23 (CDR2), and SEQ ID NO: 24 (CDR3); or

[0402] v) light chain CDRs comprising an amino acid sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:28 (CDR1), SEQ ID NO:29 (CDR2), and SEQ ID NO:30 (CDR3); or

[0403] vi) light chain CDRs comprising an amino acid sequence of SEQ ID NO:31 (CDR1), SEQ ID NO:32 (CDR2), and SEQ ID NO:33 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:34 (CDR1), SEQ ID NO:35 (CDR2), and SEQ ID NO:36 (CDR3); or

[0404] vii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:37 (CDR1), SEQ ID NO:38 (CDR2), and SEQ ID NO:39 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:40 (CDR1), SEQ ID NO:41 (CDR2), and SEQ ID NO:42 (CDR3); or

[0405] viii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:43 (CDR1), SEQ ID NO:44 (CDR2), and SEQ ID NO:45 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:46 (CDR1), SEQ ID NO:47 (CDR2), and SEQ ID NO:48 (CDR3); or

[0406] ix) light chain CDRs comprising an amino acid sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3); or

[0407] x) light chain CDRs comprising an amino acid sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3); or

[0408] xi) light chain CDRs comprising an amino acid sequence of SEQ ID NO:61 (CDR1), SEQ ID NO:62 (CDR2), and SEQ ID NO:63 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:64 (CDR1), SEQ ID NO:65 (CDR2), and SEQ ID NO:66 (CDR3); or

[0409] xii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:67 (CDR1), SEQ ID NO:68 (CDR2), and SEQ ID NO:69 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:70 (CDR1), SEQ ID NO:71 (CDR2), and SEQ ID NO:72 (CDR3); or

[0410] xiii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:73 (CDR1), SEQ ID NO:74 (CDR2), and SEQ ID NO:75 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:76 (CDR1), SEQ ID NO:77 (CDR2), and SEQ ID NO:78 (CDR3); or

[0411] xiv) light chain CDRs comprising an amino acid sequence of SEQ ID NO:79 (CDR1), SEQ ID NO:80 (CDR2), and SEQ ID NO:81 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:82 (CDR1), SEQ ID NO:83 (CDR2), and SEQ ID NO:84 (CDR3); or

[0412] xv) light chain CDRs comprising an amino acid sequence of SEQ ID NO:85 (CDR1), SEQ ID NO:86 (CDR2), and SEQ ID NO:87 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:88 (CDR1), SEQ ID NO:89 (CDR2), and SEQ ID NO:90 (CDR3); or

[0413] xvi) light chain CDRs comprising an amino acid sequence of SEQ ID NO:91 (CDR1), SEQ ID NO:92 (CDR2), and SEQ ID NO:93 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:94 (CDR1), SEQ ID NO:95 (CDR2), and SEQ ID NO:96 (CDR3); or

[0414] xvii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:97 (CDR1), SEQ ID NO:98 (CDR2), and SEQ ID NO:99 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:100 (CDR1), SEQ ID NO:101 (CDR2), and SEQ ID NO:102 (CDR3); or

[0415] xviii) light chain CDRs comprising an amino acid sequence of SEQ ID NO:103 (CDR1), SEQ ID NO:104 (CDR2), and SEQ ID NO:105 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:106 (CDR1), SEQ ID NO:107 (CDR2), and SEQ ID NO:108 (CDR3); or

[0416] xix) light chain CDRs comprising an amino acid sequence of SEQ ID NO:109 (CDR1), SEQ ID NO:110 (CDR2), and SEQ ID NO:111 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO:112 (CDR1), SEQ ID NO:113 (CDR2), and SEQ ID NO:114 (CDR3); or

[0417] xx) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 115 (CDR1), SEQ ID NO: 116 (CDR2), and SEQ ID NO: 117 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 118 (CDR1), SEQ ID NO: 119 (CDR2), and SEQ ID NO: 120 (CDR3); or

[0418] xxi) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 121 (CDR1), SEQ ID NO: 122 (CDR2), and SEQ ID NO: 123 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 124 (CDR1), SEQ ID NO: 125 (CDR2), and SEQ ID NO: 126 (CDR3); or

[0419] xxii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 127 (CDR1), SEQ ID NO: 128 (CDR2), and SEQ ID NO: 129 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 130 (CDR1), SEQ ID NO: 131 (CDR2), and SEQ ID NO: 132 (CDR3); or

[0420] xxiii) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 133 (CDR1), SEQ ID NO: 134 (CDR2), and SEQ ID NO: 135 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 136 (CDR1), SEQ ID NO: 137 (CDR2), and SEQ ID NO: 138 (CDR3); or

[0421] xxiv) light chain CDRs comprising an amino acid sequence of SEQ ID NO: 139 (CDR1), SEQ ID NO: 140 (CDR2), and SEQ ID NO: 141 (CDR3), and heavy chain CDRs comprising an amino acid sequence of SEQ ID NO: 142 (CDR1), SEQ ID NO: 143 (CDR2), and SEQ ID NO: 144 (CDR3).

[0422] The antibodies i) to xxiv) as outlined 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. A sequence listing is provided herein which readily allows the identification of the individual amino acid sequences of the specific antibodies directed to the present application.

[0423] In some embodiments, the anti-B7H6 antibodies of the present application are chimeric, humanized, human or optimized antibody molecules. In some embodiments, the antibody molecules are monoclonal antibody Fabs, F(ab)2, Fv or scFv. In some embodiments, the anti-B7H6 antibody molecules of the present application comprise a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions. In some embodiments, the light chain constant region of the anti-B7H6 antibody molecules of the present application is kappa or lambda.

[0424] In some embodiments, the anti-B7H6 antibodies of the present application have a heavy chain variable domain comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 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 the 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.

[0425] 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.

[0426] 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.

[0427] 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: 153 (B7H6#5); or vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155 (B7H6#6); or vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157 (B7H6#7); or viii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 (B7H6#8); or ix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161 (B7H6#9); or x) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163 (B7H6#10); or xi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 165 (B7H6#11); or xii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 167 (B7H6#12); or xiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 169 (B7H6#13).xv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 174 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 173 (B7H6#15); or xvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 176 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 175 (B7H6#16); or xvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 178 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 177 (B7H6#17); or xviii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 180 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 179 (B7H6#18); or xix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 182 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 181 (B7H6#19); or xx) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 184 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 183 (B7H6#20); or xxi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 186 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 185 (B7H6#21); or xxii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 188 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 187 (B7H6#22); or xxiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 190 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 189 (B7H6#23); or xxiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 192 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 191 (B7H6#24).

[0428] In some embodiments, the anti-B7H6 antibody of the application is a mouse monoclonal antibody. In the context of the present application, a mouse monoclonal antibody includes an antibody whose VH and VL are obtained from immunizing a mouse with a human B7H6 protein, followed by selecting suitable VH and VL sequences that bind with specific affinity to human B7H6, and then further joining the VH and VL sequences with constant domains derived from a mouse (e.g., mouse IgG2a) by recombinant technology; and which is produced by recombinant expression in a host cell. The present application further encompasses chimeric antibodies, e.g., comprising variable and constant regions from different species. In some embodiments, the antibody molecule of the present application is a chimeric antibody comprising VH and VL domains derived from a mouse 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 VH and VL domains derived from a mouse 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., mouse) comprising human IgG sequences, thus comprising human VH and VL sequences, and further comprises constant domains derived from another species. In any of the embodiments of a chimeric antibody as outlined above, the heavy chain constant region is a mouse, human, rabbit, rat, goat, or donkey heavy chain region.

[0429] In some embodiments, the anti-B7H6 antibody molecule of the present application has a constant domain selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgA, and IgE constant domains. In preferred embodiments, the anti-B7H6 antibody has a constant domain of IgG2a. In some embodiments, the anti-B7H6 antibody molecule has a light chain constant domain that is kappa or lambda, preferably the light chain constant domain is a kappa light chain constant domain, preferably comprising the sequence of SEQ ID NO: 247.

[0430] The B7H6-specific antibodies provided herein can be used for labeling, localization, identification or targeting of cells expressing B7H6 (e.g., in an ELISA assay, a FACS assay, immunohistology, or the like) by linking a dye, a drug or another molecule having binding specificity for a different antigen.

[0431] Another aspect of the present application provides an isolated nucleic acid molecule encoding a heavy chain variable domain and / or a light chain variable domain of an anti-B7H6 antibody molecule of the present application.

[0432] Preferably, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain of any one of SEQ ID NOs: 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 the light chain variable domain of any one of SEQ ID NOs: 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.

[0433] A further aspect of the application provides an expression vector containing a DNA molecule comprising a nucleotide sequence encoding the heavy chain variable domain and / or the light chain variable domain of an anti-B7H6 antibody molecule of the application.

[0434] Preferably, the expression vector comprises, in addition to the nucleic acid molecule, a DNA molecule encoding the constant domain of the heavy chain and / or the constant domain of the light chain, preferably a DNA molecule encoding the heavy chain variable domain and / or the light chain variable domain, preferably linked to the nucleic acid molecule.

[0435] 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, which can then be transfected into a host cell to express the recombinant protein.

[0436] Preferably, the expression vector will be a vector comprising the nucleic acid molecule(s) operably linked to at least one regulatory sequence, wherein the regulatory sequence can be a promoter, enhancer or terminator sequence, and most preferably a heterologous promoter, enhancer or terminator sequence.

[0437] In another aspect, the application relates to a host cell having an expression vector encoding the heavy chain of an anti-B7H6 antibody molecule of the application and an expression vector encoding the light chain of an anti-B7H6 antibody molecule of the application.

[0438] According to 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 are yeast cells or other fungal cells.

[0439] 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 the expression of heterologous proteins can also be used.

[0440] manufacturing and purification methods

[0441] The present application further provides a method of manufacturing a multispecific binding protein of the present application, said method typically comprising the steps of:

[0442] - culturing a host cell comprising an expression vector comprising a nucleic acid encoding a binding protein of the present application under conditions allowing the formation of a binding protein of the present application; and

[0443] - recovering the binding protein expressed by the host cell from the culture; and

[0444] - optionally further purifying and / or modifying and / or formulating the binding protein of the present application.

[0445] The present application further provides a method of manufacturing an anti-B7H6 antibody of the present application, said method typically comprising the steps of:

[0446] - culturing a host cell comprising an expression vector comprising a nucleic acid encoding an antibody molecule of the present application under conditions allowing the formation of an antibody molecule of the present application; and

[0447] - recovering the antibody molecule expressed by the host cell from the culture; and

[0448] - optionally further purifying and / or modifying and / or formulating the antibody molecule of the present application.

[0449] The nucleic acid of the present application can be, for example, a DNA molecule comprising a coding sequence as well as regulatory sequences and optionally natural or artificial introns, or can be a cDNA molecule. It can have its original codons or can have optimized codon usage, which has been specifically engineered for expression in the intended host cell or host organism. According to one embodiment of the present application, the nucleic acid of the present application is in substantially isolated form as defined above.

[0450] The nucleic acid of the present application can be prepared or obtained in a manner known per se, for example by automated DNA synthesis and / or recombinant DNA techniques, based on the information about the amino acid sequence of the protein of the present application given herein.

[0451] The nucleic acid of the present application is typically incorporated into an expression vector, i.e. a vector which, when transfected into a suitable host cell or other expression system, can provide for protein expression.

[0452] For the manufacture of a binding protein or antibody of the present application, the skilled person can choose from a variety of expression systems well known in the art, for example those reviewed by Kipriyanow and Le Gall, 2004.

[0453] Expression vectors include plasmids, retroviruses, cosmids, EBV-based episomes, and the like. Expression vectors and expression control sequences compatible with the host cell are selected. The nucleotide sequence encoding the first antigen binding unit of the B7H6 / CD3 binding protein (e.g., the B7H6 specific single chain Fab or full length B7H6 chain of the binding protein of the application) and the nucleotide sequence encoding the second antigen binding unit (e.g., the CD3 specific single chain Fab or full length CD3 chain of the binding protein of the application) can be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector. The nucleotide sequence encoding the light chain of the B7H6 antibody and the nucleotide sequence encoding the heavy chain of the B7H6 antibody can be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector.

[0454] Convenient vectors are those that encode a functionally complete human CH (constant heavy chain) immunoglobulin sequence, engineered with appropriate restriction sites such that any antigen binding unit (e.g., single chain Fab sequence) or any heavy / light chain variable domain can be readily inserted and expressed, as described above. For the antibody heavy chain, it can be, but is not limited to, any IgG isotype (IgGl, IgG2, IgG3, IgG4) or other immunoglobulin, including allelic variants.

[0455] The recombinant expression vector can also encode a signal peptide that facilitates secretion of the full length CD3 or B7H6 chain from a host cell or the light / heavy chain of the anti-B7H6 antibody. The 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 the mature full length chain DNA. The signal peptide can be an immunoglobulin signal

[0456] In addition to the DNA sequences encoding the B7H6 / CD3 chain or the DNA sequences encoding the heavy / light chain of the B7H6 antibody, the recombinant expression vectors will typically also carry regulatory sequences, optionally heterologous regulatory sequences, including a promoter, an enhancer, termination and polyadenylation signals, and other expression control elements that control the expression of the protein chain in a host cell. Examples of promoter sequences, illustrative for expression in mammalian cells, are promoters and / or enhancers derived from CMV, (e.g., CMV simian virus 40 (SV40) promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters, such as the natural immunoglobulin and actin promoters. Examples of polyadenylation signals are BGH poly A, SV40 late or early poly A; or the 3' UTR of an immunoglobulin gene or the like can be used.

[0457] The recombinant expression vector can also carry sequences for amplifying replication of the vector in the host cell (e.g., an origin of replication) and a selection marker gene. The nucleic acid molecule encoding the full length chain having the first antigen binding unit (single chain Fab and Fc domain) or an antigen binding portion thereof and / or the full length chain having the second antigen binding unit (single chain Fab and Fc domain) or an antigen binding portion thereof, and the vector comprising the DNA molecule can be introduced into a host cell (e.g., a bacterial cell or a higher eukaryotic cell, such as a mammalian cell) according to transfection procedures well known in the art, including liposome- mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer by viral vectors.

[0458] Preferably, the DNA molecules encoding the B7H6 and CD3 chains of the proteins of the application are present on two expression vectors co-transfected into a host cell, preferably a mammalian cell.

[0459] Mammalian cell lines useful as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO) cells, NS0, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma 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) can be used.

[0460] The proteins of the application are produced by culturing host cells for a period of time sufficient to allow for expression of the protein in the host cells. The protein molecules are preferably recovered from the culture medium as secreted polypeptides, or, if expressed, for example, without a secretion signal, can be recovered from host cell lysates. The protein molecules need to be purified in a manner that uses standard protein purification methods for recombinant proteins and host cell proteins to obtain a substantially homogeneous protein preparation. For example, the state of the art purification methods that can be used to obtain the protein molecules of the application include, as a first step, removal of cells and / or particulate cell debris from the culture medium or lysate. The protein is then purified from contaminating soluble proteins, polypeptides, and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation-exchange resin. As the final step in a process for obtaining a preparation of the protein molecules, the purified protein molecules can be dried, for example, lyophilized, for use in therapeutic applications, as described below.

[0461] The present invention relates to binding proteins having binding specificity for at least two different targets. With respect to the present invention, the binding molecules are derived from antibodies. Techniques for making binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin chains having 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, e.g., U.S. Patent No. 5,731,168; Atwell et al., JMB, 1997, 270, 26-35). Binding proteins of the present invention can also be made by engineering an electrostatic steering effect for the production of antibody Fc-heterodimeric molecules (WO 2009 / 089004 Al); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific proteins (see, e.g., Kostelny et al., Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., Immunol., 152:5368 (1994)); and making trispecific antibodies as described in, e.g., Tutt et al. Immunol. 147:60 (1991).

[0462] The compositions (e.g., multispecific binding proteins and anti-B7H6 antibodies) and methods disclosed herein encompass polypeptides and nucleic acids having a specified sequence or a sequence that is substantially identical or similar to it (e.g., a sequence that is at least 85%, 90%, 95% identical or higher to the specified sequence). In the context of amino acid sequences, the term "substantially identical" is used herein to mean that a first amino acid contains a sufficient or minimum number of i) amino acid residues identical to the aligned amino acid residues in a second amino acid sequence, or ii) conservative substitutions of the aligned amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences can have a common structural domain and / or a 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., a sequence provided herein). In the context of nucleotide sequences, the term "substantially identical" is used herein to mean that a first nucleic acid sequence contains a sufficient or minimum number of nucleotides identical to the aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or common functional polypeptide activity, e.g., a nucleotide sequence that is at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence.

[0463] Nucleic acid molecules of the present application include, but are not limited to, DNA molecules encoding the polypeptide sequences shown in the Sequence Listing. Furthermore, the present application also relates to nucleic acid molecules that hybridize to the DNA molecules encoding the polypeptide sequences shown in the Sequence Listing under high stringency binding and washing conditions, as defined in WO 2007 / 042309. Preferred molecules (from the 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 to one of the DNA molecules described herein. For example, in view of expression of the antibodies in eukaryotic cells, the DNA sequences shown in the Sequence Listing have been designed to match the codon usage in eukaryotic cells. If expression of the antibodies in E. coli is desired, the sequences can be altered to match the E. coli codon usage. Variants of the DNA molecules of the present application can be constructed in several different ways, as described, for example, in WO 2007 / 042309.

[0464] The proteins of the present application can have a modified N-terminal sequence, e.g. deletion of one or more N-terminal amino acids, or exchange of e.g. the first N-terminal amino acid (e.g. glutamate to alanine) to optimize the molecule for expression by using certain expression systems (e.g. specific vectors or host cells), or for expression in inclusion bodies or in soluble form, or secretion into the culture medium or periplasmic space or inclusion into the cell, or to produce a more homogeneous product. The polypeptides of the present application can have a modified C-terminal sequence (e.g. additional alanine), and / or further amino acid exchanges at defined positions within the C-terminal part or within any of the framework regions, as explained e.g. in WO2012 / 175741, WO2011 / 075861 or WO2013 / 024059, to e.g. further enhance the stability or reduce the immunogenicity of the polypeptide.

[0465] For the avoidance of doubt, all embodiments relating to the pharmaceutical compositions, kits, methods of treatment, medical uses, combinations, methods of administration and doses described herein contemplate use with any of the multispecific binding proteins described herein, alone or in combination with other therapeutic agents (as explained in more detail below).

[0466] Pharmaceutical compositions, methods of administration, doses

[0467] The present application further relates to pharmaceutical compositions for use in the treatment of a disease (as explained in more detail below), wherein the composition comprises at least one multispecific binding protein of the present application. The present application further encompasses a method of treatment of a disease (as explained in more detail below) using at least one multispecific binding protein of the present application or a pharmaceutical composition as described below, and further encompasses a medicament for the treatment of such a disease prepared by using such a binding protein or pharmaceutical composition of the present application.

[0468] The binding proteins of the application (e.g., any 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#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, as defined by the sequences shown in Table 1) and / or compositions comprising the same can be administered to a patient in need thereof in any appropriate way according to the particular pharmaceutical formulation or composition to be used. Thus, the binding proteins of the application and / or compositions comprising the same can be administered, for example, intravenously (i.v.), subcutaneously (s.c.), intramuscularly (i.m.), intraperitoneally (i.p.), transdermally, orally, sublingually (e.g., in the form of a sublingual tablet, a spray or drop placed under the tongue and absorbed into the capillary network under the mucosa of the tongue), nasally (intra- nasally) (e.g., in the form of a nasal spray and / or as an aerosol), topically, by means of a suppository, by inhalation or any other appropriate way in an effective amount or dose. The binding proteins can be administered by infusion, bolus or injection. In preferred embodiments, administration is by intravenous infusion or subcutaneous injection.

[0469] The binding proteins of the application and / or compositions comprising the same are administered according to a treatment regimen appropriate to treat and / or ameliorate the disease, disorder or condition to be treated or ameliorated. The clinician generally will determine an appropriate treatment regimen according to factors such as the disease, disorder or condition to be treated or ameliorated, the severity of the disease, the severity of symptoms, the specific binding protein of the application to be used, the particular route of administration and pharmaceutical formulation or composition to be used, age, sex, body weight, diet, general condition of the patient, and similar factors well known to the clinician. Generally, the treatment regimen will comprise administering one or more binding proteins of the application, or one or more compositions comprising the same, in a therapeutically effective amount or dose.

[0470] Generally, for the treatment and / or alleviation of the diseases, disorders and conditions mentioned herein, and depending on the particular disease, disorder or condition to be treated, the potency of the specific binding protein of the application to be used, the particular route of administration and the particular pharmaceutical formulation or composition to be used, the binding protein of the application is generally administered in an amount of between 0.005 and 20.0 mg per kg body weight and preferably in a dose of between 0.05 and 10.0 mg / kg / dose, either continuously (e.g. by infusion) or, more preferably, as a single dose (e.g. biweekly, weekly, every two or three weeks or monthly doses; see below), but can vary significantly, in particular depending on the parameters mentioned before. Thus, in some cases, it can already be sufficient to use less than the minimum dose given above, while in other cases it can be necessary to exceed the upper limit. When administered in larger amounts, it is recommended to divide it into several smaller doses distributed over a certain period of time (e.g. two or more days).

[0471] The specific binding proteins according to the application and their 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 weeks or every three weeks, monthly and the like. The administration regimen can include chronic treatment. By "chronic" is meant a duration of at least two weeks, and preferably several months or years.

[0472] The potency of the multispecific binding proteins of the application and compositions comprising the same can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se or any combination thereof, depending on the particular disease involved. Suitable assays and animal models will be clear to the person skilled in the art and include, for example, the assays and animal models used in the following examples.

[0473] Formulations

[0474] For pharmaceutical use, the binding proteins of the present application can be formulated as pharmaceutical preparations comprising (i) at least one binding protein of the present application (e.g. any 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#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 polypeptides and / or compounds.

[0475] "Pharmaceutically acceptable" means that the respective material does not show any biologically or other undesired effects when administered to a subject, and does not interact in a deleterious manner with any of the other components of a pharmaceutical composition comprising it (e.g. the pharmacologically active ingredient). For specific embodiments, reference is made to standard handbooks, e.g. Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990). For example, the binding proteins of the present application can be formulated and administered in any of the ways known per se for conventional antibodies and antibody fragments and other pharmacologically active proteins. Thus, according to a further embodiment, the present application relates to a pharmaceutical composition or formulation containing at least one binding protein of the present application and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and / or stabilizer, and optionally one or more other pharmacologically active substances, in the form of a lyophilized or other dry formulation or an aqueous or non-aqueous solution or suspension.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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 1.0-100 mg / ml, e.g., 1.0 mg / ml (IV administration) or 100 mg / ml (SC administration)) and an aqueous buffer, said buffer being, for example:

[0480] -Phosphate-buffered saline, pH 7.4,

[0481] - Other phosphate buffers, pH 6.2 to 8.2,

[0482] - acetate buffer, pH 3.2 to 7.5, preferably pH 4.8 to 5.5

[0483] - histidine buffer, pH 5.5 to 7.0,

[0484] - succinate buffer, pH 3.2 to 6.6, and

[0485] - citrate buffer, pH 2.1 to 6.2,

[0486] and optionally, salts (e.g. NaCl) and / or sugars (e.g. sucrose and trehalose) and / or other polyols (e.g. mannitol and glycerol) for providing isotonicity of the solution.

[0487] In addition, other agents can be included in the solution (e.g. detergents, e.g. 0.02% TWEEN TM 20 or TWEEN TM -80). Formulations for subcutaneous application can include significantly higher concentrations of the antibody of the application, e.g. up to 100 mg / ml or even higher than 100 mg / ml. However, it will be clear to a person skilled in the art that the ingredients given above and their amounts represent only one preferred choice. Alternatives and variations therefor will be immediately apparent to a person skilled in the art or can easily be thought of starting from the above disclosure. The above formulations can optionally be provided in the form of a lyophilized formulation to be reconstituted in a solution, e.g. in water for injection (WFI).

[0488] According to yet another aspect of the application, the binding protein of the application can be used in combination with a device for protein administration, e.g. a syringe, an injection pen, a micro pump or other device.

[0489] Method of treatment

[0490] A further aspect of the application provides a method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the binding protein of the application.

[0491] A further aspect of the application provides the binding protein of the application for use in a method of treating cancer.

[0492] A further aspect of the application is the use of the binding protein of the application for the manufacture of a pharmaceutical composition for the treatment of cancer.

[0493] For the avoidance of doubt, the medical use aspect of the application can comprise any of the specific binding proteins of the application as described hereinabove (e.g. any 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#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).

[0494] The term "cancer" as used herein is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathologic type or stage of invasion.

[0495] Exemplary cancers whose growth can be inhibited using the multispecific binding proteins described herein are any tumors expressing B7H6, preferably colorectal cancer (e.g. metastatic colorectal cancer, mCRC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC).

[0496] Exemplary cancers whose growth can be inhibited using the multispecific binding proteins described herein are any tumors expressing B7H6, including (but not limited to) T-cell lymphoma, myeloid leukemia, breast cancer; ovarian cancer, oral squamous carcinoma and gastrointestinal cancer. Gastrointestinal cancer includes (but is not limited to) esophageal cancer (e.g. gastroesophageal junction cancer), stomach (gastric) cancer, hepatocellular cancer, biliary tract cancer (e.g. cholangiocarcinoma), gallbladder cancer, pancreatic cancer or colorectal cancer (CRC).

[0497] In some embodiments, the following cancers, tumors and other proliferative diseases can be treated with the multispecific binding proteins of the application: 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 lymphoma); leukemia (chronic or acute myeloid leukemia, non-lymphocytic leukemia) or multiple myeloma.

[0498] In preferred embodiments of the application, the cancer is mCRC.

[0499] All cancers, tumors, neoplasms, etc. mentioned above, characterized by their specific location / source in the body are intended to include both primary tumors and metastatic tumors derived therefrom.

[0500] If a patient has a cancer characterized by having a high expression of B7H6, the patient is more likely to respond to treatment with a binding protein of the application (as described herein). Thus, in some embodiments, the cancer to be treated with a binding protein of the application is a cancer having a high expression of B7H6, for example higher than the average expression in cancer cells of a patient population having the same type of cancer expressing B7H6.

[0501] The binding proteins of the application can be used in a treatment regimen in first line, second line or any other line of treatment and maintenance therapy.

[0502] The binding proteins of the application can be used for the prevention, short-term or long-term treatment of the diseases mentioned above, optionally also in combination with radiotherapy, one or more additional therapeutic agents and / or surgery.

[0503] In preferred embodiments, the protein of the application is used in combination 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 by the sequences in Table A below) and in WO2017 / 198741 (incorporated herein by reference). Preferably, the anti-PDL-1 antibody is selected from the group consisting of atezolizumab, avelumab and durvalumab. In particularly preferred embodiments, the binding protein of the application (preferably any 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) is combined with PD1-1 for the treatment of cancer. In particularly preferred embodiments, the binding protein of the application (preferably any 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) is combined with PD1-2 for the treatment of cancer.In a particularly preferred embodiment, a binding protein of the application (preferably any 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) is combined with PD1-3 for the treatment of cancer. In a particularly preferred embodiment, a binding protein of the application (preferably any 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) is combined with PD1-4 for the treatment of cancer. In a particularly preferred embodiment, a binding protein of the application (preferably any 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) is combined with PD1-5 for the treatment of cancer.

[0504] Table A: Amino acid sequences of the heavy and light chain sequences of the anti-PD1 antibodies PD1-1, PD1-2, PD1-3, PD1-4, PD1-5 and the SEQ ID NO.

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511] According to the preferred embodiment and any other aspect of the application, the 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 as shown in Table A above.

[0512] Thus, PD1-1 has a heavy chain comprising the amino acid sequence of SEQ ID NO: 331 and a light chain comprising the amino acid sequence of SEQ ID NO: 332;

[0513] PD1-2 has a heavy chain comprising the amino acid sequence of SEQ ID NO: 333 and a light chain comprising the amino acid sequence of SEQ ID NO: 334;

[0514] PD1-3 has a heavy chain comprising the amino acid sequence of SEQ ID NO: 335 and a light chain comprising the amino acid sequence of SEQ ID NO: 336;

[0515] PD1-4 has a heavy chain comprising the amino acid sequence of SEQ ID NO: 337 and a light chain comprising the amino acid sequence of SEQ ID NO: 338; and

[0516] PD1-5 has a heavy chain comprising the amino acid sequence of SEQ ID NO: 339 and a light chain comprising the amino acid sequence of SEQ ID NO: 340.

[0517] The above also includes the use of the binding proteins of the application in various methods of treating the above-mentioned diseases by administering a therapeutically effective dose to a patient in need thereof, as well as the use of the binding proteins for the manufacture of a medicament for the treatment of the diseases mentioned, as well as pharmaceutical compositions comprising the binding proteins of the application, as well as the preparation and / or manufacture of a medicament comprising the binding proteins of the application and the like.

[0518] Combinations with other active substances or treatments

[0519] The binding proteins of the application (e.g., any 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#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) can be used alone or in combination with other cancer therapies (e.g., surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, or a combination thereof). For example, the binding proteins of the application can be used in combination with one or more additional therapeutic agents, in particular with cytotoxic or cytostatic chemotherapeutic agents, therapeutically active compounds that inhibit angiogenesis, signal transduction pathway inhibitors (e.g., EGFR inhibitors, immunomodulators, immune checkpoint inhibitors, mitotic checkpoint inhibitors, or hormone therapy agents) for the treatment of cancer.

[0520] The additional therapeutic agent can optionally be administered simultaneously as a component of the same pharmaceutical formulation with the B7H6 / CD3 binding protein, or before or after administration thereof.

[0521] Cell growth inhibitory and / or cytotoxic active substances that can be administered in combination with the binding molecules of the application include, but are not limited to, hormones, hormone analogs and anti-hormones, aromatase inhibitors, LHRH agonists and antagonists, growth factors (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, e.g. HER2, HER3, HER4) and hepatocyte growth factor (HGF) and the like growth factor) inhibitors, inhibitors being for example (anti)growth factor antibodies, (anti)growth factor receptor antibodies and tyrosine kinase inhibitors, e.g. cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib and trastuzumab; antimetabolites (e.g. antifolates, e.g. methotrexate, raltitrexed, pyrimidine analogs, e.g. 5-fluorouracil (5-FU), FOLFOX (a combination regimen of leucovorin, 5-FU and oxaliplatin), FOLFIRI (a combination regimen of leucovorin, 5-FU and irinotecan), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine and gemcitabine, purine and adenosine analogs, e.g. mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine (ara 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);anti-mitotic agents (e.g. Vinca alkaloids, such as vinblastine, vindesine, vinorelbin and vincristine; and taxanes, such as paclitaxel, docetaxel); angiogenesis inhibitors (including bevacizumab, ramucirumab and aflibercept), microtubulin inhibitors;DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g. epipodophyllotoxins such as etoposide and 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, PI3K alpha inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (e.g. volasertib), CDK inhibitors (including CDK9 inhibitors, Aurora kinase inhibitors), tyrosine kinase 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 analogues (e.g. everolimus, temsirolimus, ridaforolimus, sirolimus), androgene synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutics, e.g. immune checkpoint inhibitors (e.g. CTLA4, PD1, PD-L1, LAG3 and TIM3 binding molecules / immunoglobulins, e.g. ipilimumab, nivolumab, pembrolizumab), and various chemotherapeutics, e.g. amifostin, anagrelid, clodronat, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer; proteasome inhibitors (e.g. Bortezomib); Smac and BH3 mimetics; agents that restore p53 function, including mdm2-p53 antagonists;Wnt / beta-catenin signaling pathway inhibitors; and / or cyclin-dependent kinase 9 inhibitors.

[0522] Particularly preferred is treatment with a combination of a binding molecule of the application and one or more immunotherapeutic agents, including anti-PD-1 and anti-PD-L1 agents and anti-LAG3 agents: Exemplary anti-PD1 agents include, but are not limited to, the anti-PD-1 antibodies PDR-001, pembrolizumab, nivolumab, pidilizumab, and PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5, as disclosed herein (Table A) and in WO2017 / 198741. Exemplary anti-PDL-1 agents include, but are not limited to, atezolizumab, avelumab, and durvalumab. In preferred embodiments, a binding molecule of the application (preferably any 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) is combined with PD1-1. In preferred embodiments, a binding molecule of the application (preferably any 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) is combined with PD1-2.In preferred embodiments, the binding molecules of the application (preferably any 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) are combined with PD1-3. In preferred embodiments, the binding molecules of the application (preferably any 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) are combined with PD1-4. In preferred embodiments, the binding molecules of the application (preferably any 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) are combined with PD1-5.

[0523] In certain embodiments, the additional therapeutic agent can be other immunotherapeutic agents, for example modulators of TIM-1, TIM-3, TIM-4, PD-L2, LAG3, CTLA-4, Galectin 9, Galectin-1, CD69, CD113, GPR56, CD48, GARP, CAECAM-1, BTLA, TIGIT, CD160, LAIR1, 2B4, CEACAM, CD39, TGF, 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 receptors, SIRPa (CD47), ILT-2, ILT-4, IDO, CD39, Arginase, CD73 HHLA2, Butyrophilin or A2aR.

[0524] In some embodiments, the additional immunotherapeutic agent is a member of the TNF family of molecules that bind to cognate TNF receptor family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-lBBL, CD137, CD137 / FAP, GITR, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin alpha / TNFbeta, TNFR2, TNFalpha, LTbetaR, Lymphotoxin alpha1beta2, FAS, FASL, RELT, DR6, TROY, NGFR. Preferably, the additional immunotherapeutic agent is CD137 / FAP.

[0525] In some embodiments, 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 stimulate the immune response (e.g. IL2), for example for the treatment of proliferative diseases, e.g. cancer.

[0526] In some embodiments, the additional immunotherapeutic agent is an agonist of a protein that stimulates T cell activation, such as CD28, GITRL, OX40L, CD27, and CD28H or a STING agonist.

[0527] In some embodiments, 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).

[0528] Kit

[0529] The present application also encompasses a kit comprising at least one multispecific binding protein of the present application (such as any one 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#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 optionally one or more additional components selected from the group consisting of other drugs for treating the diseases and conditions described above.

[0530] In one embodiment, the kit comprises a composition comprising an effective amount of a binding protein of the present application in unit dosage form.

[0531] The present application also encompasses a kit comprising at least one multispecific binding protein of the present application and one or more additional components selected from the group consisting of other drugs for treating the diseases and conditions described above.

[0532] In one embodiment, the kit comprises a composition comprising an effective amount of a multispecific binding protein of the application (preferably any 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) in unit dosage form. In another embodiment, the kit comprises both a composition comprising an effective amount of a multispecific binding protein of the application (preferably any 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) in unit dosage form and a composition comprising 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 in WO2017 / 198741) in unit dosage form.

[0533] In some embodiments, the kit comprises a sterile container containing the composition; the container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. The container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. Furthermore, the kit can comprise the pharmaceutical compositions in a first container having the binding protein of the application in lyophilized form and a second container having a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used to reconstitute or dilute the binding protein.

[0534] If desired, the multispecific binding proteins of the application are provided with instructions for administering the multispecific binding proteins to an individual having cancer. The instructions typically include information on using the composition to treat or prevent cancer. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treating or preventing cancer or symptoms thereof; precautions; warnings; indications; contraindications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container, where present, or applied thereto by a label, or in a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0535] The proposed kit components can be packaged in a manner that is routine to those of skill in the art. For example, the proposed kit components can be provided in solution or as a liquid dispersion or the like.

[0536] Examples

[0537] The following examples illustrate the application. The examples should not be construed as limiting the scope of the application.

[0538] Example 1: Design and construction of B7H6 / CD3 binding proteins

[0539] The present inventors have developed multispecific binding proteins that bind to B7H6 and CD3 and induce T cell activation, leading to lysis of tumor cells expressing B7H6. The molecules used are designed with an IgG antibody scaffold and IgG-like structure. They feature a knob-into-hole technology in the Fc for heterodimerization of the knob and hole arms. In addition, the binding proteins have a flexible peptide sequence between the light chain and the respective heavy chain of each arm. Thus, the binding proteins comprise two arms, one binding to CD3 and the other to B7H6, each arm comprising a single chain Fab and an Fc region (see Figure 1 ).

[0540] Preferably, the binding molecule is bispecific and bivalent (monovalent for each of the two targets).

[0541] Binding domains recognizing B7H6 and CD3 were prepared using high-throughput V gene recovery from hybridomas and single B cells.

[0542] To obtain anti-B7H6 binding agents, hybridomas or single B cells derived from B7H6 immunized wild type and ALIVAMAB TM humanized mice (ALIVAMAB MOUSE TM platform with human immunoglobulin loci) were cultured in vitro. Binding domains recognizing B7H6 and CD3 were prepared using high-throughput V gene recovery from hybridomas and single B cells. Immunoassay kits (PerkinElmer, Waltham, MA, USA) were used to screen supernatants conjugated with recombinant human B7H6. Flow cytometry was then used to screen NCI-H716 cells expressing human B7H6. The supernatant bound to CCL-251TM was also screened for supernatant bound to cynomolgus monkey B7-H6 recombinantly expressed on CHO cells.

[0543] 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 Cells are precipitated and used as source material. For single B cells, 100 to 500 cells expanded from a single isolated B cell are used as source material. RNA was isolated using a mini RNA extraction kit (Qiagen, Hilden, Germany). Then, it was used... cDNA synthesis was performed using a 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. Subsequently, VH and VL fragments were amplified using a two-step PCR amplification process 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 and 25 μl... The PCR program consisted of Max DNA polymerase premix (Clontech), 2 μl of unpurified cDNA, and 21 μl of double-distilled H2O. The cycling program started at 94°C for 3 min, followed by 35 cycles (94°C for 30 sec, 50°C for 1 min, 68°C for 1 min), and ended at 72°C for 7 min. The second round of PCR was performed using the VL and VH second-round primers, which contained a 15 bp complementary extension overlapping with specific regions in their respective pTT5 parent vectors (VH and VL). The second round of PCR was performed using the same cycling program.

[0544] In- The HD Cloning Kit (Clontech, USA) is used for the directed cloning of the VL gene into the pTT5huIgK vector and the directed cloning of the VH gene into the pTT5huIgG1KO vector. This is to promote In- HD cloning, purify PCR products and treat with Cloning Enhancer prior to In-Fusion HD cloning. Cloning and transformation were performed according to the manufacturer's protocol (Clontech, U.S.A.). Miniprep DNA was subjected to Sanger sequencing to confirm that complete V-gene fragments were obtained.

[0545] Using this approach, Ig VH and VL gene pairs encoding binding domains specific for B7H6 were prepared. Recombinant antibodies were produced by transient transfection of CHO-E37 cells with the corresponding heavy and light chain encoding plasmids.

[0546] To obtain additional anti-CD3 binding agents, immunization of WT mice was performed using the huCD3 epsilon peptide 1-27 construct. Hybridoma supernatants were screened for binding to recombinant huCD3E+G-Fc protein and to recombinant cyCD3E+G-Fc protein, as well as for binding to huCD3 positive and cyCD3 positive cells. Variable regions of positive clones were recovered and cloned as IgG or IgG-like bispecific constructs for further evaluation.

[0547] Humanization / optimization of B7H6 and CD3 binding agents

[0548] The sequences of the above described B7H6 or CD3 binding agents, as well as of CD3 binding agents described in the literature (Pessano et al., EMBO J. 1985 Feb;4(2):337-44; Salmerón A et al., J Immunol. 1991 Nov 1;147(9):3047-52) were subjected to humanization and / or optimization. Sequence optimization / humanization of antibodies is a method to engineer antibodies produced in non-human species (against a specific antigen / epitope) for use as therapeutics similar to antibodies produced in humans and thereby eliminate potential adverse effects (e.g. immunogenicity) while retaining specificity. The sequence optimization / humanization method utilized herein is described by Singh et al., 2015 (Singh S et al., mAbs 2015:7(4):778-91). Briefly, closely matching human germlines are identified in silico and optimization / humanized variants are evaluated using phage screening methods. Based on binding, percent human score and Final lead candidate sequences were selected based on in silico prediction of potential immunogenicity (RANKER).

[0549] Construction of bispecific proteins binding B7H6 and CD3

[0550] The variable regions of B7H6 and CD3 binders were cloned into the expression vector pTT5 (National Research Council, Canada) using conventional molecular biology techniques to form a bispecific binding protein having one B7H6 specific binding arm comprising a single chain Fab and Fc region that binds to B7H6 (this binding unit is also referred to herein as "B7H6 arm" or "B7H6 chain") and one CD3 specific binding arm comprising a single chain Fab and Fc region that binds to CD3 (this binding unit is also referred to herein as "CD3 arm" or "CD3 chain). The Fc regions of 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 W or SAV chains. For multi-fragment DNA assembly, Gibson-assembly and HiFi DNA assembly method following the manufacturer's protocol (New England Biolabs, Ipswich, MA, USA). DNA minipreps were sequenced.

[0551] Each expression vector contains a eukaryotic promoter cassette for the chain encoding genes (B7H6 or CD3 arms / chains), i.e. genes encoding the signal sequence and light and heavy chains, an expression cassette for a prokaryotic selection marker gene (e.g. ampicillin), and an origin of replication. The DNA plasmids were propagated in ampicillin resistant E. coli colonies and cultures and purified.

[0552] Example 2: Expression and purification of bispecific binding proteins binding B7H6 and CD3

[0553] Bispecific molecules binding B7H6 and CD3 were produced by transient transfection of CHO-E cells with pTT5 vectors carrying B7H6 / CD3 chain encoding genes (one chain being a W chain and the other chain being a SAV chain). Briefly, transfected CHO-E cells grown in suspension in serum-free medium were cultivated in shake flasks at 140 rpm agitation, 37°C and 5% CO2 and kept under exponential growth conditions. On the day of transfection, Mirus Bio TransIT The cells were chemically transfected with a mass ratio of 1 :3 of W chain plasmid and SAV chain plasmid using Transfection Reagent. The cells were then seeded at 1 to 2 x 10^6 cells / ml in 1 L FreeStyle TM CHO expression medium (Life Technologies, NY, US). Then on day 7, the cells were fed for 10 days with a one-time feed and 200 ml of a commercial feed solution under orbital shaking to optimize the expression of the protein. The cells were harvested using The instrument (Pall ForteBio, CA, US) and the ProtA biosensor tip were used to determine the antibody titer in the cell culture supernatant according to the manufacturer's instructions.

[0554] Using GE Healthcare Life Sciences The pure protein purification system purified recombinant B7H6 / CD3 binding protein from culture supernatant in a two-step process. First, MabSelect was used... TM The column (GE Healthcare) captures samples from harvested cell culture medium via protein A affinity chromatography. At neutral pH, proteins bind to protein A and are washed with high salt (1M NaCl) to remove cell culture medium components and any proteins or components not specifically bound to protein A. Antibody or antibody-like construct samples are lysed in isocratic mode using 30 mM sodium acetate (pH 3.5). The eluted sample is neutralized to pH 5.0 using 1% 3M sodium acetate solution (pH 9.0). The neutralized protein is aseptically filtered using a 0.22 μm filtration system. Concentrations are measured via UV280 using a nanodrop 8000 spectrophotometer. In the second purification, POROS is used. TM 50HS cation exchange resin column (Applied Biosystems, Carlsbad, CA, USA) for cation exchange chromatography or using 26 / 600 Size exclusion chromatography was applied using a 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 comprised two-step purified material with approximately 95% to 99% monomer content.

[0555] 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:

[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]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633] Example 3: Generation of recombinant proteins

[0634] • Human B7H6-His

[0635] The complete extracellular domain of human-B7H6 was expressed using the pTT vector (encoding human B7H6-His, SEQ ID NO: 317) with His 6-tag by transient transfection using Lenti-X TM Gibco Opti-MEM® medium (Thermo Fisher Scientific) and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubated at 37°C for 24 hours. The medium was then replaced with fresh Opti-MEM® medium and the cells were incubTM Freestyle TM In F17 expression medium (Thermo Fisher Scientific), 1.6 × 10⁻⁶ ppm was used for molecular transfection. 6 HEK 293F cells / ml (Thermo Fisher). Pre-incubate with a 1:3 DNA:PEI complex and 1 mg / L DNA for 5 minutes, filter, and pre-incubate again at room temperature for 15 minutes before adding to cells. Culture cells at 37°C, 5% CO2, and 140 rpm with shaking. 24 hours post-transfection, add tryptone N1 to the cells to a final concentration of 0.5%. 48 hours post-transfection, refeed cells with 2 mM glutamine and 2 g / L glucose. Simultaneously, lower the temperature to 33°C. 120 hours post-transfection, add 2 mM glutamine and 1 g / L glucose to the final feed. 144 hours post-transfection, harvest cells by centrifugation at 6000 rpm for 15 minutes. Clarify the supernatant using a G4 filter.

[0636] Protein purification was performed in two steps. First, affinity chromatography was performed using a Ni-NTA column. The protein was precipitated for 10 CVs with 1×PBS (pH 7.2) + 10 mM imidazole wash buffer, followed by another 10 CVs with 1×PBS (pH 7.2) + 20 mM imidazole wash buffer, maintaining a 4-60% PBS (pH 7.2) precipitation gradient supplemented with 0.5 M imidazole. The fractions were collected and analyzed by SDS-PAGE before pooling and concentration. Next, using… Gel filtration chromatography (GLC) was performed using 200, 16 / 600, and 120 mL columns (GE Healthcare Life Sciences). 5 mL of the concentrated pooled product from affinity chromatography was loaded onto the column at a flow rate of 0.5 mL / min. A buffer solution of 20 mM HEPES, 100 mM NaCl, and 5% sucrose, pH 7.4, was prepared. The fractions were collected and analyzed by SDS-PAGE prior to pooling, and then sterilized using a 0.2 μm filter.

[0637] ·Cyno B7H6-His

[0638] By using Lenti-X TM Transient transfection with a lentiviral system (Clontech) using a His6-tag via a pTT vector (encoding cyno B7H6-His, SEQ ID NO:320) expresses the complete extracellular domain of Cyno-B7H6. In Gibco... TM Freestyle TMIn F17 expression medium (Thermo Fisher Scientific), 1.6 × 10⁻⁶ ppm was used for molecular transfection. 6 HEK 293F cells / ml (Thermo Fisher). Pre-incubate with a 1:3 DNA:PEI complex and 1 mg / L DNA for 5 minutes, filter, and pre-incubate again at room temperature for 15 minutes before adding to cells. Culture cells at 37°C, 5% CO2, and 140 rpm with shaking. 24 hours post-transfection, add tryptone N1 to the cells to a final concentration of 0.5%. 48 hours post-transfection, refeed cells with 2 mM glutamine and 2 g / L glucose. Simultaneously, lower the temperature to 33°C. 120 hours post-transfection, add 2 mM glutamine and 1 g / L glucose to the final feed. 144 hours post-transfection, harvest cells by centrifugation at 6000 rpm for 15 minutes. Clarify the supernatant using a G4 filter.

[0639] Protein purification was performed in two steps. First, affinity chromatography was performed using a Ni-NTA column. The protein was precipitated for 10 CVs with a wash buffer of 1×PBS, 0.2M sucrose, 0.01% CHAPS, 5% glycerol (pH 7.2) + 10mM imidazole. Then, the protein was precipitated for another 10 CVs with a wash buffer of 1×PBS (pH 7.2) + 20mM imidazole, using a precipitate gradient of 4-60% of 1×PBS, 0.2M sucrose, 0.01% CHAPS, 5% glycerol (pH 7.2), supplemented with 0.5M imidazole. The fractions were collected and analyzed by SDS-PAGE before pooling and concentration. Next, using… Gel filtration chromatography (GE Healthcare LifeSciences) was performed at 200, 16 / 600. 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 buffer solution was prepared as follows: 1X PBS, 0.2 M sucrose, 0.01% CHAPS, 5% glycerol, pH 7.2. The fractions were collected and analyzed by SDS-PAGE prior to pooling, and then sterilized using a 0.2 μm filter.

[0640] Human CD3 E+G HuFc-6xHis (E+G indicating εγ subunit)

[0641] Using HEK-293 cells (Thermo Fisher) and Lenti-X TMA 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) were used to generate a cell line for producing human CD3 E+G HuFc-6xHis. For expression, freestyle blotting was performed at 37°C, humidified with 8% CO2, and shaking at 135 rpm. TM Cells were cultured and expanded in 293 medium (Thermo Fisher Scientific). On day 6, the conditioned culture supernatant was harvested 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 (overnight incubation at 4°C followed by elution with 250 mM imidazole); then... Size exclusion chromatography was performed on a 200 column (GE Healthcare Life Sciences) in PBS containing 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, and 1 mM TCEP (pH 7.2). A 10K MWCO PES membrane was used for final analysis and storage. The collected substances were concentrated using a centrifuge. The purified substances were then characterized by mass spectrometry and analytical ultracentrifugation.

[0642] • Cyno CD3 E+G HuFc-6xHis (E+G indicates the εγ subunit)

[0643] Using HEK-293 cells (Thermo Fisher) and Lenti-X TM A lentiviral system (Clontech) and plasmids encoding CynoCD3 E+G HuFc-6xHis (cynomolgus monkey CD3E accession number: Q95LI5<, cynoCD3 E+G huFc-His: SEQ ID NO:323) were used to generate a cell line for producing CynoCD3 E+G HuFc-6xHis. For expression, freestyle cynoCD3 E+G HuFc-6xHis was performed at 37°C, humidified with 8% CO2, and shaking at 135 rpm. TMCells were cultured and expanded in Expi293 medium (Thermo Fisher Scientific). Conditioned culture supernatant was harvested at day 6 by centrifugation at 9300xg for 30 minutes. Expression was monitored by SDS-PAGE and Western blotting. Conditioned culture supernatant was conditioned 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 (overnight incubation at 4°C and elution with 250 mM imidazole); followed by size exclusion chromatography on a HiLoad 26 / 600 Superdex 200 column (GE Healthcare Life Sciences) in target buffer PBS with 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, 1 mM TCEP (pH 7.2). The pooled material was concentrated using a 100 kDa MWCO centrifugal device. The purified material was qualitatively analyzed by mass spectrometry and analytical ultracentrifugation. 200 column (GE Healthcare Life Sciences) in target buffer PBS with 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, 1 mM TCEP (pH 7.2). The pooled material was concentrated using a 100 kDa MWCO centrifugal device. The purified material was qualitatively analyzed by mass spectrometry and analytical ultracentrifugation. 100 centrifugal device. The pooled material was concentrated using a 100 kDa MWCO centrifugal device. The purified material was qualitatively analyzed by mass spectrometry and analytical ultracentrifugation.

[0644] • Fc-His tagged human B7H6 ECD

[0645] In this construct, huB7H6 ECD is followed by a GS linker, then by a huIgGl-Fc domain and a C-terminal His6 tag (SEQ ID NO: 318). The construct was expressed by transient transfection of HEK293-6E cells using a 1 :3 DNA:PEI ratio and 1 mg DNA / L culture. The PEI reagent was linear PEI MAX [Mw 40,000] (Polysciences: Cat. No. 24765-2). Transfected cells were incubated at 37°C, 5% CO2 and 130 rpm. Twenty-four hours post-transfection, tryptone N1 (Organotechnie; Cat. No. 19553) and glucose were added to a final concentration of 0.5% and 1 g / L, respectively. Cells were harvested after 5 days. After centrifugation, the supernatant was filtered through a 0.2 pm membrane filter. The huB7H6-ECD-Fc-His protein was purified in two steps: first by affinity chromatography on a Ni NTA agarose matrix and second by size exclusion chromatography on a HiLoad 26 / 600 Superdex 200, 26 / 600 column (GE Healthcare Life Sciences). The pooled fractions were filtered and stored in a 1x PBS, 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, pH-7.2 conditioning buffer. 200, 26 / 600 column (GE Healthcare Life Sciences). The pooled fractions were filtered and stored in a 1x PBS, 0.2 M sucrose, 5% glycerol, 0.01% CHAPS, pH-7.2 conditioning buffer.

[0646] • Fc-His tagged human Ala mutated B7H6 ECD (NKp30 interaction site aa35-38 and aa102-105 replaced by Ala).

[0647] This construct (SEQ ID NO: 319) has huB7H6 with Ala substitutions at positions 35-38 and 102-105, so it does not bind to NKp30. huB7H6-Ala-ECD is followed by a GS linker, then a huIgGl Fc domain and a C-terminal His6 tag. This construct was expressed in HEK293-6E cells by transient transfection, purified with a two-step purification method, and stored as described above for the Fc-His tagged huB7H6-ECD construct.

[0648] • Human B7H1-Fc

[0649] This construct (SEQ ID NO: 324) contains huB7H1 with a cMyc tag, a thrombin cleavage site, and a huFc domain. Constructs were expressed by transient transfection using HEK293f cells, a 1 : 1.5 DNA:PEI ratio, and 1 mg DNA / L of culture. Flasks were incubated at 37°C in a humidified 8% C02 environment at 135 rpm shaking. Cells were harvested after 3 days. After centrifugation of the cells, the protein was purified from the supernatant. First, the nProtein A 4 Fast Flow media (GE Healthcare, #17-5280-03) was used for affinity purification, and the eluate was dialyzed in 20 mM Tris, 100 mM NaCl, 10% glycerol, 1 mM TCEP, 3 mM CaCl2(pH 8.0). Second, the sample was incubated with thrombin CleanCleave TM resin (1 mL, Sigma). Third, the pool from the previous step was again incubated with nProtein A 4 Fast Flow media. The unbound material was saved, polished by further gel filtration on a HiTrap® 75 (GE Healthcare) column, and concentrated.

[0650] Example 4: SPR-based determination of affinity and interspecies cross-reactivity of recombinant B7H6 and CD3 epsilon gamma subunit

[0651] To determine the affinity of human and cynomolgus monkey B7H6 and human B7H1 to the B7H6 / CD3 binding protein, Biacore TMExperiments were performed on a Biacore® 8K instrument (GE Healthcare Life Sciences). Briefly, B7H6 / CD3 binding proteins were captured via protein A / G. Running buffer for this experiment and all serial dilutions were prepared in HBS-EP+. CM5 sensor chips were activated with an equal mixture of EDC / NHS at a flow rate of 10 pL / min for 420 s through both flow cells, and protein A / G (50 pg / ml in 10 mM NaOAc, pH 4.5) was immobilized at a flow rate of 10 pL / min for 420 s through all flow cells to generate approximately 2500 RU of protein A / G on the surface. Sensor chips were inactivated with 1 M ethanolamine-HCl at a flow rate of 10 pL / min for 420 s through all flow cells.

[0652] Approximately 700 RU of B7H6 / CD3 binding proteins were captured on flow cell 2 of the protein A / G surface at a flow rate of 10 pL / min for 60 s. Analytes HuB7H6, CyB7H6, and HuB7H1 were injected over the captured B7H6 / CD3 binding proteins at a flow rate of 30 pL / min for 300 s, dissociation for 1200 s, through both flow cells. Concentrations of HuB7H6 and CyB7H6 were 0 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. Concentration of HuB7H1 was 0 nM and 1 pM. Surfaces were regenerated by injecting 10 mM glycine-HCl, pH 1.5, at a flow rate of 30 pL / min for 20 s through both flow cells.

[0653] Reference flow cell 1 (interaction with sensor surface) and blank (HBS-EP+ or 0 nM analyte) were subtracted from raw data. Sensorgrams were globally fitted to a 1 : 1 Langmuir binding using Biacore® 8K evaluation software to provide association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) values. TM

[0654] To determine the affinity of B7H6 / CD3 binding proteins 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 immobilized on a CM5 sensor chip at a flow rate of 30 pL / min for 180 s. B7H6 / CD3 binding proteins were injected over the immobilized HuCD3E+G-hFc and CyCD3E+G at a flow rate of 30 pL / min for 300 s, dissociation for 1200 s. Concentrations of B7H6 / CD3 binding proteins were 0 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. Surfaces were regenerated by injecting 10 mM glycine-HCl, pH 1.5, at a flow rate of 30 pL / min for 20 s through both flow cells. TM Experiments were performed on a Biacore® 8K instrument (GE Healthcare Life Sciences). Briefly, B7H6 / CD3 binding proteins were captured via protein A / G. Running buffer for this experiment and all serial dilutions were prepared in HBS-EP+. CM5 sensor chips were activated with an equal mixture of EDC / NHS at a flow rate of 10 pL / min for 420 s through both flow cells, and protein A / G (50 pg / ml in 10 mM NaOAc, pH 4.5) was immobilized at a flow rate of 10 pL / min for 420 s through all flow cells to generate approximately 2500 RU of protein A / G on the surface. Sensor chips were inactivated with 1 M ethanolamine-HCl at a flow rate of 10 pL / min for 420 s through all flow cells. TM ​GLM sensor chips (Bio-Rad) were amine coupled and B7H6 / CD3 binding proteins were flowed over the immobilized surface. Running buffer and all serial dilutions for this experiment were prepared in HBS-EP+. GLM sensor chips were normalized according to Bio-Rad's recommendations. Sensor chips were activated with an equal mixture of EDC / s-NHS at a flow rate of 30 μL / min for 300 s in the horizontal direction. HuCD3E+G-hFc was immobilized at 0.4 μg / mL, 0.2 μg / mL, and 0.1 μg / mL in 10 mM acetate (pH 4.5) at a flow rate of 30 μL / min for 300 s in the vertical direction to L1, L2, and L3, respectively, resulting in approximately 100 RU of HuCD3E+G-hFc on L1, approximately 40 RU of HuCD3E+G-hFc on L2, and approximately 0 RU of HuCD3E+G-hFc on L3. CyCD3E+G-hFc was immobilized at 0.4 μg / mL, 0.2 μg / mL, and 0.1 μg / mL in 10 mM acetate (pH 4.5) at a flow rate of 30 μL / min for 300 s in the vertical direction to L4, L5, and L6, respectively, resulting in approximately 385 RU of HuCD3E+G-hFc on L4, approximately 170 RU of CyCD3E+G-hFc on L5, and approximately 50 RU of CyCD3E+G-hFc on L6. Sensor chips were inactivated with 1 M ethanolamine-HCl at a flow rate of 30 μL / min for 300 s in the horizontal direction. Sensor chips were regenerated twice with 0.85% phosphoric acid for 18 s at a flow rate of 100 μL / min horizontally and twice vertically.

[0655] B7H6 / CD3 binding protein analytes were injected horizontally over the immobilized surface at a flow rate of 30 μL / min for 300 s and dissociated for 600 s. The concentrations of B7H6 / CD3 binding proteins used were 0 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM. The surface was regenerated twice with 0.85% phosphoric acid for 18 s at a flow rate of 100 μL / min horizontally.

[0656] The median point (interaction with the sensor surface) and the blank (HBS-EP+ or 0 nM analyte) were subtracted from the raw data. Sensorgrams were globally fit to a 1 : 1 Langmuir binding using Bio-Rad ProteOn TM Management software to provide association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) values.

[0657] The affinities 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, 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, respectively) are shown in Table 2, as described above.

[0658] Table 2: Affinities (KD) of B7H6 / CD3 binding proteins to human B7H6, cyno B7H6, human CD3 epsilon gamma subunit, cyno CD3 epsilon gamma subunit, and human B7H1 as determined by SPR analysis. No detectable binding is indicated by “nb”.

[0659]

[0660]

[0661] nb: no binding

[0662] Example 5: Generation of recombinant CHO-K1 cell lines expressing cynomolgus B7H6 extracellular domain on the cell surface

[0663] To generate stable CHO-K1 cells expressing the extracellular domain of cynomolgus B7H6 on the cell surface (NCBI: XP_005578557), the respective coding sequence (aa 25 to 262 of XP_005578557.1) was cloned into pcDNA3.1 (Thermo Fisher Scientific). The construct contains an N-terminal mouse IgG Vk leader sequence followed by a 6-His-myc-tag and the extracellular domain of cynomolgus B7H6 (aa 25-262 of NCBI XP_005578557.1). To ensure cell surface localization of the B7H6 extracellular domain, the construct is followed by a linker and the transmembrane and intracellular domain of EpCAM (Uniprot P16422). 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, a schematic of the construct is shown in Figure 2

[0664] Table 3: Amino acid sequences of B7H6 subdomains expressed on CHO-K1 cells

[0665]

[0666]

[0667] Example 6: Binding of exemplary B7H6 binding proteins to recombinant human B7H6 extracellular domain protein

[0668] To evaluate the binding of B7H6 / CD3 binding proteins to recombinant human Fc-His tagged B7H6 ECD and human Fc-His tagged Ala-mutated B7H6 extracellular protein, MediSorp TM plates (Nunc, 467320) were coated with 2 μg / ml recombinant protein over night at 4°C. The next day, plates were blocked with 0.5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 hour at room temperature (RT). Plates were then washed with OBS containing 0.05% Tween®20 and B7H6 / CD3 binding proteins were incubated at concentrations ranging from 0.00001 to 10 μg / ml. After an additional washing step, bound B7H6 / CD3 binding proteins were detected by peroxidase-conjugated goat anti-human IgG F(ab’)2 specific secondary antibody (Jackson Immunoresearch) and visualized by TMB substrate solution (Bender MedSystems). Figure 3 ​​A+B and 4A+B show exemplary B7H6 / CD3 binding proteins (B7H6 / CD3 binding proteins comprising 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: 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 the CD3 chain of SEQ ID NO: 311, 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, respectively, and recombinant human B7H6 ECD Figure 3 A+B) and human Ala mutated B7H6 ECD Figure 4 A+B) proteins.

[0669] All exemplary B7H6 / CD3 binding proteins tested show comparable binding to recombinant human B7H6 ECD Figure 3B7H6 / CD3 binding proteins comprising a 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 a CD3 chain of SEQ ID NO: 311 and B7H6 binding proteins comprising a B7H6 chain of SEQ ID NO: 230 or SEQ ID NO: 239 and a CD3 chain of SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, or SEQ ID NO: 316, respectively, showed strong binding to human Fc-His tagged Ala mutated B7H6 extracellular protein, wherein the NKp30 binding site was mutated to alanine. B7H6 / CD3 binding proteins comprising a B7H6 chain of 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 a CD3 chain of SEQ ID NO: 311 showed only weak binding or no binding to Fc-His tagged Ala mutated B7H6 extracellular protein at high concentrations.

[0670] Example 7: Binding to B7H6 positive HCT cells

[0671] B7H6 / CD3 binding proteins were tested for binding to HCT-15 (human (colorectal carcinoma) CRC cell line) by flow cytometry. In previous experiments, HCT-15 cells were confirmed to express B7-H6 on the RNA level as well as on the protein level, expressing approximately 8,000 B7-H6 receptors on the cell surface (data not shown). B7H6 / CD3 binding proteins were produced as described in Example 2. HCT-15 cells were stained with increasing concentrations of two-step purified B7H6 / CD3 binding proteins in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). Bound molecules were detected with PE-conjugated anti-human secondary antibody (Sigma-Aldrich, #P8047). Figure 5A+B shows binding of exemplary B7H6 / CD3 binding proteins (B7H6 / CD3 binding proteins comprising 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: 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 the CD3 chain of SEQ ID NO: 311, 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, respectively, and human HCT-15 cells.

[0672] Example 8: Cross-reactivity with cynomolgus monkey B7H6

[0673] B7H6 / CD3 binding proteins were tested for binding to recombinant CHO-K1 cells expressing cynomolgus monkey B7H6 by flow cytometry. B7H6 / CD3 binding proteins were produced as described in Example 2. The recombinant cell line expressing cynomolgus monkey B7H6 was generated as described in Example 5. Cells were stained with increasing concentrations of two-step purified B7H6 / CD3 binding proteins in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). Bound molecules were detected with a PE-conjugated anti-human secondary antibody (Sigma-Aldrich, #P8047). Figure 6Binding 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, 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, respectively, and CD3 binding proteins comprising 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 the B7H6 chain of SEQ ID NO: 230 or SEQ ID NO: 239, respectively) to recombinant cells expressing cynomolgus monkey B7H6.

[0674] Example 9: Binding to human T cells

[0675] B7H6 / CD3 binding proteins were tested for binding to purified human T cells by flow cytometry. B7H6 / CD3 binding proteins were produced as described in Example 2. T cells were isolated from buffy coats obtained from the Austrian Red Cross. For all buffy coats, informed consent was obtained in accordance with the Declaration of Helsinki and cantonal ethical committee in Austria.

[0676] Using Human peripheral blood mononuclear cells (PBMCs) were prepared using Paque density gradient medium (GE Healthcare Lifesciences) followed by centrifugation. Human peripheral blood mononuclear cells (PBMCs) are derived from a buffy coat preparation, which is a byproduct of blood banks that collect blood for transfusion. Thus, by Mononuclear cells were isolated by density gradient centrifugation (35 min, no brake, at 1400 rpm) and extensive washing with PBS. Dead cells were removed by incubation in ACK lysis buffer (Thermo Fisher Scientific, A1049201) for 3 min, followed by washing in PBS and then suspended in RPMI 1640 GlutaMAX TMThe remaining red blood cells were removed by incubation in analysis medium (Gibco # 61870-010), 5% human AB serum AB (Gemini, GemCell Cat. # 100-512 LOT # H56500I) + 1% MEM-NEAA (Gibco # 11140-035), 10 mM HEPES (Affymetrix # 7365-49-9), 10 μΜ β-mercaptoethanol (Gibco # 21985-023) and sodium pyruvate (Gibco # 11360-039).

[0677] T cells were isolated by negative selection using Pan T cell isolation kit II (Miltenyi Biotec # 130-091-156). Briefly, cells were resuspended in 40 μΐ buffer PBS / 0,5% BSA (Gibco ref # 041-94553M) / 2 mM EDTA (Invitrogen ref # 15575-038) / 10 million cells and incubated with 10 μΐ biotin-antibody cocktail / 10 million cells for 5 min at 4°C. Then, 30 μΐ buffer and 20 μΐ anti-biotin microbeads / 10 million cells were added and incubated for 10 min at 4°C. Then, the mixture was placed in a pre- rinsed 25 LS column (Miltenyi Biotec # 130-042-401) in a magnetic field of a magnetic cell separator (Miltenyi Biotec). The flow-through was collected and washed in analysis medium.

[0678] T cells were stained with increasing concentrations of two-step purified B7H6 / CD3 binding proteins in FACS buffer (PBS / 0.5% BSA / 0.05% sodium azide). Bound molecules were detected with PE-conjugated anti-human secondary antibody (Sigma-Aldrich, # P8047). Figure 7 ​​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, 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, respectively) to human T cells.

[0679] Example 10: Binding selectivity

[0680] B7H6 / CD3 binding proteins were tested for binding to B7H6 and CD3 negative CH...

Claims

1. A protein comprising a first antigen binding unit that specifically binds to B7H6 and a second antigen binding unit that specifically binds to CD3, wherein the first antigen binding unit that specifically binds to B7H6 is selected from: xiii) an antigen binding unit that comprises a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 73, a CDR-L2 as depicted in SEQ ID NO: 74 and a CDR-L3 as depicted in SEQ ID NO: 75 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 76, a CDR-H2 as depicted in SEQ ID NO: 77 and a CDR-H3 as depicted in SEQ ID NO: 78; xiv) an antigen binding unit that comprises a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 79, a CDR-L2 as depicted in SEQ ID NO: 80 and a CDR-L3 as depicted in SEQ ID NO: 81 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 82, a CDR-H2 as depicted in SEQ ID NO: 83 and a CDR-H3 as depicted in SEQ ID NO: 84; xv) an antigen binding unit that comprises a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 85, a CDR-L2 as depicted in SEQ ID NO: 86 and a CDR-L3 as depicted in SEQ ID NO: 87 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 88, a CDR-H2 as depicted in SEQ ID NO: 89 and a CDR-H3 as depicted in SEQ ID NO: 90; xvi) an antigen binding unit that comprises a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 91, a CDR-L2 as depicted in SEQ ID NO: 92 and a CDR-L3 as depicted in SEQ ID NO: 93 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 94, a CDR-H2 as depicted in SEQ ID NO: 95 and a CDR-H3 as depicted in SEQ ID NO: 96; or xvii) an antigen binding unit that comprises a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 97, a CDR-L2 as depicted in SEQ ID NO: 98 and a CDR-L3 as depicted in SEQ ID NO: 99 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 100, a CDR-H2 as depicted in SEQ ID NO: 101 and a CDR-H3 as depicted in SEQ ID NO: 102, and wherein the second antigen binding unit that specifically binds to CD3 comprises: xviii) an antigen binding unit that comprises a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 103, a CDR-L2 as depicted in SEQ ID NO: 104 and a CDR-L3 as depicted in SEQ ID NO: 105 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 106, a CDR-H2 as depicted in SEQ ID NO: 107 and a CDR-H3 as depicted in SEQ ID NO:

108. a light chain variable domain comprising a CDR-L1 as shown in SEQ ID NO: 257, a CDR-L2 as shown in SEQ ID NO: 258, and a CDR-L3 as shown in SEQ ID NO: 259, and a heavy chain variable domain comprising a CDR-H1 as shown in SEQ ID NO: 260, a CDR-H2 as shown in SEQ ID NO: 261, and a CDR-H3 as shown in SEQ ID NO:

262.

2. The protein of claim 1, wherein the first antigen binding unit that specifically binds to B7H6 is an antigen binding unit comprising a light chain variable domain comprising a CDR-L1 as shown in SEQ ID NO: 79, a CDR-L2 as shown in SEQ ID NO: 80, and a CDR-L3 as shown in SEQ ID NO: 81, and a heavy chain variable domain comprising a CDR-H1 as shown in SEQ ID NO: 82, a CDR-H2 as shown in SEQ ID NO: 83, and a CDR-H3 as shown in SEQ ID NO:

84.

3. The protein of claim 1 or 2, wherein i) the first antigen binding unit that specifically binds to B7H6 comprises, from its N- terminus to 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 comprises, from its N- terminus to 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.

4. The protein of claim 3, wherein the first light chain constant domain and the second light chain constant domain comprise a human kappa or lambda domain.

5. The protein of claim 3, further comprising first and second Fc domains, the first Fc domain being covalently attached to the first antigen binding unit, and the second Fc domain being covalently attached to the second antigen binding unit.

6. The protein of claim 3, further comprising first and second Fc domains, the first Fc domain being covalently attached to the C-terminus of the first antigen binding unit, and the second Fc domain being covalently attached to the C-terminus of the second antigen binding unit.

7. The protein of claim 5, wherein i) the first Fc domain comprises a tyrosine at position 366, and the second Fc domain comprises a threonine at position 407, or ii) the first Fc domain comprises a tryptophan at position 366, and the second Fc domain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, or iii) the second Fc domain comprises a tyrosine at position 366, and the first Fc domain comprises a threonine at position 407, or iv) the second Fc domain comprises a tyrosine at position 366, and the first Fc domain comprises a serine at position 366, an alanine at position 368, and a valine at position 407. iv) The second Fc domain contains tryptophan at position 366, and the first Fc domain contains serine at position 366, alanine at position 368, and valine at position 407. The amino acid positions are numbered according to the Eu numbering system.

8. The protein of claim 7, wherein the first or second Fc domain further comprises arginine at position 435 and phenylalanine at position 436, wherein the amino acid positions are numbered according to the Eu numbering system.

9. The protein according to any one of claims 5 to 8, wherein the first and / or the second Fc domain comprises alanine at position 234 and alanine at position 235, wherein the amino acid positions are numbered according to the Eu numbering system.

10. The protein of claim 5, wherein 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.

11. A set of isolated nucleic acid molecules, wherein a first isolated nucleic acid molecule encodes a first antigen-binding unit of a protein according to any one of claims 1 to 10, optionally further encoding a first Fc domain, and a second isolated nucleic acid molecule encodes a second antigen-binding unit of a protein according to any one of claims 1 to 10, optionally further encoding a second Fc domain.

12. A set of expression vectors, wherein a first expression vector comprises a first isolated nucleic acid molecule from a set of isolated nucleic acid molecules according to claim 11, and a second expression vector comprises a second isolated nucleic acid molecule from a set of isolated nucleic acid molecules according to claim 11.

13. A host cell transfected with one of the expression vectors according to claim 12.

14. A method for manufacturing the protein according to any one of claims 1 to 10, the method comprising: i) The host cells according to claim 13 are cultured under conditions that allow protein expression according to any one of claims 1 to 10; and ii) Recover the protein; and iii) Optionally, further purification and / or modification and / or formulation of the protein.

15. A protein generated by the method according to claim 14.

16. A protein 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 peptide linker, and a first heavy chain, and the second polypeptide chain comprises a second light chain, a second peptide linker, and a second heavy chain. The first polypeptide chain that specifically binds to B7H6 comprises: xiii) a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 73, a CDR-L2 as depicted in SEQ ID NO: 74 and a CDR-L3 as depicted in SEQ ID NO: 75 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 76, a CDR-H2 as depicted in SEQ ID NO: 77 and a CDR-H3 as depicted in SEQ ID NO: 78; xiv) a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 79, a CDR-L2 as depicted in SEQ ID NO: 80 and a CDR-L3 as depicted in SEQ ID NO: 81 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 82, a CDR-H2 as depicted in SEQ ID NO: 83 and a CDR-H3 as depicted in SEQ ID NO: 84; xv) a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 85, a CDR-L2 as depicted in SEQ ID NO: 86 and a CDR-L3 as depicted in SEQ ID NO: 87 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 88, a CDR-H2 as depicted in SEQ ID NO: 89 and a CDR-H3 as depicted in SEQ ID NO: 90; xvi) a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 91, a CDR-L2 as depicted in SEQ ID NO: 92 and a CDR-L3 as depicted in SEQ ID NO: 93 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 94, a CDR-H2 as depicted in SEQ ID NO: 95 and a CDR-H3 as depicted in SEQ ID NO: 96; or xvii) a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 97, a CDR-L2 as depicted in SEQ ID NO: 98 and a CDR-L3 as depicted in SEQ ID NO: 99 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 100, a CDR-H2 as depicted in SEQ ID NO: 101 and a CDR-H3 as depicted in SEQ ID NO: 102, and wherein the second polypeptide chain specifically binding to CD3 comprises: a light chain variable domain comprising a CDR-L1 as depicted in SEQ ID NO: 257, a CDR-L2 as depicted in SEQ ID NO: 258 and a CDR-L3 as depicted in SEQ ID NO: 259 and a heavy chain variable domain comprising a CDR-H1 as depicted in SEQ ID NO: 260, a CDR-H2 as depicted in SEQ ID NO: 261 and a CDR-H3 as depicted in SEQ ID NO:

262.

17. The protein of claim 16, wherein the C-terminus of the first light chain is covalently linked to the N-terminus of the first heavy chain via the first peptide linker, and wherein the C-terminus of the second light chain is covalently linked to the N-terminus of the second heavy chain via the second peptide linker.

18. The protein of claim 16, wherein the first and the second polypeptide chains are linked via a disulfide bond, forming a bispecific, bivalent and heterodimeric protein.

19. The protein of claim 16, wherein the first polypeptide chain that specifically binds to B7H6 comprises a light chain variable domain comprising a CDR-L1 as set forth in SEQ ID NO: 79, a CDR-L2 as set forth in SEQ ID NO: 80, and a CDR-L3 as set forth in SEQ ID NO: 81, and a heavy chain variable domain comprising a CDR-H1 as set forth in SEQ ID NO: 82, a CDR-H2 as set forth in SEQ ID NO: 83, and a CDR-H3 as set forth in SEQ ID NO:

84.

20. The protein of claim 16, wherein the first light chain and the second light chain independently comprise a human kappa or lambda domain.

21. The protein of claim 16, wherein i) the first heavy chain comprises a tyrosine at position 366 and the second heavy chain comprises a threonine at position 407, or ii) the first heavy chain comprises a tryptophan at position 366 and the second heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, or iii) the second heavy chain comprises a tyrosine at position 366 and the first heavy chain comprises a threonine at position 407, or iv) the second heavy chain comprises a tryptophan at position 366 and the first heavy chain comprises a serine at position 366, an alanine at position 368, and a valine at position 407, wherein the amino acid positions are numbered according to the Eu numbering system.

22. The protein of claim 21, wherein the first or the second heavy chain further comprises an arginine at position 435 and a phenylalanine at position 436, wherein the amino acid positions are numbered according to the Eu numbering system.

23. The protein of any one of claims 16-22, wherein the first and / or the second heavy chain comprises an alanine at position 234 and an alanine at position 235, wherein the amino acid positions are numbered according to the Eu numbering system.

24. The protein of claim 16, wherein the first heavy chain comprises an Fc domain having the amino acid sequence of SEQ ID NO: 242 and the second heavy chain comprises an Fc domain having the amino acid sequence of SEQ ID NO:

243.

25. A set of isolated nucleic acid molecules, wherein a first isolated nucleic acid molecule encodes a first polypeptide chain of a protein according to any one of claims 16 to 24, and a second isolated nucleic acid molecule encodes a second polypeptide chain of a protein according to any one of claims 16 to 24.

26. A set of expression vectors, wherein a first expression vector comprises a first isolated nucleic acid molecule of the set of isolated nucleic acid molecules of claim 25, and a second expression vector comprises a second isolated nucleic acid molecule of the set of isolated nucleic acid molecules of claim 25.

27. A host cell transfected with the set of expression vectors of claim 26.

28. A method of manufacturing a protein of any one of claims 16 to 24, the method comprising: i) culturing a host cell according to claim 27 under conditions permitting expression of a protein of any one of claims 16 to 24; and ii) recovering the protein; and iii) optionally further purifying and / or modifying and / or formulating the protein.

29. A protein produced by the method of claim 28.

30. A pharmaceutical composition comprising a protein of any one of claims 1 to 10, 15 to 24 and 29 and a pharmaceutically acceptable carrier.

31. Use of a protein of any one of claims 1 to 10, 15 to 24 and 29 in the manufacture of a pharmaceutical composition for the treatment of colorectal cancer.

32. The use of claim 31, wherein the colorectal cancer is metastatic colorectal cancer.

33. The use of claim 31, wherein the protein is used in combination with a cytotoxic or cytostatic chemotherapeutic agent, a therapeutically active compound that inhibits angiogenesis, a signal transduction pathway inhibitor, an immunomodulatory agent, an immune checkpoint inhibitor, a mitotic checkpoint inhibitor or a hormonal therapy agent.

34. The use of claim 33, wherein the protein is used in combination with an immune checkpoint inhibitor.

35. The use of claim 34, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-Ll antibody.

36. The use of claim 35, wherein the anti-PD-1 antibody is selected from the group consisting of PD1-1 comprising a heavy chain as set forth in SEQ ID NO: 331 and a light chain as set forth in SEQ ID NO: 332, PD1-2 comprising a heavy chain as set forth in SEQ ID NO: 333 and a light chain as set forth in SEQ ID NO: 334, PD1-3 comprising a heavy chain as set forth in SEQ ID NO: 335 and a light chain as set forth in SEQ ID NO: 336, PD1-4 comprising a heavy chain as set forth in SEQ ID NO: 337 and a light chain as set forth in SEQ ID NO: 338, and PD1-5 comprising a heavy chain as set forth in SEQ ID NO: 339 and a light chain as set forth in SEQ ID NO: 340.

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