Novel multispecific antibodies and uses thereof

Abstract

The present invention provides multispecific molecules specific for CD276 and PD-L1, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and uses thereof.

Description

[Technical Field]

[0001] The present disclosure relates generally to novel multispecific antibodies that target CD276 and one or more additional targets. [Background technology]

[0002] B7-H3 (CD276, human amino acid sequence UniProt ID: Q5ZPR3, mouse amino acid sequence UniProt ID: Q8VE98) is a newly discovered, important immune checkpoint member of the B7 and CD28 families. It is a type I transmembrane costimulatory molecule and exists in two isoforms determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans, it consists of one pair (2Ig-B7-H3) or two identical pairs (4Ig-B7-H3) due to exon duplication. B7-H3 mRNA is widely distributed in most tissues; in contrast, B7-H3 protein expression is very limited in normal tissues due to post-transcriptional regulation by microRNAs. However, the B7-H3 protein is frequently expressed in many different types of cancer (60% of all cancers) (“B7-H3: an attractive target for antibody-based immunotherapy”. Clinical Cancer Research: clincanres.2584.2020).

[0003] The function of B7-H3 remains controversial. It has been classified as either a costimulatory molecule for T cell activation, inhibiting tumor antigen-specific immune responses, or as a non-immunological role, such as promoting migration, tumor growth, invasion, metastasis, malignant stage, recurrence rate, angiogenesis, chemoresistance, epithelial-mesenchymal transition, and tumor cell metabolism. The receptor for B7-H3 is reported to be triggering receptor expressed on myeloid cell (TREM)-like transcript 2 (TLT-2, or TREML2), found on myeloid cells, which binds to B7-H3 and costimulates activation of CD8 T cells in particular. B7-H3 has also been reported to be an inhibitor of NK cells and osteoblasts by ligating an unknown receptor (The contrasting role of B7-H3, PNAS July 29, 2008, 105 (30) 10277-10278).

[0004] Based on the clinical success of inhibitory immune checkpoint blockade (CTLA-4, PD-1, and PD-L1), mAb antibodies against CD276 appear to be a promising therapeutic strategy worthy of development. Due to its selective expression on solid tumors, several groups have generated anti-CD276 antibodies, such as enoblituzumab (MGA271), omburtamab, MGD009, MGC018, DS-7300a, and CAR T cells ("B7-H3: an attractive target for antibody-based immunotherapy". Clinical Cancer Research: clincanres.2584.2020), and have observed tumor growth inhibition in vitro and in vivo. CD276 has also been reported to be expressed in hematologic tumor cells (see Wei Zhang et al., B7 Family Members in Lymphoma: Promising Novel Targets for Tumor Immunotherapy? Front. Oncol., 31 March 2021), indicating that CD276 may also be a potential target for treating hematologic cancers.

[0005] Despite the development of therapeutics that target CD276, there is a great need for antibodies that target CD276 and one or more additional targets, such as PD-L1. Summary of the Invention

[0006] Throughout this disclosure, the articles "a," "an," and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or multiple antibodies.

[0007] The present disclosure provides novel anti-CD276 antibody molecules, their amino acid and nucleotide sequences, and uses thereof.

[0008] In one aspect, the present disclosure provides a multispecific antibody, or antigen-binding fragment thereof, comprising a first binding domain and a second binding domain, wherein the first binding domain specifically binds to CD276 and the second binding domain specifically binds to a second target other than CD276.

[0009] In some embodiments, the first binding domain is selected from the group consisting of SEQ ID NOs: 1 to 3, 9 to 11, 17 to 19, 25 to 27, 33 to 35, 41 to 43, 49 to 51, 57 to 59, 65 to 67, 73 to 75, 81 to 83, 89 to 91, 97 to 99, 105 to 107, 113 to 115, 121 to 123, 129 to 131, 137 to 139, 145 to 147, 153 to 155, 161 to 163, 169 to 171, 177 to 179, 185 to 188, 190 to 1910, 200 to 2010, 2010 to 2020, 210 to 2120, 212 to 2140, 214 to 2160, 216 to 2180, 218 to 2190, 220 to 2212, 222 to 2230, 224 to 2250, 226 to 2270, 228 to 2290, 230 to 2312, 232 to 2330, 234 to 2350, 236 to 2370, 238 to 2390, 240 to 2410, 242 to 2430, 244 to 2450, 246 to 2470, 248 to 2490, 250 to 2510, 252 to 2530, 25 87, 193-195, 201-203, 209-211, 217-219, 225-227, 233-235, 241-243, 249-251, 257-259, 265-267, 273-275, 281-283, 289-291, 297-299, 305-307, 313-315, 321-323, 329-331, 337-339, and 374-375. Determinant DNA fragment (CDR) sequences, and / or SEQ ID NOs: 4 to 6, 12 to 14, 20 to 22, 28 to 30, 36 to 38, 44 to 46, 52 to 54, 60 to 62, 68 to 70, 76 to 78, 84 to 86, 92 to 94, 100 to 102, 108 to 110, 116 to 118, 124 to 126, 132 to 134, 140 to 142, 148 to 150, 156 to 158, 164 to 166, 172 to 174, 180 to 181, 188 to 190, 196 and 376-377.

[0010] In some embodiments, the first binding domain comprises: a) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 1 to 3; b) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 9 to 11; c) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 17 to 19; d) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 25 to 27; and e) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 33 to 35; f) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 41 to 43; g) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 49 to 51; h) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 57 to 59; i) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 65 to 67; j) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 73 to 75; k) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 81 to 83; l) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 89 to 91; m) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 97 to 99; n) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 105 to 107; o) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 113 to 115; p) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 121 to 123; q) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 129 to 131; r) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 137 to 139; s) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 145 to 147; t) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 153 to 155; u) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 161 to 163; v) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 169 to 171; w) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 177 to 179; x) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 185 to 187; y) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 193 to 195; z) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 201 to 203; aa) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 209 to 211; bb) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 217 to 219; cc) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 225 to 227; dd) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 233 to 235; and ee) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 241 to 243; ff) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 249 to 251; gg) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 257 to 259; hh) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 265 to 267; ii) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 273 to 275; and jj) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 281 to 283; kk) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 289 to 291; ll) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 297 to 299; mm) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 305 to 307; nn) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 313 to 315; oo) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 321 to 323; pp) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 329 to 331; qq) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 337 to 339; rr) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 9, 374, and 375; The heavy chain variable region comprises a heavy chain variable region selected from the group consisting of:

[0011] In some embodiments, the first binding domain provided herein comprises: a) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 4 to 6; b) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 12 to 14; and c) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 20 to 22; and d) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 28 to 30; and e) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 36 to 38; f) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 44 to 46; and g) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 52 to 54; h) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 60 to 62; i) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 68 to 70; j) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 76 to 78; k) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 84 to 86; l) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 92 to 94; m) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 100 to 102, 108 to 110; n) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 108 to 110; o) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 116 to 118; p) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 124 to 126; q) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 132 to 134; r) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 140 to 142; s) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 148 to 150; t) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 156 to 158; u) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 164 to 166; v) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 172 to 174; w) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 180 to 181; x) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 188 to 190; y) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 196 to 198; z) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 204 to 206; aa) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 212 to 214; bb) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 220 to 222; cc) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 228 to 230; dd) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 236 to 238; and ee) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 244 to 246; ff) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 252 to 254; gg) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 260 to 262; hh) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 268 to 270; ii) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 276 to 278; and jj) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 284 to 286; kk) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 292 to 294; ll) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 300 to 302; mm) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 308 to 310; nn) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 316 to 318; oo) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 324 to 326; pp) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 332 to 334; qq) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 340 to 342; rr) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 376, 13, and 14; ss) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 377, 45, and 46; The light chain variable region comprises a light chain variable region selected from the group consisting of:

[0012] In some embodiments, the first binding domain comprises: a) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; b) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; c) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; d) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; e) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38; f) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46; g) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54; h) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; i) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70; j) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78; k) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86; l) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94; m) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102; n) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110; o) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118; p) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126; q) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 134; r) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142; s) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150; t) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 153, SEQ ID NO: 154, and SEQ ID NO: 155, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158; u) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166; v) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174; w) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 179, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 180, SEQ ID NO: 181, and SEQ ID NO: 182; x) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 187, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 189, and SEQ ID NO: 190; y) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 198; z) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 204, SEQ ID NO: 205, and SEQ ID NO: 206; aa) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 209, SEQ ID NO: 210, and SEQ ID NO: 211, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 214; bb) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222; cc) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 225, SEQ ID NO: 226, and SEQ ID NO: 227, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 228, SEQ ID NO: 229, and SEQ ID NO: 230; dd) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 236, SEQ ID NO: 237, and SEQ ID NO: 238; ee) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 241, SEQ ID NO: 242, and SEQ ID NO: 243, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 244, SEQ ID NO: 245, and SEQ ID NO: 246; ff) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 249, SEQ ID NO: 250, and SEQ ID NO: 251, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 252, SEQ ID NO: 253, and SEQ ID NO: 254; gg) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 257, SEQ ID NO: 258, and SEQ ID NO: 259, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 260, SEQ ID NO: 261, and SEQ ID NO: 262; hh) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 265, SEQ ID NO: 266, and SEQ ID NO: 267, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 268, SEQ ID NO: 269, and SEQ ID NO: 270; ii) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 273, SEQ ID NO: 274, and SEQ ID NO: 275, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278; jj) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 281, SEQ ID NO: 282, and SEQ ID NO: 283, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 284, SEQ ID NO: 285, and SEQ ID NO: 286; kk) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 289, SEQ ID NO: 290, and SEQ ID NO: 291, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 292, SEQ ID NO: 293, and SEQ ID NO: 294; ll) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 297, SEQ ID NO: 298, and SEQ ID NO: 299, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 300, SEQ ID NO: 301, and SEQ ID NO: 302; mm) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 305, SEQ ID NO: 306, and SEQ ID NO: 307, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310; nn) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 313, SEQ ID NO: 314, and SEQ ID NO: 315, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 316, SEQ ID NO: 317, and SEQ ID NO: 318; oo) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 321, SEQ ID NO: 322, and SEQ ID NO: 323, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 324, SEQ ID NO: 325, and SEQ ID NO: 326; pp) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 329, SEQ ID NO: 330, and SEQ ID NO: 331, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 332, SEQ ID NO: 333, and SEQ ID NO: 334; qq) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 337, SEQ ID NO: 338, and SEQ ID NO: 339, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 340, SEQ ID NO: 341, and SEQ ID NO: 342; or rr) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:9, SEQ ID NO:374, and SEQ ID NO:375, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:376, SEQ ID NO:13, and SEQ ID NO:14; or ss) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 377, SEQ ID NO: 45, and SEQ ID NO: 46; Includes.

[0013] In some embodiments, the first binding domain is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:119, SEQ ID NO:127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:199, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO: 231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:347, and SEQ ID NO:349 and homologous sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0014] In some embodiments, the first binding domain is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:120, SEQ ID NO:128, SEQ ID NO:136, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:1756, SEQ ID NO:184, SEQ ID NO:192, SEQ ID NO:200, SEQ ID NO:208, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO: SEQ ID NO:232, SEQ ID NO:240, SEQ ID NO:248, SEQ ID NO:256, SEQ ID NO:264, SEQ ID NO:272, SEQ ID NO:280, SEQ ID NO:288, SEQ ID NO:296, SEQ ID NO:304, SEQ ID NO:312, SEQ ID NO:320, SEQ ID NO:328, SEQ ID NO:336, SEQ ID NO:344, SEQ ID NO:348, and SEQ ID NO:350, and homologous sequences sharing at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0015] In some embodiments, the first binding domain is SEQ ID NO: 378 (EVQLVESGGGLXQPGXSLRLSCXTSGFTLSDYYMSWVRQXPGKGLEWVXFMRNKANXYTTEYSASVRGRFTISRDTSKSXIYLQMNSLXXEDTAVYYCVRDRXGRPFAYWGQGTLVTVSS), where X at position i (i=12, 16, 23, 40, 49, 57, 80, 89, 90, and 103) of SEQ ID NO: 378 is Hi It is called X H12 is V or I, and X H16 is G or R, and X H23 is A or T, and X H40 is A or P, and X H49 is G or S, and X H57 is A or G, and X H80 is I or T, and X H89 is R or K, and X H90 is A or T, and X H103is D or E), and SEQ ID NO: 379 (DIXMTQSPXSLXXXXGXXXXIXCKSSQSLLNXINQKNFLTWYXQKPGXXPXLLIYWASTRESGVPXRFSGSGSGTDFTLXISXXXXEDLXXYYCQNDYTYPLTFGQGTKLEIK), where X at position i (i=3, 9, 12, 13, 14, 15, 17, 18, 19, 20, 22, 32, 43, 48, 49, 51, 66, 80, 83, 84, 85, 86, 90, and 91) of SEQ ID NO: 379 is X Li It is called X L3 is V or Q, X L9 is D, L or S, and X L12 is A, S or P, and X L13 is A or V, and X L14 is S or T, and X L15 is L, V or P, and X L17 is D or E, and X L18 is R or P, and X L19 is A or V, and X L20 is S or T, and X L22 is N, T or S, and X L32 is A or S, and X L43 is Q or L, and X L48 is Q or K, and X L49 is A, P or S, and X L51 is K or Q, and X L66 is S or D, and X L80 is K or T, and X L83 is R or S, and X L84 is L or V, and X L85 is Q or E, and X L86 is A or P, and X L90 is A or G, and X L91 is T or V), and

[0016] In some embodiments, the first binding domain is SEQ ID NO: 380 (QVQLQESGPGLVKPSXTLSLTCXVXGYSITSDYAWNWIRQXPGKGLEWIGYISHSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSLGRRWYFDVWGQGTTVTVSS), where X at position i (i=16, 23, 25, and 41) of SEQ ID NO: 380 is X Hi It is called X H16 is E or Q, and X H23 is A or T, and X H25 is S or Y, and X H41 is H or P), and a heavy chain variable region comprising SEQ ID NO:381 (DIXMTQSPXSLXXXXGXXXXIXCKSSQSLLXSSTQKNYLAWYXQKPGXXPXLLIYFASTRDSGVPXRFSGSGSGTDFTLXISXXXXEDLXXYFCQQHYIIPFTFGQGTKLEIK), where X at position i of SEQ ID NO:381 (i=3, 9, 12, 13, 14, 15, 17, 18, 19, 20, 22, 31, 43, 48, 49, 51, 66, 80, 83, 84, 85, 86, 90, and 91) is X Li It is called X L3 is V or Q, and X L9 is D, L or S, and X L12 is A, S or P, and X L13 is A or V, and X L14 is S or T, and X L15 is L, V or P, and X L17 is D or E, and X L18 is R or P, and X L19 is A or V, and X L20 is S or T, and X L22 is N, T or S, and X L31 is N or Q, and X L43 is Q or L, and X L48 is Q or K, and X L49 is A, P or S, and X L51 is K or Q, and X L66is S or D, and X L80 is K or T, and X L83 is R or S, and X L84 is L or V, and X L85 is Q or E, and X L86 is A or P, and X L90 is A or G, and X L91 is T or V), and

[0017] In some embodiments, the first binding domain comprises (i) a heavy chain variable region comprising one, two, or three CDR sequences set forth as SEQ ID NO: 9, SEQ ID NO: 10, or 374 (MRNKANAYTT) and SEQ ID NO: 11 or 375 (VRDREGRPFAY), respectively, and a kappa light chain variable region comprising one, two, or three CDR sequences set forth as SEQ ID NO: 12 or 376 (QSLLNAINQKNF), SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or (ii) a heavy chain variable region comprising one, two, or three CDR sequences set forth as SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively, and a kappa light chain variable region comprising one, two, or three CDR sequences set forth as SEQ ID NO: 44 or 377 (QSLLQSSTQKNY), SEQ ID NO: 45, and SEQ ID NO: 46, respectively.

[0018] In some embodiments, the first binding domain comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 351, 353, 355, 357, 358, 360, 362, 364, 365, 367, and 370; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 352, 354, 356, 359, 361, 363, 366, 368, 369, 371, 372, and 373.

[0019] In some embodiments, the first binding domain comprises: a) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8; b) a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 16; c) a heavy chain variable region comprising SEQ ID NO: 23 and a light chain variable region comprising SEQ ID NO: 24; d) a heavy chain variable region comprising SEQ ID NO: 31 and a light chain variable region comprising SEQ ID NO: 32; e) a heavy chain variable region comprising SEQ ID NO: 39 and a light chain variable region comprising SEQ ID NO: 40; f) a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 48; g) a heavy chain variable region comprising SEQ ID NO: 55 and a light chain variable region comprising SEQ ID NO: 56; h) a heavy chain variable region comprising SEQ ID NO: 63 and a light chain variable region comprising SEQ ID NO: 64; i) a heavy chain variable region comprising SEQ ID NO: 71 and a light chain variable region comprising SEQ ID NO: 72; j) a heavy chain variable region comprising SEQ ID NO: 79 and a light chain variable region comprising SEQ ID NO: 80; k) a heavy chain variable region comprising SEQ ID NO: 87 and a light chain variable region comprising SEQ ID NO: 88; l) a heavy chain variable region comprising SEQ ID NO: 95 and a light chain variable region comprising SEQ ID NO: 96; m) a heavy chain variable region comprising SEQ ID NO: 103 and a light chain variable region comprising SEQ ID NO: 104; n) a heavy chain variable region comprising SEQ ID NO: 111 and a light chain variable region comprising SEQ ID NO: 112; o) a heavy chain variable region comprising SEQ ID NO: 119 and a light chain variable region comprising SEQ ID NO: 120; p) a heavy chain variable region comprising SEQ ID NO: 127 and a light chain variable region comprising SEQ ID NO: 128; q) a heavy chain variable region comprising SEQ ID NO: 135 and a light chain variable region comprising SEQ ID NO: 136; r) a heavy chain variable region comprising SEQ ID NO: 143 and a light chain variable region comprising SEQ ID NO: 144; s) a heavy chain variable region comprising SEQ ID NO: 151 and a light chain variable region comprising SEQ ID NO: 152; t) a heavy chain variable region comprising SEQ ID NO: 159 and a light chain variable region comprising SEQ ID NO: 160; u) a heavy chain variable region comprising SEQ ID NO: 167 and a light chain variable region comprising SEQ ID NO: 168; v) a heavy chain variable region comprising SEQ ID NO: 175 and a light chain variable region comprising SEQ ID NO: 176; w) a heavy chain variable region comprising SEQ ID NO: 183 and a light chain variable region comprising SEQ ID NO: 184; x) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 192; y) a heavy chain variable region comprising SEQ ID NO: 199 and a light chain variable region comprising SEQ ID NO: 200; z) a heavy chain variable region comprising SEQ ID NO: 207 and a light chain variable region comprising SEQ ID NO: 208; aa) a heavy chain variable region comprising SEQ ID NO: 215 and a light chain variable region comprising SEQ ID NO: 216; bb) a heavy chain variable region comprising SEQ ID NO: 223 and a light chain variable region comprising SEQ ID NO: 224; cc) a heavy chain variable region comprising SEQ ID NO: 231 and a light chain variable region comprising SEQ ID NO: 232; dd) a heavy chain variable region comprising SEQ ID NO: 239 and a light chain variable region comprising SEQ ID NO: 240; ee) a heavy chain variable region comprising SEQ ID NO: 247 and a light chain variable region comprising SEQ ID NO: 248; ff) a heavy chain variable region comprising SEQ ID NO: 255 and a light chain variable region comprising SEQ ID NO: 256; gg) a heavy chain variable region comprising SEQ ID NO: 263 and a light chain variable region comprising SEQ ID NO: 264; hh) a heavy chain variable region comprising SEQ ID NO: 271 and a light chain variable region comprising SEQ ID NO: 272; ii) a heavy chain variable region comprising SEQ ID NO: 279 and a light chain variable region comprising SEQ ID NO: 280; jj) a heavy chain variable region comprising SEQ ID NO: 287 and a light chain variable region comprising SEQ ID NO: 288; kk) a heavy chain variable region comprising SEQ ID NO: 295 and a light chain variable region comprising SEQ ID NO: 296; ll) a heavy chain variable region comprising SEQ ID NO: 303 and a light chain variable region comprising SEQ ID NO: 304; mm) a heavy chain variable region comprising SEQ ID NO: 311 and a light chain variable region comprising SEQ ID NO: 312; nn) a heavy chain variable region comprising SEQ ID NO: 319 and a light chain variable region comprising SEQ ID NO: 320; oo) a heavy chain variable region comprising SEQ ID NO: 327 and a light chain variable region comprising SEQ ID NO: 328; pp) a heavy chain variable region comprising SEQ ID NO: 335 and a light chain variable region comprising SEQ ID NO: 336; qq) a heavy chain variable region comprising SEQ ID NO: 343 and a light chain variable region comprising SEQ ID NO: 344; rr) a heavy chain variable region comprising SEQ ID NO: 347 and a light chain variable region comprising SEQ ID NO: 348; or ss) a heavy chain variable region comprising SEQ ID NO: 349 and a light chain variable region comprising SEQ ID NO: 350; Includes.

[0020] In some embodiments, the first binding domain comprises: a) a heavy chain variable region comprising SEQ ID NO: 351 and a light chain variable region comprising SEQ ID NO: 352; b) a heavy chain variable region comprising SEQ ID NO: 353 and a light chain variable region comprising SEQ ID NO: 354; c) a heavy chain variable region comprising SEQ ID NO: 355 and a light chain variable region comprising SEQ ID NO: 352; d) a heavy chain variable region comprising SEQ ID NO: 355 and a light chain variable region comprising SEQ ID NO: 356; e) a heavy chain variable region comprising SEQ ID NO: 357 and a light chain variable region comprising SEQ ID NO: 352; f) a heavy chain variable region comprising SEQ ID NO: 357 and a light chain variable region comprising SEQ ID NO: 354; g) a heavy chain variable region comprising SEQ ID NO: 358 and a light chain variable region comprising SEQ ID NO: 359; h) a heavy chain variable region comprising SEQ ID NO: 360 and a light chain variable region comprising SEQ ID NO: 361; i) a heavy chain variable region comprising SEQ ID NO: 362 and a light chain variable region comprising SEQ ID NO: 359; j) a heavy chain variable region comprising SEQ ID NO: 362 and a light chain variable region comprising SEQ ID NO: 363; k) a heavy chain variable region comprising SEQ ID NO: 364 and a light chain variable region comprising SEQ ID NO: 359; l) a heavy chain variable region comprising SEQ ID NO: 364 and a light chain variable region comprising SEQ ID NO: 361; m) a heavy chain variable region comprising SEQ ID NO: 365 and a light chain variable region comprising SEQ ID NO: 366; n) a heavy chain variable region comprising SEQ ID NO: 367 and a light chain variable region comprising SEQ ID NO: 368; o) a heavy chain variable region comprising SEQ ID NO: 367 and a light chain variable region comprising SEQ ID NO: 369; p) a heavy chain variable region comprising SEQ ID NO: 367 and a light chain variable region comprising SEQ ID NO: 366; q) a heavy chain variable region comprising SEQ ID NO: 370 and a light chain variable region comprising SEQ ID NO: 369; r) a heavy chain variable region comprising SEQ ID NO: 370 and a light chain variable region comprising SEQ ID NO: 366; s) a heavy chain variable region comprising SEQ ID NO: 365 and a light chain variable region comprising SEQ ID NO: 371; t) a heavy chain variable region comprising SEQ ID NO: 367 and a light chain variable region comprising SEQ ID NO: 372; u) a heavy chain variable region comprising SEQ ID NO: 367 and a light chain variable region comprising SEQ ID NO: 373; v) a heavy chain variable region comprising SEQ ID NO: 367 and a light chain variable region comprising SEQ ID NO: 371; w) a heavy chain variable region comprising SEQ ID NO: 370 and a light chain variable region comprising SEQ ID NO: 373; or x) a heavy chain variable region comprising SEQ ID NO: 370 and a light chain variable region comprising SEQ ID NO: 371; Includes.

[0021] In some embodiments, the first binding domain further comprises one or more amino acid residue substitutions or modifications while retaining specific binding affinity to human CD276, in some embodiments, the substitutions are in one or more CDR sequences and / or in one or more of the VH or VL sequences but not in any of the CDR sequences.

[0022] In some embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises an activating receptor binding domain, and optionally a human Ig constant region, or optionally a human IgG constant region. In some embodiments, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0023] In some embodiments, the second target is PD-L1.

[0024] In some embodiments, the second binding domain comprises one, two, or three heavy chain complementarity determining region (CDR) sequences selected from the group consisting of SEQ ID NOs: 384 to 386, and / or one, two, or three light chain CDR sequences selected from the group consisting of SEQ ID NOs: 387 to 389. In some embodiments, the second binding domain comprises a heavy chain variable region comprising the three CDR sequences set forth in SEQ ID NOs: 384, 385, and 386, respectively, and a light chain variable region comprising the three CDR sequences set forth in SEQ ID NOs: 387, 388, and 389, respectively.

[0025] In some embodiments, the second binding domain comprises a heavy chain variable region set forth in SEQ ID NO:382 and / or a light chain variable region set forth in SEQ ID NO:383.

[0026] In some embodiments, the second binding domain further comprises one or more amino acid residue substitutions or modifications while retaining specific binding affinity to human PD-L1, in some embodiments, the substitutions are in one or more CDR sequences and / or in one or more of the VH or VL sequences but not in any of the CDR sequences.

[0027] In some embodiments, the second binding domain comprises a VH-linker-VL scFv structure, in some embodiments, the linker comprises the sequence (GGGGS)3 (SEQ ID NO: 390).

[0028] In some embodiments, the N-terminus of the second binding domain is operably linked to the C-terminus of the activating receptor binding domain.

[0029] In some embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein are bispecific or trispecific antibodies.

[0030] In some embodiments, the multispecific antibody or antigen-binding fragment is linked to one or more conjugates. In some embodiments, the conjugate is covalently attached directly or via a linker. In some embodiments, the conjugate comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or another anti-cancer agent.

[0031] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein.

[0032] In one aspect, the present disclosure provides a vector comprising an isolated polynucleotide provided herein.

[0033] In one aspect, the present disclosure provides a host cell comprising the vector provided herein.

[0034] In one aspect, the present disclosure provides a pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof provided herein, or a polynucleotide encoding said multispecific antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0035] In one aspect, the present disclosure provides a method of expressing a multispecific antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which a vector provided herein is expressed.

[0036] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject that is benefited from modulation of CD276 activity, comprising administering to the subject a therapeutically effective amount of a multispecific antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein.

[0037] In some embodiments, the disease or condition is a CD276-associated disease or condition.

[0038] In some embodiments, the disease or condition is cancer, an adaptive immune disorder, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0039] In some embodiments, the cancer is an adrenal gland tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid globe tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplasia ossificans, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, or cancer), gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, pancreatic islet tumors, Kaposi's sarcoma, kidney cancer, leukemia, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare blood disorders, metastatic kidney cancer, rhabdomyosarcoma-like tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer, and optionally, the cancer is chemotherapy-resistant.

[0040] In some embodiments, the disease or condition is a hematological cancer selected from a B-cell lymphoma, e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma (MZL), mantle cell lymphoma (MCL), Richter's syndrome, Burkitt's lymphoma, or follicular lymphoma.

[0041] In some embodiments, the subject is a human.

[0042] In some embodiments, the methods provided herein comprise administering to the subject a therapeutically effective amount of one or more therapeutic agents, which in some embodiments are chemotherapeutic agents, radiation therapy agents, hormone therapy agents, toxins, or immunotherapy agents.

[0043] In some embodiments, the administration is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular.

[0044] In some embodiments, the method further comprises administration of one or more additional cancer treatments selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, hormone therapy, and surgery.

[0045] In one aspect, the present disclosure provides a method of modulating CD276 activity in a CD276-expressing cell, the method comprising exposing the CD276-expressing cell to an antibody or antigen-binding fragment thereof provided herein.

[0046] In one aspect, the disclosure provides a method of modulating PD-1 / PD-L1 pathway activity in a PD-L1-expressing cell, the method comprising exposing the PD-L1-expressing cell to a multispecific antibody, or antigen-binding fragment thereof, provided herein.

[0047] In one aspect, the disclosure provides use of a multispecific antibody, or antigen-binding fragment thereof, provided herein in the manufacture of a medicament for treating a CD276- and / or PD-L1-associated disease or condition in a subject.

[0048] In some embodiments, the pharmaceutical agent further comprises a second therapeutic agent, which in some embodiments is a chemotherapeutic agent, a radiation therapy agent, a hormone therapy agent, a toxin, or an immunotherapy agent. [Brief explanation of the drawings]

[0049] The drawings are for purposes of illustration only and not limitation. [Figure 1] FIG. 1 shows the binding affinity of anti-CD276 antibodies provided herein to SKOV3 cells as measured by FACs analysis. [Figure 2] 2A-2E show the binding of 6-D8-E7-A11 to several cancer cell lines expressing B7H3, as measured by FACS analysis. [Figure 3] 3A-3C show the ADCC effect of anti-CD276 antibodies provided herein on SKOV3 cells. [Figure 4] Figures 4A and 4B show the CDC effect of anti-CD276 antibodies provided herein on CHO-S-hCD276 cells. [Figure 5(1)] 5A-5E show the indirect ADC cytotoxic effect of anti-CD276 antibodies provided herein on SKOV3 cells. [Figure 5(2)] (continuation) [Figure 5(3)] (continuation) [Figure 6] FIG. 6 shows the in vivo efficacy of anti-CD276 antibodies provided herein in inhibiting tumor growth in a mouse model inoculated with MC-38-hCD276 (B7H3) tumor cells. [Figure 7] FIG. 7 shows the effect on tumor growth in the subcutaneous calu-6 model in balb / c nude mice (mean±standard error). [Figure 8] FIG. 8 shows IL2 release upon T cell activation in an MLR assay. [Figure 9] FIG. 9 shows IFNγ release upon T cell activation in an MLR assay. [Figure 10] FIG. 10 shows the binding affinity of humanized antibodies derived from 30-C7-C11-D4 to SKOV3. [Figure 11] FIG. 11 shows the binding affinity of humanized antibodies derived from 10-G6-C4-B2 to SKOV3. [Figure 12]FIG. 12 shows the effect on tumor growth in the subcutaneous Calu-6 model in Balb / c nude mice (mean±standard error). [Figure 13] FIG. 13 shows the effect of test articles on tumor growth in the subcutaneous MC-38-hCD276(B7H3) model in C57BL / 6 mice (mean±standard error). [Figure 14] FIG. 14 shows the effect of test articles on tumor growth in the subcutaneous MC-38-hCD276(B7H3) model in C57BL / 6 mice (mean±standard error). [Figure 15] FIG. 15 shows the effect of test articles on tumor growth in the subcutaneous MC-38-hCD276(B7H3) model in C57BL / 6 mice (mean±standard error). DETAILED DESCRIPTION OF THE INVENTION

[0050] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0051] definition The term "antibody," as used herein, includes any immunoglobulin, monoclonal, polyclonal, multivalent, divalent, or monovalent antibody that binds to a specific antigen. A naturally occurring, intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain contains a variable region (V H ) and the first, second, and third constant regions (C H1 , C H2 , C H3 Mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (VL) and constant regions. Antibodies are "Y" shaped, with the tail of the Y consisting of the second and third constant regions of two heavy chains joined together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain joined to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of antibodies and antigen-binding domains disclosed herein may be defined or identified according to the Kabat, IMGT, AbM, Chothia, or Al-Lazikani definitions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); NR Whitelegg et al, Protein Engineering, v13(12), 819-824 (2000); Chothia, C. et al., Nature. Dec 21-28;342(6252):877-83 (1989), Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991), Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003), Marie-Paule Lefranc et al, Immunome Research, 1(3), (2005), Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)).The three CDRs are interposed between adjacent sections known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified into classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0052] As used herein, the term "antibody molecule" refers to an antigen-binding protein or polypeptide comprising at least one antibody fragment (such as a CDR and / or variable region sequence). Antibody molecules include, for example, monoclonal antibodies, antibody fragments or domains, fusion proteins comprising antibody fragments or domains, and polypeptide complexes comprising antibody fragments or domains.

[0053] As used herein, the term "antigen-binding domain" (e.g., a CD276-binding domain or a PD-L1-binding domain) refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding domains include, without limitation, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, single-chain antibody molecules (scFv), camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding domain is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding domain may comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies. For more detailed formatting of antigen-binding domains, see Spiess et al., 2015 and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated by reference in their entirety.

[0054] "Fab," with respect to an antibody, refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0055] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0056] "F(ab')2" refers to a Fab' dimer.

[0057] "Fragment difficult (Fd)" with respect to antibodies refers to the amino-terminal half of a heavy chain fragment that can combine with a light chain to form Fab. For example, an Fd fragment can consist of the VH and CH1 domains.

[0058] With respect to antibodies, "Fv" refers to the smallest antibody fragment that contains an intact antigen-binding site. An Fv fragment consists of the variable region of a single light chain linked to the variable region of a single heavy chain. Numerous Fv designs are available, including dsFv, in which the association between the two domains is strengthened by an introduced disulfide bond, and scFv, which may be formed by linking the two domains together as a single polypeptide with a peptide linker. Fv constructs containing the variable domain of an immunoglobulin heavy or light chain linked to the variable and constant domains of the corresponding immunoglobulin heavy or light chain have also been produced.

[0059] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other either directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879(1988)).

[0060] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. H The moieties are linked by a peptide linker (e.g., a long flexible linker) and each of the two V L In some embodiments, the dsFv-dsFv' is bispecific, with each disulfide paired heavy and light chain having a different antigen specificity.

[0061] "Fc," with respect to an antibody, refers to the portion of an antibody consisting of the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0062] "Camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to a camelized single domain antibody consisting of two V H It refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38 (1999), Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001), WO94 / 04678, WO94 / 25591, U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from camelids (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies possess a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428):446-8 (1993); Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr; 54(1):39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1):93-101 (2003)). The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0063] "Nanobody" refers to an antibody fragment consisting of a VHH domain and two constant domains CH2 and CH3 from a heavy chain antibody.

[0064] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently linked with a peptide linker to create a bivalent or multivalent domain antibody. HThe domains may target the same or different antigens.

[0065] The term "chimeric," as used herein, refers to an antibody or antigen-binding domain in which a portion of the heavy and / or light chain is derived from one species and the remainder of the heavy and / or light chain is derived from another species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0066] The term "humanized," as used herein, means that an antibody or antigen-binding domain contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

[0067] The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without spacers or linkers or intervening sequences, in a relationship that permits them to function in their intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have its intended biological function. For example, an antibody variable region may be operably linked to a constant region to provide a stable product with antigen-binding activity. As another example, an antigen-binding domain may be operably linked to another antigen-binding domain via an intervening sequence between them, which may be a spacer or may comprise a much longer sequence such as an antibody constant region. This term may also be used with respect to polynucleotides. As an example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows regulated expression of the polypeptide from the polynucleotide.

[0068] The terms "fusion" or "fused," when used in reference to an amino acid sequence (e.g., a peptide, polypeptide, or protein), refer to the joining of two or more amino acid sequences into a single non-naturally occurring amino acid, for example, by chemical conjugation or recombinant means. A fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and expressed by methods in which a construct containing the recombinant polynucleotide is introduced into a host cell.

[0069] As used herein, "antigen" refers to a compound, composition, peptide, polypeptide, protein, or substance capable of stimulating the production of antibodies or a T-cell response in a cell culture or an animal, and includes compositions (such as those containing cancer-specific proteins) that are added to cell cultures (such as hybridomas) or injected or absorbed into animals. Antigens react with the products of specific humoral or cellular immunity (such as antibodies), including those induced by heterologous antigens.

[0070] As used herein, the term "CD276 protein" or "B7-H3 protein" is intended to encompass any form of CD276, for example, 1) the native, unprocessed CD276 molecule, a "full-length" CD276 chain or a naturally occurring variant of CD276, including, for example, a splice variant or allelic variant; 2) any form of CD276 that results from processing in the cell; or 3) full-length, fragmented (e.g., truncated forms, extracellular / transmembrane domain) or modified (e.g., mutated, glycosylated / PEGylated, His-tagged / immunofluorescence fused) forms of a CD276 subunit produced by recombinant methods.

[0071] The terms "anti-CD276 antibody," "anti-CD276 binding domain," or "CD276 binding domain" refer to an antibody or antigen-binding domain capable of specifically binding to CD276 (e.g., human, monkey, or mouse CD276).

[0072] As used herein, "PD-L1" refers to programmed cell death-ligand 1 (PD-L1; see, e.g., Freeman et al. (2000) J. Exp. Med. 192:1027). A representative amino acid sequence of human PD-L1 is published under NCBI accession number NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is provided under NCBI accession number NM_014143.3. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, and fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to its receptors, PD-1 or B7-1, which are expressed on activated T cells, B cells, and myeloid cells. Binding of PD-L1 to its receptor induces signaling that suppresses TCR-mediated activation of cytokine production and T cell proliferation. PD-L1 is therefore thought to play a major role in suppressing the immune system during certain events, such as pregnancy, autoimmune diseases, and tissue allotransplantation, allowing tumor or cancer cells to evade immunological checkpoints and escape the immune response.

[0073] As used herein, the terms "specific binding" or "specifically bind" refer herein to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen.

[0074] In certain embodiments, the antibody molecules or antigen-binding domains provided herein specifically bind to CD276 and / or PD-L1 and have a binding affinity (K D ) is ≦10 -6 M (e.g., ≦5x10 -7 M, ≤2x10 -7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤4x10 -9 M). The K used here D is the ratio of the dissociation rate to the association rate (k off / k on) and can be determined by conventional methods known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). In certain embodiments, K D Values ​​can be suitably determined by flow cytometry.

[0075] Binding affinity to CD276 and / or PD-L1 is measured using the "half maximal effective concentration" (EC), which is the concentration of antibody at which 50% of the maximal effect (e.g., binding or inhibition) is observed. 50 ) value. 50 Values ​​can be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blot, flow cytometry assays, and other binding assays. In certain embodiments, the antibodies and fragments thereof provided herein have an EC50 of 0.05 nM or less, 0.06 nM or less, 0.07 nM or less, 0.08 nM or less, 0.09 nM or less, 0.1 nM or less, 0.2 nM or less, 0.3 nM or less, 0.4 nM or less, 0.5 nM or less, 0.6 nM or less, 0.7 nM or less, 0.8 nM or less, 0.9 nM or less, 1 nM or less, 1.5 nM or less, 2 nM or less, 2.5 nM or less, 3.5 nM or less, 3 nM or less, 4 nM or less, 4.5 nM or less, 5 nM or less, 6 nM or less, 7 nM or less, 8 nM or less, 9 nM or less, or 10 nM or less by flow cytometry assay. 50 (i.e., 50% binding concentration) specifically binds to CD276 and / or PD-L1.

[0076] The ability to "block binding" or "compete for the same epitope," as used herein, refers to the ability of an antibody or antigen-binding domain to inhibit the binding interaction between two molecules (e.g., human CD276 and its binding ligand, e.g., TLT-2) to any detectable degree. In certain embodiments, an antibody or antigen-binding domain that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, this inhibition can be greater than 85% or greater than 90%.

[0077] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids (also called linear or contiguous epitopes) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitopes). Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on a protein, and small segments of contiguous amino acids can be digested from antigens bound to major histocompatibility complex (MHC) molecules or retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically contains at least three, more usually at least five, about seven, or about eight to ten amino acids in a unique spatial conformation. Two antibodies may bind to the same or closely related epitopes within an antigen if they exhibit competitive binding for the antigen. For example, an antibody or antigen-binding domain can be considered to bind to the same / closely related epitope as the reference antibody if it blocks binding of the reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%.

[0078] With respect to amino acid sequences, a "conservative substitution" refers to the replacement of an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), acidic side chains (e.g., Asp, Glu), basic side chains (e.g., His, Lys, and Arg), or aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not significantly alter the conformational structure of a protein, thereby preserving the biological activity of the protein.

[0079] The terms "homologue" and "homologous," as used herein, refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence that is interchangeable and has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0080] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be accomplished by publicly available tools such as, for example, BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website; see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by these tools or customize the parameters for alignment, for example, by selecting a suitable algorithm.

[0081] As used herein, "effector function" refers to a biological activity resulting from the binding of an antibody's Fc region to its effector, such as the C1 complex, an Fc receptor, and an effector cell (e.g., a macrophage). Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cellular cytotoxicity (ADCC), which is induced by the binding of an antibody's Fc region to an Fc receptor on an effector cell; and antibody-dependent cellular phagocytosis (ADCP), which is induced by the binding of an antibody's Fc region to a phagocytic cell. It is well established that specific glycan structures associated with conserved biantennary glycans within the Fc-CH2 domain can strongly influence the interaction with FcγRs that mediate ADCC and ADCP, as well as the interaction with Clq binding, the initial binding event that leads to CDC (see Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015; 25: 1325-34).

[0082] "Treating" a symptom, as used herein, includes preventing or alleviating the symptom, delaying the onset or rate of progression of the symptom, reducing the risk of developing the symptom, preventing or delaying the onset of symptoms associated with the symptom, reducing or terminating symptoms associated with the symptom, causing complete or partial regression of the symptom, curing the symptom, or some combination thereof.

[0083] The terms "subject" or "individual" or "animal" or "patient," as used herein, refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, monkeys, domestic animals, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, and bears.

[0084] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to express that protein. A vector can be used to transform, transduce, or transfect a host cell to express the genetic elements carried by the vector within the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector may contain an origin of replication. A vector may also contain materials that aid in entry into a cell, including, but not limited to, a viral particle, a liposome, or a protein coating. A vector may be an expression vector or a cloning vector.

[0085] The term "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector can be or has been introduced.

[0086] "CD276-associated disease or condition," as used herein, refers to any disease or condition caused by, exacerbated by, or otherwise associated with, increased or decreased expression or activity of CD276. In some embodiments, the CD276-associated condition is an immune-related disorder, such as cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0087] A "PD-L1-associated" disease or condition, as used herein, refers to any disease or condition that is caused by, exacerbated by, or otherwise associated with, increased or decreased expression or activity of PD-L1. In some embodiments, the PD-L1-associated disease or condition involves suppression of the immune system. In some embodiments, the PD-L1-associated disease or condition is an immune-related disorder, such as cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0088] "Cancer," as used herein, refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, invasion, or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. Examples of cancers or tumors include adrenal gland tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid globe tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossificans, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, and the like. Cancer), gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, pancreatic islet tumors, Kaposi's sarcoma, kidney cancer, leukemia, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumors, childhood cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare blood disorders, metastatic kidney cancer, rhabdomyosarcoma-like tumors, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

[0089] In certain embodiments, the hematological malignancy comprises a B-cell lymphoma, optionally Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma (MZL), mantle cell lymphoma (MCL), Richter's syndrome, Burkitt's lymphoma, or follicular lymphoma.

[0090] In certain embodiments, the cancer is selected from gastric cancer, breast cancer, head and neck cancer, pancreatic cancer, and colon cancer. In certain embodiments, the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0091] In certain embodiments, the cancer is chemotherapy-resistant. The term "chemoresistant cancer," as used herein, refers to a type of cancer that does not respond to the effects of chemotherapy. For example, a cancer that has been responding to chemotherapy or a combination of different chemotherapy drugs may suddenly begin to grow, which may be referred to as chemotherapy-resistant cancer.

[0092] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the subject to which it is administered.

[0093] Multispecific antibodies or antigen-binding fragments thereof In one aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof comprising multiple antigen-binding domains and an activating receptor-binding domain.

[0094] i. Multiple antigen-binding domains In some embodiments, the plurality of antigen-binding domains comprises at least a first binding domain that targets a first antigen and a second binding domain that targets a second antigen, wherein one of the first antigen and the second antigen is an immune checkpoint molecule or a tumor antigen, and the other is one or more additional targets.

[0095] In some embodiments, the one or more additional targets include a target that is another immune checkpoint molecule (e.g., PD-L1). In some other embodiments, the one or more additional targets include a target associated with immune cell (e.g., T cell or B cell) function. In some embodiments, the one or more additional targets include a target selected from the group consisting of CD40, CD3, CD28, etc.

[0096] In some embodiments, the first binding domain targets CD276 while the second binding domain targets one or more additional targets. In some embodiments, the second binding domain targets CD276 while the first binding domain targets one or more additional targets. In some embodiments, the first binding domain targets CD276 while the second binding domain targets PD-L1. In some other embodiments, the first binding domain targets PD-L1 while the second binding domain targets CD276.

[0097] CD276-binding domain In certain embodiments, the CD276 binding domain is selected from the group consisting of anti-CD276 antibodies 9-E8-F9-C10, 10-G6-C4-B2, 18-F9-D8-G7, 9-G2-H6-E4, 20-F8-B5-G2, 30-C7-C11-D4, 23-F10-G4-F11, 6-H11-G5-D8, 27-E7-D8-C7, 30-E2- G7-G7, 5-D1-G6-D9, 3-C2-C3-E7, 11-G10-B4-B11, 16-C6-F7-F5, 22-E11-C3-F2, 24-C10-F 9-G7, 25-C8-D7-C5, 4-D5-B9-B11, 10-B9-D10-A12, 15-G1-D1-E3, 8-B4-F5-E11, 6-F3-G2- G1, 9-B9-H11-G7, 9-G12-D6-A11, 13-A8-C4-G1, 15-C8-B5-G7, 28-G2-E6-B10, 3-G7-D8-D3 , 14-E7-G9-D4, 20-C5-D7-D3, 8-C3-E3-F3, 2-A7-B10-A3, 26-D2-D6-B12, 11-C12-F4-F6, 1 The first binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of 6-G3-D10-C10, 27-F8-E10-E11, 7-E1-F8-F6, 25-D3-G4-C6, 13-E4-G9-A4, 20-A2-D10-G8, 3-F2-E7-F9, 6-D8-E7-A11, and 21-B3-B1-H5. In certain embodiments, the first binding domain is capable of specifically binding to CD276. Optionally, the CD276 is derived from a human, monkey, or mouse. In certain embodiments, the CD276 is recombinant CD276 or CD276 expressed on a cell surface.

[0098] All 43 anti-CD276 antibodies provided herein are murine monoclonal antibodies. Table 1 shows the CDR sequences of these 43 anti-CD276 antibodies according to the IMGT numbering system. The heavy and light chain variable region sequences are also shown below. [Table 1-1] [Table 1-2] Table 1-3 Table 1-4

[0099] Table 2は, 9-E8-F9-C10, 10-G6-C4-B2, 18-F9-D8-G7, 9-G2-H6-E4, 20-F8-B5-G2, 30-C7- C11-D4, 23-F10-G4-F11, 6-H11-G5-D8, 27-E7-D8-C7, 30-E2-G7-G7, 5-D1-G6-D9, 3 -C2-C3-E7, 11-G10-B4-B11, 16-C6-F7-F5, 22-E11-C3-F2, 24-C10-F9-G7, 25-C8-D 7-C5, 4-D5-B9-B11, 10-B9-D10-A12, 15-G1-D1-E3, 8-B4-F5-E11, 6-F3-G2-G1, 9-B 9-H11-G7, 9-G12-D6-A11, 13-A8-C4-G1, 15-C8-B5-G7, 28-G2-E6-B10, 3-G7-D8-D3 , 14-E7-G9-D4, 20-C5-D7-D3, 8-C3-E3-F3, 2-A7-B10-A3, 26-D2-D6-B12, 11-C12-F 4-F6, 16-G3-D10-C10, 27-F8-E10-E11, 7-E1-F8-F6, 25-D3-G4-C6, 13-E4-G9-A4, 2 The layout of the 0-A2-D10-G8, 3-F2-E7-F9, 6-D8-E7-A11, and 21-B3-B1-H5 antibodies is shown below. Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 [Table 2-6] [Table 2-7]

[0100] Although CDRs are known to be involved in antigen binding, it has been discovered that not all six CDRs are essential or invariant. In other words, the anti-CD276 antibodies 9-E8-F9-C10, 10-G6-C4-B2, 18-F9-D8-G7, 9-G2-H6-E4, 20-F8-B5-G2, 30-C7-C11-D4, 23-F10-G4-F11, 6-H11-G5-D8, 27-E7-D8-C7, 30-E2- G7-G7, 5-D1-G6-D9, 3-C2-C3-E7, 11-G10-B4-B11, 16-C6-F7-F5, 22-E11-C3-F2, 24-C10-F9-G 7, 25-C8-D7-C5, 4-D5-B9-B11, 10-B9-D10-A12, 15-G1-D1-E3, 8-B4-F5-E11, 6-F3-G2-G1, 9-B9 -H11-G7, 9-G12-D6-A11, 13-A8-C4-G1, 15-C8-B5-G7, 28-G2-E6-B10, 3-G7-D8-D3, 14-E7-G9- D4, 20-C5-D7-D3, 8-C3-E3-F3, 2-A7-B10-A3, 26-D2-D6-B12, 11-C12-F4-F6, 16-G3-D10-C10, 2 It is possible to replace, change or modify one or more CDRs in a CD276 binding domain comprising the CDRs from 7-F8-E10-E11, 7-E1-F8-F6, 25-D3-G4-C6, 13-E4-G9-A4, 20-A2-D10-G8, 3-F2-E7-F9, 6-D8-E7-A11, or 21-B3-B1-H5.

[0101] In certain embodiments, the CD276 binding domain is selected from the group consisting of anti-CD276 antibodies 9-E8-F9-C10, 10-G6-C4-B2, 18-F9-D8-G7, 9-G2-H6-E4, 20-F8-B5-G2, 30-C7-C11-D4, 23-F10-G4-F11, 6-H11-G5-D8, 27-E7-D8-C7, 3 0-E2-G7-G7, 5-D1-G6-D9, 3-C2-C3-E7, 11-G10-B4-B11, 16-C6-F7-F5, 22-E11-C3-F2, 24-C10-F9-G7, 25-C8-D7-C5, 4-D5-B9-B11, 10-B9-D10-A12, 15-G1-D1-E3, 8-B4-F5-E1 1, 6-F3-G2-G1, 9-B9-H11-G7, 9-G12-D6-A11, 13-A8-C4-G1, 15-C8-B5-G7, 28-G2-E6-B 10, 3-G7-D8-D3, 14-E7-G9-D4, 20-C5-D7-D3, 8-C3-E3-F3, 2-A7-B10-A3, 26-D2-D6-B12 , 11-C12-F4-F6, 16-G3-D10-C10, 27-F8-E10-E11, 7-E1-F8-F6, 25-D3-G4-C6, 13-E4-G9-A4, 20-A2-D10-G8, 3-F2-E7-F9, 6-D8-E7-A11, and 21-B3-B1-H5. In specific embodiments, the CD276 binding domain comprises a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283, 291, 299, 307, 315, 323, 331, and 339. The heavy chain CDR3 region is centrally located in the antigen-binding site and is therefore thought to make the most contacts with the antigen and provide the most free energy for the affinity of the antibody for the antigen. Heavy chain CDR3 is also considered to be by far the most diverse CDR among the antigen-binding sites in terms of length, amino acid composition, and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81).Diversity in the heavy chain CDR3 is sufficient to confer most antibody specificity (Xu JL, Davis MM. Immunity. 13:37-45) and desirable antigen-binding affinity (Schier R, et al. J Mol Biol. 263:551-67).

[0102] In certain embodiments, the CD276-binding domain comprises suitable framework region (FR) sequences, so long as the antibody and / or antigen-binding fragment thereof is capable of specifically binding to CD276. The CDR sequences shown in Table 1 are derived from a mouse antibody, but can be grafted to suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, etc., by suitable known methods, such as recombinant techniques.

[0103] In certain embodiments, the sequence of the CD276-binding domain is PTM-optimized. As used herein, PTM-optimized refers to post-translational modifications aimed at avoiding potential aggregation, loss of activity, or other risks. Exemplary PTM-optimized CD276-binding domains include mVH5-mVL4-10 or mVH-mVL1-30, derived from 10-G6-C4-B2 and 30-C7-C11-D4, respectively. The variable region sequences of mVH5-mVL4-10 and mVH-mVL1-30 are shown in Table 3, with all CDR regions underlined. [Table 3]

[0104] In certain embodiments, the CD276-binding domain is humanized. Humanized antibodies or antigen-binding fragments are desirable because they are less immunogenic in humans. Humanized antibodies are chimeric in the variable region because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be essentially achieved by substituting non-human (e.g., murine) CDR genes for the corresponding human CDRs in a human immunoglobulin gene (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0105] Suitable human heavy and light chain variable domains can be selected to achieve this goal by methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting non-human CDR sequences (see, e.g., Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, a framework derived from the consensus sequence of all human antibodies can be used for grafting non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623). In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences, except for CDR sequences, which are non-human. In some embodiments, the variable region FRs, and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be derived from different human immunoglobulin genes, e.g., the FR sequences are derived from one human antibody and the constant region is derived from another human antibody.

[0106] Table 4 below shows hVH2-hVL1-10, hVH3-hVL3-10, hVH4-hVL1-10, hVH4-hVL2-10, hVH5-hVL1-1. 0, hVH5-hVL3-10, 10-G6-C4-B2_hVH2-VL1_PTM, 10-G6-C4-B2_hVH3-VL3_PTM, 10-G 6-C4-B2_hVH4-VL1_PTM, 10-G6-C4-B2_hVH4-VL2_PTM, 10-G6-C4-B2_hVH5-VL1_PT M, 10-G6-C4-B2_hVH5-VL3_PTM, hVH1-hVL5-30, hVH2-hVL1-30, hVH3-hVL2-30, hVH3 -hVL5-30, hVH4-hVL2-30, hVH4-hVL5-30, 30-C7-C11-D4_hVH1-hVL5_PTM, 30-C7-C 11-D4_hVH2-hVL1_PTM, 30-C7-C11-D4_hVH3-hVL2_PTM, 30-C7-C11-D4_hVH3-hVL5_ Figure 1 shows the amino acid sequences of the heavy and light chain variable regions of the humanized antibodies 10-G6-C4-B2 and 30-C7-C11-D4, designated as 10-G6-C4-B2_hVH4-hVL2_PTM, 30-C7-C11-D4_hVH4-hVL2_PTM, and 30-C7-C11-D4_hVH4-hVL5_PTM, with all CDR regions underlined. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0107] In certain embodiments, the humanized CD276 binding domain is composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be derived from different human immunoglobulin genes, e.g., the FR sequences are derived from one human antibody and the constant region is derived from another human antibody. In some embodiments, the humanized CD276 domain comprises human heavy chain HFR1-4 and / or light chain LFR1-4.

[0108] In some embodiments, the human-derived FR region may comprise the same amino acid sequence as the human immunoglobulin from which it was derived. In some embodiments, one or more amino acid residues in the human FR are substituted with the corresponding residue from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more closely resemble the non-human parent antibody structure in order to optimize binding characteristics (e.g., improve binding affinity). In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all of the FR sequences of the heavy or light chain variable domain. In some embodiments, such amino acid residue changes may occur only in the heavy chain FR region, only in the light chain FR region, or in both chains. In certain embodiments, one or more amino acids in the human FR sequence are randomly mutated to improve binding affinity. In certain embodiments, one or more amino acids of the human FR sequences are backmutated to the corresponding amino acid of the parent non-human antibody to improve binding affinity.

[0109] PD-L1 binding domain In some embodiments, the variable region of the PD-L1-binding domain contained in the multispecific antibodies or antigen-binding fragments thereof provided herein may be any heavy chain variable region and light chain variable region of a PD-L1 antibody known in the art. In a specific embodiment, the PD-L1-binding domain comprises the heavy chain variable region and light chain variable region from YN035, cited in WO2019196309A1. [Table 5] [Table 6]

[0110] ii. Activating receptor binding domain The multispecific antibody or antigen-binding fragment thereof further comprises an activating receptor-binding domain. As used herein, the term "activating receptor" refers to a receptor (e.g., FcγR) expressed on immune effector cells (e.g., phagocytes such as macrophages) that, upon activation, e.g., by binding to an Fc domain, mediates at least one effector function or proinflammatory response of the immune effector cell (e.g., phagocytes such as macrophages). In certain embodiments, the immune effector cells provided herein co-express CD276 and one or more additional targets (e.g., PD-L1).

[0111] In a specific embodiment, the activating receptor is an FcγR, and the activating receptor-binding domain is an Fc domain. The Fc domain activates Fc receptors (FcRs) on macrophages, triggering a phosphorylation cascade propagated by the receptor's immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are conserved sequences present in the cytoplasmic tails of several activating receptors on immune effector cells, such as FcRs, T cell receptors, and immunoglobulins (Ig). ITAMs can be characterized by conserved amino acid sequence motifs separated by a predetermined interval of 6 to 8 amino acids, consisting of paired YXXL / I motifs (where Y represents tyrosine, L represents lysine, and I represents isoleucine). Phosphorylation of residues within the ITAM recruits several signaling molecules for phagocytic activation. Therefore, an activating receptor can be any receptor expressed on immune effector cells that can be bound, activated, and induce phagocytosis via an intracellular phagocytic signaling domain containing an ITAM.

[0112] In other embodiments, the activating receptor is a receptor involved in various phagocytic signaling or mechanisms, such as the Akt-mediated signaling cascade (via CD19, CD28, CSFR, or PDGFR receptors), clustering of receptors on immune effector cells (e.g., macrophages) that enhance phagocytosis (e.g., via integrins or selectins), or antigen-mediated cytotoxicity (via FcDR1 (CD89) receptor or CD206).

[0113] For example, activating receptors associated with effector functions such as phagocytosis include fragment crystallizable gamma receptors (FcγRs), TREM2, lectins, scavenger receptor Al (SRA1), MARCO, CD36, CD163, CD68, CD205, CD206, FcDRl, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CD64, CD32a, CD16a, CD89, CD19, CD28, CSFR, PDGFR, MSR1, and S The activating receptor may be CARA3, COLEC12, SCARA5, SCARB1, SCARB2, Dectin-1, RAGE (SR-E1), LRP1, LRP2, ASGP, SR-PSOX, CXCL16, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor, or a complement receptor such as CR1 and CR3. In a specific embodiment, the activating receptor is an FcγR.

[0114] In certain embodiments, activating receptors capable of generating an inflammatory signal upon activation include, without limitation, PI3K, FcγRl, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, BAH.Tyro3, ​​Axl, Traf6, Syk, MyD88, Zap70, FcεRl, FcαRl, BAFF-R, DAP12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.

[0115] The term "activating receptor-binding domain," as used herein, refers to a domain (e.g., a portion of an antibody) that is capable of specifically binding to an activating receptor on an immune effector cell, such binding resulting in receptor activation and its downstream signaling (e.g., immune cell effector function or an inflammatory response). For example, the activating receptor-binding domain comprises an Fc domain of an antibody or a variant thereof. In certain embodiments, the Fc domain may be derived from IgG1 or IgG4.

[0116] In certain embodiments, the activating receptor-binding domain of the multispecific molecules provided herein is selected from the group consisting of fragment crystallizable gamma receptor (FcγR), TREM2, lectin, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, CD68, CD205, CD206, FcDR1, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CD64, CD32a, CD16a, CD89, CD19, CD28, CSFR, PDGFR, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, Dectin 1, RAGE (SR-E1), LRP1, LRP2, ASGP, SR-PS It binds to and activates an activating receptor selected from the group consisting of OX, CXCL16, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor or complement receptors (such as CR1 and CR3), PI3K, FcγRl, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, BAH.Tyro3, ​​Axl, Traf6, Syk, MyD88, Zap70, FcεRl, FcαRl, BAFF-R, DAP12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.

[0117] In certain embodiments, the activating receptor-binding domain of the multispecific molecules provided herein comprises an Fc domain or a variant thereof and activates an Fc receptor (FcR), such as FcγRII, on macrophages.

[0118] In some embodiments, the activating receptor-binding domain comprises a heavy and / or light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ region.

[0119] In some embodiments, the activating receptor-binding domain is derived from an immunoglobulin (Ig), optionally a human Ig, optionally a human IgG. In some embodiments, the activating receptor-binding domain is derived from a human IgG1, IgG2, IgG3, or IgG4.

[0120] Human IgG isotypes (subclasses of mature gamma globulin class G antibodies: IgG1, IgG2, IgG3, and IgG4) exhibit different abilities to recruit effector functions. For example, ADCC is promoted by IgG1 and IgG3, ADCP is promoted by IgG1, IgG2, IgG3, and IgG4, and CDC is promoted by IgG1 and IgG3. Isotype-specific involvement of these effector functions is based on selectivity for Fc receptors on different immune cells and their ability to bind C1q, thereby activating membrane attack complex (MAC) assembly. Among the various isotypes, IgG1 and IgG3 have high relative affinity for Fcγ receptors, including FcγRI, FcγRIIa / b / c, and FcγRIIIa / b. However, the affinity of Fcγ for IgG2 is significantly lower, except for the FcγRIIa H131 polymorphism, and IgG4 only has measurable affinity for FcγRI.

[0121] In certain embodiments, the activating receptor-binding domain is derived from the constant region of a human IgG1 isotype, or an IgG4 or IgG2 isotype with reduced or depleted effector functions, which can induce ADCC, CDC, or ADCP. Effector functions, such as ADCC and CDC, can result in cytotoxicity to cells expressing tumor antigens (e.g., CD276). Effector functions can be assessed by various assays, such as Fc receptor binding assays, C1q binding assays, and cytolytic assays.

[0122] In a specific embodiment, the activating receptor-binding domain is derived from the constant region of a mouse IgG2 isotype capable of inducing ADCC, CDC, or ADCP.

[0123] iii. Construction of multispecific antibodies or antigen-binding fragments thereof In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein are recombinant proteins comprising multiple antigen-binding domains as described throughout the specification, each of which has specific binding affinity for a respective target antigen, and which are connected to each other by one or more linkers. The one or more linkers may comprise cognate peptides that bind to each other in a complementary manner. For example, the multispecific antibodies or antigen-binding fragments thereof provided herein may comprise multiple antigen-binding domains as described throughout the specification, each of which has specific binding affinity for a respective target antigen, and which are connected to each other by one or more linkers. Multispecific antibodies or antigen-binding fragments thereof is fused to the first peptide of a pair of cognate peptides, the The second binding domain is fused to the second peptide of the pair of cognate peptides, such that the first binding domain and the second binding domain can be connected by the pair of cognate peptides through complementary bonds between each of the pair of cognate peptides.

[0124] In certain embodiments, the pair of cognate peptides comprises two heavy chains of an antibody or any complementary portions thereof, a pair of complementary light and heavy chains of an antibody or any complementary portions thereof, leucine zipper domains that exhibit complementary binding to each other (e.g., zipper sequences in the binding regions in c-Fos and c-June proteins), or synthetic peptides designed to specifically bind to each other via a synthetic connector.

[0125] The multiple antigen-binding domains (e.g., first and second binding domains) and activating receptor-binding domains of the multispecific antibodies or antigen-binding fragments thereof provided herein may be connected by chemical bonds such as crosslinking (e.g., using a BS2G crosslinker (bis[sulfosuccinimidyl]glutarate), a BS3 crosslinker (bis[sulfosuccinimidyl]suberate), sulfo-DSS, a DST crosslinker (disuccinimidyl tartrate), a BMPS (N-(B-maleimidopropyloxy)succinimide ester), a MBS crosslinker (maleimidobenzoyl-N-hydroxysuccinimide ester), or a PDPH crosslinker (3-[2-pyridyldithio]propionylhydrazide)).

[0126] In certain embodiments, the first binding domain (e.g., the CD276-binding domain or the PD-L1-binding domain) is linked to the N-terminus of an activating receptor-binding domain (e.g., an Fc domain). In certain embodiments, the first binding antibody domain (e.g., the CD276-binding domain or the PD-L1-binding domain) comprises a Fab domain, and optionally, the Fab domain comprises a heavy chain linked to one of the N-terminus of an activating receptor-binding domain (e.g., an Fc domain).

[0127] In certain embodiments, the first binding domain and the second binding domain both comprise Fab domains, and optionally, each of the Fab domains comprises a heavy chain linked to the respective N-terminus of an activating receptor-binding domain (e.g., an Fc domain).

[0128] In certain embodiments, the second binding domain (e.g., a CD276-binding domain or a PD-L1-binding domain) is linked to the activating receptor-binding domain (e.g., an Fc domain) or the first binding domain. In certain embodiments, the second binding domain comprises an scFv structure.

[0129] In certain embodiments, the second binding domain is linked to the C-terminus of the light chain of the first binding domain (e.g., a first binding domain comprising a Fab domain). In certain embodiments, the second binding domain is linked to the N-terminus of the light chain of the first binding domain (e.g., a first binding domain comprising a Fab domain). In certain embodiments, the second binding domain is linked to the C-terminus of the heavy chain of the first binding domain (e.g., a first binding domain comprising a Fab domain). In certain embodiments, the second binding domain is linked to the N-terminus of the heavy chain of the first binding domain (e.g., a first binding domain comprising a Fab domain).

[0130] As used herein, the term "linked to" refers to a covalent or non-covalent interaction (e.g., hydrogen bonding, ionic bonding, van der Waals interactions, and hydrophobic bonding) between two components.

[0131] In certain embodiments, the second binding domain is linked to the C-terminus of the activating receptor-binding domain (e.g., Fc domain). In certain embodiments, the first binding domain (e.g., a first binding domain comprising a Fab domain) is linked to the N-terminus of the activating receptor-binding domain, and the second binding domain is linked to the C-terminus of the activating receptor-binding domain. In such embodiments, the first binding domain (e.g., a CD276-binding domain or a PD-L1-binding domain) and the activating receptor-binding domain (e.g., an Fc domain) of the multispecific antibody or antigen-binding fragment thereof disclosed herein together form an intact antibody structure. In some embodiments, the second binding domain comprises an N- to C-terminal scFv structure comprising a second heavy chain variable region, a first linker, and a second light chain variable region. In certain embodiments, the first linker has the sequence of (GGGGS)3 (SEQ ID NO: 390).

[0132] In some embodiments, the N-terminus of the second binding domain is linked to the C-terminus of the activating receptor binding domain via a second linker. In certain embodiments, the second linker has the sequence (GGGGS)3 (SEQ ID NO: 390).

[0133] In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof disclosed herein comprise two second binding domains, each operably linked to the respective C-terminus of the activating receptor-binding domain.

[0134] In some embodiments, the multispecific antibody or antigen-binding fragment thereof comprises four chains having the following structure from N-terminus to C-terminus: Chains 1 and 4: VL1-CL. Chains 2 and 3: VH1-CH1-hinge region-CH2-CH3-linker2-VH2-linker1-VL2.

[0135] Here, VH1 represents the heavy chain variable region of the first binding domain, VL1 represents the light chain variable region of the first binding domain, VH2 represents the heavy chain variable region of the second binding domain, and VL2 represents the light chain variable region of the second binding domain, Linker1 represents the first linker, and Linker2 represents the second linker.

[0136] In some specific embodiments, the first binding domain is a CD276-binding domain and the second binding domain is a PD-L1-binding domain. In some other specific embodiments, the first binding domain is a PD-L1-binding domain and the second binding domain is a CD276-binding domain.

[0137] Antibody variants The present disclosure also encompasses various variants of the multispecific antibodies and / or antigen-binding fragments thereof provided herein. In certain embodiments, the present disclosure encompasses various types of variants of the exemplary antibodies provided herein, i.e., antibodies having a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283, 291, 299, 307, 315, 323, 331, 339, or 375.

[0138] In certain embodiments, antibody variants comprise one or more CDR sequences set forth in Tables 1, 3, or 5, one or more FR sequences set forth in Tables 2, 4, or 6, heavy or light chain variable region sequences, and / or one or more modifications or substitutions in the activating receptor binding domain (e.g., Fc region). Such variants retain the specific binding affinity of the parent antibody to CD276 and / or PD-L1, but have one or more desirable properties conferred by the modification or substitution. For example, antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or eliminated effector function, improved FcRn receptor binding, and increased pharmacokinetic half-life, pH sensitivity, and / or conjugation compatibility (e.g., one or more introduced cysteine ​​residues).

[0139] The parent antibody sequence may be screened by methods known in the art, such as "alanine systematic mutagenesis," to identify suitable or preferred residues to modify or replace (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) may be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibodies are produced and screened for properties of interest. If substitution at a particular amino acid position exhibits a functional change of interest, this position can be identified as a potential residue for modification or replacement. Potential residues may be further evaluated by substituting them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0140] Affinity variants Affinity variants may contain modifications or substitutions in one or more CDR sequences shown in Tables 1, 3 or 5, one or more FR sequences shown in Tables 2, 3, 4 or 6, or heavy or light chain variable region sequences. Affinity variants retain the specific binding affinity of the parent antibody to CD276 and / or PD-L1, and thus have a specific binding affinity for CD276 and / or PD-L1 that exceeds that of the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences comprise conservative substitutions.

[0141] Those skilled in the art will understand that one or more amino acid residues in the CDR and FR sequences shown in Tables 1 or 5 may be substituted, while the resulting antibody or antigen-binding fragment still retains its binding affinity to CD276 and / or PD-L1, and thus may have improved binding affinity. Various methods known in the art can be used to achieve this goal. For example, phage display technology can be used to generate and express a library of antibody variants (such as Fab or scFv variants), which can then be screened for binding affinity to CD276 and / or PD-L1. As another example, computer software can be used to virtually simulate the binding of an antibody to CD276 and / or PD-L1 to identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided for substitution to prevent a decrease in binding affinity, or may be targeted for substitution to provide stronger binding.

[0142] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences, hi certain embodiments, affinity variants comprise a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR and / or FR sequences.

[0143] In certain embodiments, multispecific antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with those listed in Tables 1 or 5, while retaining a similar level of binding affinity to CD276 and / or PD-L1 as, or even higher than, the parent antibody.

[0144] In certain embodiments, multispecific antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those listed in Tables 2, 4, or 6, while retaining a similar, or even higher, level of binding affinity to CD276 and / or PD-L1 as the parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a sequence selected from those listed in Table 1. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., within the FRs).

[0145] Glycosylation variants The multispecific antibodies and antigen-binding fragments provided herein also encompass glycosylation variants that can be obtained to increase or decrease the degree of glycosylation of the antibodies or antigen-binding fragments thereof.

[0146] Multispecific antibodies or antigen-binding fragments thereof may comprise one or more amino acid residues having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, such as an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence to substitute one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) or an existing serine or threonine residue (for O-linked glycosylation sites). Similarly, new glycosylation sites can be created by introducing such tripeptide sequences or serine or threonine residues.

[0147] Cysteine ​​engineered variants The multispecific antibodies and antigen-binding fragments provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0148] A free cysteine ​​residue is one that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugation at the engineered cysteine ​​site, e.g., via maleimide or haloacetyl, with, for example, cytotoxic and / or imaging compounds, labels, or radioisotopes. Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine ​​residues are known in the art; see, e.g., WO2006 / 034488.

[0149] Fc variants The multispecific antibodies and antigen-binding fragments provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region.

[0150] In certain embodiments, the multispecific antibodies or antigen-binding fragments provided herein comprise one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). By binding to FcRn at acidic pH, such variants can avoid lysosomal degradation and subsequently be translocated and released extracellularly, thereby increasing pharmacokinetic half-life. Methods for engineering antibodies and antigen-binding fragments thereof to improve their binding affinity to FcRn are well known in the art; see, for example, Vaughn, D. et al., Structure, 6(1): 63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176:346-356 (2006).

[0151] In certain embodiments, the multispecific antibodies or antigen-binding fragments provided herein comprise one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC). Specific amino acid residues in the CH2 domain of the Fc region may be substituted to enhance ADCC activity. Alternatively, or additionally, carbohydrate structures on the antibody may be altered to enhance ADCC activity. Methods for altering ADCC activity by antibody engineering have been described in the art, for example, Shields R L. et al., J. Biol. Chem. 2001, 276(9): 6591-604; Idusogie E E. et al., J. Immunol. 2000, 164(8): 4178-84; Steurer W. et al., J. Immunol. 1995, 155(3): 1165-74; Idusogie E E. et al., J. Immunol. 2001, 166(4): 2571-5; Lazar G A. et al., PNAS, 2006, 103(11): 4005-4010; Ryan M C. et al., Mol. Cancer Ther., 2007, 6: 3009-3018; Richards See JO,. et al., Mol Cancer Ther. 2008, 7(8): 2517-27; Shields RL et al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J. Biol. Chem, 2003, 278: 3466-3473.

[0152] In certain embodiments, the multispecific antibody or antigen-binding fragment comprises one or more amino acid substitutions that alter complement dependent cytotoxicity (CDC), for example, by increasing or decreasing C1q binding and / or CDC (see, e.g., WO99 / 51642, Duncan & Winter Nature 322:738-40 (1988), U.S. Pat. No. 5,648,260, U.S. Pat. No. 5,624,821, and WO94 / 29351 for other examples of Fc region variants).

[0153] In certain embodiments, multispecific antibodies or antigen-binding fragments thereof comprise one or more amino acid substitutions at the interface of the Fc region to promote and / or enhance heterodimerization. These modifications include the introduction of a protrusion into a first Fc polypeptide and a cavity into a second Fc polypeptide, where the protrusion may be positioned within the cavity to enhance the interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0154] Conjugates In some embodiments, multispecific antibodies and antigen-binding fragments thereof are linked to one or more conjugates, optionally covalently linked directly or via a linker. A conjugate is a non-proteinaceous moiety capable of binding to an antibody or antigen-binding fragment thereof. It is contemplated that a variety of conjugates may be linked to the antibodies or antigen-binding fragments provided herein (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be linked to the multispecific antibodies or antigen-binding fragments by covalent bonding, affinity bonding, intercalation, coordinate bonding, complex formation, association, blending, or addition, among other methods. In some embodiments, the conjugate comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer agent.

[0155] In certain embodiments, the multispecific antibodies and antigen-binding fragments disclosed herein may be engineered to contain designated sites outside the epitope-binding portion that can be utilized for binding to one or more conjugates. For example, such sites can contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent linkage to a conjugate.

[0156] In certain embodiments, the multispecific antibody may be linked indirectly to the conjugate or via another conjugate. For example, the multispecific antibody or antigen-binding fragment may be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate may be a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme substrate label).

[0157] A "toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., cyclohexyl 1-phosphate dehydrogenase), cyclohexyl 1-phosphate dehydrogenase, ... For example, including, without limitation, mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0158] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidases, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y,88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides, luminescent labels), plastid moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0159] In certain embodiments, the conjugate may be a pharmacokinetic-modifying moiety that serves to extend the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and ethylene glycol / propylene glycol copolymers. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when two or more polymers are attached, they may be the same or different molecules.

[0160] In certain embodiments, the conjugate may be a purification moiety such as a magnetic bead.

[0161] In certain embodiments, the multispecific antibodies and / or antigen-binding fragments thereof provided herein are used as substrates for conjugates.

[0162] Polynucleotides and Recombinant Methods The present disclosure provides isolated polynucleotides encoding multispecific antibodies and antigen-binding fragments thereof. DNA encoding monoclonal antibodies is readily isolated and sequenced by conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). The encoding DNA may also be obtained by synthetic methods.

[0163] The isolated polynucleotides encoding multispecific antibodies and antigen-binding fragments thereof may be inserted into vectors for further cloning (amplification of the DNA) or expression by recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0164] In some embodiments, vector systems include mammalian, bacterial, yeast systems, etc., including plasmids and other laboratory and commercially available vectors, such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMD18-T, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0165] Vectors containing polynucleotide sequences encoding antibodies or antigen-binding fragments may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein include the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacilli, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0166] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for multispecific antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are commonly available and useful herein, including Schizosaccharomyces pombe, e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickelamii (ATCC 24,178), Kluyveromyces wartii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), and Kluyveromyces thermotolerans. and Kluyveromyces marxianus; Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa; Schwanniomyces species such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora crassa, Penicillium, and Tolypocladium; and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0167] Suitable host cells for expressing the glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as the armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of the mosquito virus (NPV) and the Bm-5 strain of the silkworm NPV, are publicly available, and such viruses can be used in accordance with the present invention, particularly for transfection of armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0168] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatocellular carcinoma line (Hep G2).

[0169] Host cells are transformed with the expression or cloning vectors for producing the multispecific antibodies described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination, as known in the art.

[0170] The host cells used to produce the multispecific antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. No. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature and pH, will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0171] When using recombinant techniques, polyspecific antibodies may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate cell debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon or MilliporePellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0172] Multispecific antibodies and antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being preferred.

[0173] In certain embodiments, solid-phase-immobilized protein A is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Gusset al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody recovered, other protein purification techniques can be used, such as fractionation on an ion exchange column followed by ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0174] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer of about pH 2.5 to 4.5, preferably at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0175] Pharmaceutical Composition The present disclosure further provides pharmaceutical compositions comprising a multispecific antibody or antigen-binding fragment thereof provided herein and one or more pharmaceutically acceptable carriers.

[0176] Pharmaceutically acceptable carriers for use in pharmaceutical compositions may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or other ingredients known in the art, or various combinations thereof.

[0177] Suitable ingredients herein may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein reduces oxidation of the antibodies or antigen-binding fragments thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided comprising one or more multispecific antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further provided are methods for preventing oxidation of, extending the shelf life of, and / or improving the efficacy of, the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0178] By way of further example, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antibacterial agents used as carriers may be added to pharmaceutical compositions in multi-dose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0179] The pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0180] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for preparing a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and / or non-pyrogenic solutions prepared for injection, sterile dry soluble preparations such as lyophilized powders prepared to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions prepared for injection, sterile dry insoluble preparations prepared to be combined with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0181] In certain embodiments, unit dose parenteral preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration shall be sterile and non-pyrogenic, as known and practiced in the art.

[0182] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-CD276 antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial with a small amount (e.g., about 10%) beyond that required for the dose or set of doses may be acceptable to facilitate accurate sample draws and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0183] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the given selected therapy and can be determined empirically.

[0184] How to use The present disclosure also provides methods of treatment comprising administering a therapeutically effective amount of a multispecific antibody or antigen-binding fragment thereof as provided herein to a subject in need thereof, thereby treating or preventing a CD276-associated disease or condition. In some embodiments, the CD276- and / or PD-L1-associated disease or condition is cancer, an autoimmune disease, an inflammatory disease, an adaptive immune disease, or an infectious disease.

[0185] Examples of cancer include non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal, ductal, and lobular carcinoma), pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), fibrosarcoma, myxosarcoma, and liposarcoma. , chondrosarcoma, osteosarcoma and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphatic tumors, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin's lymphoma (CHL), primary mediastinal large B-cell lymphoma lymphoma, T-cell / histiocytic-rich B-cell lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, HHV8-associated primary inflammatory disease These include, but are not limited to, lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary CNS lymphoma, spinal axis tumors, brain stem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0186] In certain embodiments, the cancer is an adrenal gland tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid globe tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplasia ossificans, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, pancreatic islet tumor, Kaposi's sarcoma, kidney cancer, leukemia, adipose tissue tumor, or leukemia. Lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare blood disorder, metastatic renal cancer, rhabdomyosarcoma-like tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer. In certain embodiments, the cancer is chemotherapy-resistant.

[0187] In certain embodiments, the disease or condition is a hematological cancer selected from B-cell lymphomas, including, but not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma (MZL), mantle cell lymphoma (MCL), Richter's syndrome, Burkitt's lymphoma, or follicular lymphoma.

[0188] Autoimmune diseases include acquired immune deficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac sprue dermatitis herpetiformis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, juvenile dermatomyositis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, and idiopathic pulmonary fibrosis. ITP, IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic joints These include, but are not limited to, rheumatism, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also known as progressive systemic sclerosis (PSS), systemic sclerosis (SS)), Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0189] Inflammatory disorders include, for example, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergies, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy, and ventilator-induced lung injury. In some embodiments, the CD276-associated condition is an inflammatory disease, such as systemic lupus erythematosus (SLE), intestinal mucosal inflammation, colitis-associated wasting disease, multiple sclerosis, viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease and inflammatory bowel disease, psoriasis, systemic sclerosis, autoimmune diabetes, etc.

[0190] Infectious diseases include fungal infections, parasitic / protozoal infections, or chronic viral infections, such as malaria, coccidiodomycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candidiasis albicans, paracoccidioidomycosis, microsporidiosis, acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiomycosis, malaria, Baylissariasis, Chagas disease, Clonorchiasis, Cochliomyiasis, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Clonorchiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenococcosis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Tomentosa myiasis, Myiasis , onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis, helminth infection, hepatitis B (HBV), hepatitis C (HCV), herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papillomavirus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, Kaposi-West sarcoma-associated herpesvirus infection, Schingling virus (Torque teno virus), human T-lymphotrophic virus I, human T-lymphotrophic virus II, varicella-zoster, JC virus, or BK virus infection.

[0191] In certain embodiments, the subject is a human.

[0192] In another aspect, there is provided a method of treating a disease or condition in a subject that benefits from modulation of CD276 activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody, or antigen-binding fragment thereof, as provided herein. In yet another aspect, there is provided a method of treating a disease or condition in a subject that benefits from modulation of PD-1 / PD-L1 pathway activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody, or antigen-binding fragment thereof, as provided herein. As used herein, the term "disease or condition" can be used interchangeably with the term "CD276- and / or PD-L1-associated disease or condition."

[0193] A therapeutically effective amount of an antibody or antigen-binding fragment thereof as provided herein will depend on various factors known in the art, such as the subject's weight, age, past medical history, current drug treatments, health status and potential for cross-reactivity, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of disease development. Doses can be proportionally increased or decreased by one skilled in the art (e.g., a physician or veterinarian) depending on these and other circumstances or requirements.

[0194] In certain embodiments, a multispecific antibody or antigen-binding fragment thereof as provided herein may be administered in a therapeutically effective amount of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain of these embodiments, the antibody or antigen-binding fragment thereof is administered at a dose of about 50 mg / kg or less, and in certain of these embodiments, the dose is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, an initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0195] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0196] The multispecific antibodies and antigen-binding fragments thereof disclosed herein can be administered by any route known in the art, such as a parenteral route (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular or intradermal injection) or a non-parenteral route (e.g., oral, intranasal, intraocular, sublingual, rectal or topical).

[0197] In some embodiments, the multispecific antibodies or antigen-binding fragments thereof disclosed herein can be administered alone or in combination with one or more additional therapeutic procedures or agents. In some embodiments, the multispecific antibodies or antigen-binding fragments thereof disclosed herein can be administered alone or in combination with a second therapeutic agent. For example, the multispecific antibodies or antigen-binding fragments thereof disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent or an anti-cancer agent. In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof disclosed herein can be administered in combination with one or more immunosuppressive molecules, such as antagonists of CD24, CD47, SIRPα, PD-L1, or the beta-2 microglobulin subunit of major histocompatibility complex class I (B2M). The term "antagonist," as used herein, can refer to any small molecule, small molecule, or microRNA, or antibody or antigen-binding fragment thereof, that blocks or inhibits the binding of CD24, CD47, SIRPα, PD-L1, or B2M to its respective binding partner, thereby preventing the induction of immunosuppressive signals.

[0198] In certain of these embodiments, a multispecific antibody or antigen-binding fragment thereof disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents; in certain of these embodiments, the multispecific antibody or antigen-binding fragment thereof and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof that is administered "in combination with" another therapeutic agent need not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof that is administered prior to or after another agent is considered to be administered "in combination with" that agent, as that phrase is used herein, even if the antibody or antigen-binding fragment thereof and the second agent are administered by different routes. When possible, additional therapeutic agents that are administered in combination with an antibody or antigen-binding fragment thereof disclosed herein should be administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to the schedule listed in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57 th Edition; Medical Economics Company; ISBN: 1563634457; 57 th Edition (November 2002)) or according to protocols well known in the art.

[0199] In some embodiments, the present disclosure also provides use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276- and / or PD-L1-associated disease or condition in a subject.

[0200] In another aspect, the present disclosure provides a method of modulating CD276 activity in a CD276-expressing cell, the method comprising exposing the CD276-expressing cell to a multispecific antibody, or antigen-binding fragment thereof, provided herein. In yet another aspect, the present disclosure provides a method of modulating PD-1 / PD-L1 pathway activity in a PD-L1-expressing cell, the method comprising exposing the PD-L1-expressing cell to a multispecific antibody, or antigen-binding fragment thereof, provided herein.

[0201] In another aspect, the present disclosure also provides use of a multispecific antibody, or antigen-binding fragment thereof, provided herein in the manufacture of a medicament for treating a CD276- and / or PD-L1-associated disease or condition in a subject.

[0202] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are within the scope of the invention, in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention, but are merely intended to illustrate specific embodiments within the scope of the invention. Those skilled in the art will be able to develop equivalent compositions, materials, and methods without inventive effort and without departing from the scope of the invention. It will be understood that many variations can be made to the procedures described herein within the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. [Example]

[0203] Example 1 Hybridoma Development 1. method 1.1 Immunization and serum titration 1.1.1 Immunogens and Immunization Strategies Cell immunization CHO-S cells overexpressing human CD276 protein (UniProt ID: Q5ZPR3, i.e., CHO-S-hCD276) or mouse CD276 protein (UniProt ID: Q8VE98, i.e., CHO-S-hCD276) were used as immunogens.

[0204] Protein immunization Recombinant human CD276 protein (SEQ ID NO: 346): Recombinant human CD276 protein was prepared by digesting human CD276 protein with enterokinase, and the extracellular domain of human CD276 was fused with a 6xHis tag and DDDDK (SEQ ID NO: 345).

[0205] Recombinant human CD276 protein (SEQ ID NO: 346) LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAA PYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPE DPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSM TLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEAHHHHHHDDDK.

[0206] Balb / c and SJL mice were immunized as follows: Primary immunization was followed by several boosts until the animals developed sufficient antiserum titers suitable for hybridoma development. [Table A]

[0207] 1.1.2 Immunization schedule [Table B] [Table C] [Table D] [Table E] [Table F]

[0208] 1.1.3 Test blood collection antiserum analysis Screening: Test bleeds were performed and assessed by testing using FACS against CHO-S cell lines stably overexpressing human and / or mouse CD276 (CHO-S-hCD276 and / or CHO-S-mCD276).

[0209] Screening: Test bleeds were performed and evaluated by testing with the extracellular domain of recombinant human CD276 protein using an Elisa.

[0210] 1.2 Hybridoma generation and screening 1.2.1 Cell fusion and screening Fusion: Splenocyte fusion was performed on the mice that responded best to immunization as determined by test bleed FACS. Lymphocytes from the spleen and lymph nodes were fused to the Sp2 / 0 cell line using an optimized electrofusion protocol. Multiple fusions were performed to ensure successful cell fusion.

[0211] Screening and expansion: Fusions were plated in stacked 96-well plates (2 × 10 4 ~10 5Cells were monitored for growth in the plates and fed weekly. Cells were screened in the primary screening assay by FACS and / or other feasible assays, such as ELISA, over a 10-14 day period. Multiple fusions were performed and screened for each target antigen. Positive parental clones that showed positive binding to CHO-S-CD276 and positive ELISA signals from the primary screening were expanded to 24-well plates for secondary screening.

[0212] Additional antibody screening: Following the primary screen, positive parental clones expanded to 24-well plates were screened again by the assay described in the Hybridoma Screening Funnel below.

[0213] Desired hybridomas were selected and subcloning was carried out.

[0214] 1.2.2 Hybridoma subcloning, screening and cryopreservation Subcloning: Parental hybridomas with the desired reactivity and isotype from the above screening funnel were then subcloned by multiple rounds of limiting dilution or single cell sorting until monoclones were obtained.

[0215] Screening and Expansion: Subcloning plates were screened by protein or cell-based ELISA, and subclones with good binding ability were expanded to 24 wells for confirmation testing. The specificity and cross-reactivity of these subclones were confirmed by FACS analysis. Briefly, parental CHO-S cells, CHO-S-hCD276, a CHO-S cell line stably overexpressing cynomolgus monkey CD276, and a CHO-K1 S cell line stably overexpressing mouse CD276 were incubated with antibodies produced by each subclone, respectively. Binding of the primary antibody to the cells was detected using a fluorescent dye-conjugated secondary antibody. The median fluorescence intensity was measured by FACS analysis.

[0216] Cryopreservation: The desired subclonal cell lines were sequenced and further expanded in culture flasks for cryopreservation. 0.5–13.0 × 10 6 Four to six vials per cell line were initially cryopreserved at 1 cell / vial. If necessary, master and working cell banks were established for selected, most valuable cell lines.

[0217] 2. result We discovered 43 antibodies with unique sequences that positively bound to CHO-S cells stably overexpressing human CD276 protein (CHO-S-hCD276) but not to the parent CHO-S cells. These antibodies were suggested to recognize human CD276. Of these, 42 antibodies were able to bind to cynomolgus monkey CD276, and 11 antibodies were able to bind to mouse CD276 protein. The MFIs of mouse antibodies staining CHO-S, CHO-S-hCD276, CHO-S-mCD276, and CHO-S-cynomolgus monkey CD276 (CHO-S-cynoCD276), as detected by FACS, are summarized in the table below (Table 7). [Table 7]

[0218] Example 2 Antibody Characterization: Affinity 1. method 1.1 Cell-based binding affinity to SKOV3 cancer cell line

[0219] The sequences of 31 murine antibodies from Table 5 were selected to engineer and produce human IgG1 chimeric antibodies. The binding affinities of these antibodies and the benchmark antibodies enoblituzumab (see U.S. Pat. No. 8,802,091, specifically MGA271 with a structure designated hBRCA84D-2) and MGC018 (an antibody-drug conjugate in which mAb MGA017 (human IgG1) is conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxybenzamide azaindole (DUBA), an alkylating agent that can damage DNA and thereby cause cell death in both dividing and non-dividing cells) to SKOV3, a human patient-derived ovarian cancer cell line, were determined by FACS analysis.

[0220] The protocol for FACs analysis is described below.

[0221] 1. Cells were digested using trypsin (1X). The collected cells were centrifuged at 300 g for 3 minutes, and the supernatant was discarded.

[0222] 2. Cells were washed twice with FACS buffer by centrifugation at 300 g for 3 min and discarding the supernatant.

[0223] 3. Resuspend the cells and 5 Cells / well were seeded onto assay plates in 50 μl of FACS buffer, followed by the addition of 50 μl of primary antibody (final concentrations of primary antibody: 5.00, 1.67, 0.56, 0.19, 0.06, 0.02, 0.01, 0.00 μg / ml or 20.00, 6.67, 2.22, 0.74, 0.25, 0.08, 0.03, 0.00 μg / ml) and incubation at 4°C for 1 hour.

[0224] 4. Cells were washed twice under the conditions of step 2. Cells were resuspended in 100 μl / well of diluted secondary antibody and incubated in the dark at 4°C for 1 hour.

[0225] 5. Cells were washed twice according to the conditions in step 2. Cells were resuspended in 100 μl / well of FACS buffer. Cells were kept in the dark for FACS analysis.

[0226] The binding affinity of the selected antibodies to SKOV3 is higher, lower, or similar to that of the benchmark antibody enoblituzumab (see Table 8 and Figure 1). [Table 8]

[0227] 2. Protein-based affinity testing with Biacore (ChemPartner) Test conditions Analyte:B7H3 Running buffer: HBS-EP + Flow rate: 30μL / min Capture: Antibody, 10μL / min 60s Injection of serially diluted B7H3 Contact time: 180s Dissociation time: 400 s Regeneration: pH=1.5 Gly, 30μL / min 30s Method: Multiple cycle kinetics / affinity with capture Machine model: Biacore 8K(GE) Analysis temperature: 25℃ [Table 9]

[0228] 3. Expression patterns of B7H3 on several cancer cell lines B7H3 expression was detected by FACS with 6-D8-E7-A11. High expression levels of B7H3 were found on several cancer cell lines, including BxPC3 (pancreatic), MCF7 (breast), Dentroit562 (head and neck), RKO (colon), and SUN620 (gastric) (see Figure 2).

[0229] Example 3 Antibody Characterization: ADCC 1. method To determine the ADCC of anti-CD276 antibodies, 1 x 10 5 2 x 10 SKOV3 / well were then seeded into a 96-well flat-bottom sterile plate. 4 Jurkat-NFAT-luciferase-CD16 cells were added as effector cells. Serially diluted antibodies were then added to each well, and the plates were incubated at 37°C, 5% CO for 18 hours. Finally, luciferase activity was detected to assess the ADCC activity of the antibodies.

[0230] 2. result All of the antibodies of the present invention showed potent ADCC effects against SKOV3 cells (human ovarian cancer cell line). Some of the antibodies showed lower or similar EC2 activity compared to the benchmark antibody enoblituzumab. 50 (Table 10 and Figure 3), demonstrating that it is more potent than enoblituzumab (MGA271) in mediating ADCC effects against SKOV3 cells. [Table 10]

[0231] Example 4 Antibody Characterization: CDC 1. method To determine the CDC of anti-CD276 antibodies, CHO-S-hCD276 cells were resuspended in cell culture medium at 4E5 cells / mL and then added to a 96-well opaque-walled plate at 50 μL / well. Anti-CD276 antibodies were diluted in complete F-12K medium and added to a 96-well opaque-walled plate at 50 μL / well. Human serum complement was diluted in cell culture medium and added to the same plate at 50 μL / well. The mixture was incubated at 37°C in a CO2 incubator for 2 hours. CellTiter-Glo reagent, which is used to determine cytotoxicity, was added at 50 μL / well, and the mixture was incubated at room temperature for 10 minutes. The luminescence signal of live cells was recorded on a microplate reader.

[0232] 2. result All anti-CD276 antibodies showed potent CDC effects on CHO-S-hCD276 cells, with lower EC2s compared to the benchmark antibody enoblituzumab (MGA271). 50 The results are shown in Table 10 and Figure 4. [Table 11]

[0233] Example 5 Antibody Characterization: Indirect ADC Cytotoxicity 1. method Fab-ZAP is a chemical conjugate of a goat anti-human monovalent antibody (secondary antibody) and saporin, a ribosome-inactivating protein. Fab-ZAP is used to determine the internalization capacity of antibodies. In this assay, 80 μL of SKOV-3 cells were plated at 2000 cells / well in a 96-well plate and incubated overnight at 37°C. Anti-CD276 antibody was then added at 40 μL / well. Fab-ZAP human dilution was added at 40 μL / well and incubated at 37°C in a CO2 incubator for 96 hours. CellTiter-Glo reagent, used to determine cell cytotoxicity, was added at 100 μL / well and incubated at room temperature for 10 minutes. The luminescence signal of live cells was recorded on a microplate reader.

[0234] 2. result All anti-CD276 antibodies had lower or similar IC compared to the benchmark antibody MGC018. 50 showed potent indirect ADC effects on SKOV3 cells (Table 12, Figure 5), indicating that it is a potential candidate for generating ADCs. [Table 12]

[0235] Example 6 Antibody In Vivo Efficacy in Treating Subcutaneous MC-38-hCD276 Murine Colon Carcinoma in Female C57BL / 6 Mice 1. Study Design and Methods Several monoclonal antibodies with mouse IgG2a Fc were constructed and expressed for in vivo efficacy testing. Bispecific antibodies using mouse IgG2a B7H3 mAb and PD-L1 mAb (SHANGHAI ORIGINCELL MEDICAL TECHNOLOGY CO., LTD. See WO2019196309A1, YN035) were also constructed in the IgG(H)scFV format. The N-terminus of the anti-PD-L1 scFv VH followed by (GGGGS)3-VL was linked to the C-terminus of the Fc region of the B7H3 antibody. For the Bis-25-C8-D7-C5 reverse antibody, the N-terminus of the anti-B7H3 scFv VH followed by (GGGGS)3-VL was linked to the C-terminus of the Fc region of the PD-L1 antibody.

[0236] Briefly, the bispecific antibodies were named according to the following pattern:

[0237] Taking bispecific antibodies based on the CD276 antibody 25-C8-D7-C5 and PD-L1 (i.e., YN035) as an example, in the Bis-25-C8-D7-C5 antibody, the CD276-binding domain (i.e., the binding domain derived from 25-C8-D7-C5) is the first binding domain and the PD-L1-binding domain is the second binding domain, while in the Bis-25-C8-D7-C5-reverse antibody, the CD276-binding domain (i.e., the binding domain derived from 25-C8-D7-C5) is the second binding domain and the PD-L1-binding domain is the first binding domain.

[0238] Heavy chain variable region amino acid sequence of YN035 (CDR regions are underlined) QVQLVQSGAEVKKPGSSVKVSCKASRGPFS TYAIS WVRQAPGQGLEWMG RIIPILGIANYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR TMEGYGFGNFDY WGQGTLVTVSS (SEQ ID NO: 382)

[0239] Light chain variable region amino acid sequence of YN035 (CDR regions are underlined) QSALTQPASVSGSPGQSITISC TGTVSEVGGYNEVS WYQQHPGKAPKLMIY GNSNRPS GVSNRFSGSKSSGNTASLTISGLQAEDEADYYC QSYDSSLSGTV FGGGTKLTVLG (SEQ ID NO: 383)

[0240] 1. MC-38-hCD276 (B7H3) tumor cells were maintained in vitro in DMEM medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Cells in the logarithmic growth phase were harvested and quantified using a cell counter before tumor inoculation.

[0241] 2. Each mouse was inoculated subcutaneously in the right hind flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 10^6) in 0.1 ml of PBS for tumor development. The day of randomization was designated as day 0, and treatment began on day 0.

[0242] 3. The average tumor size is approximately 50-60 mm 3 Once the NIH score reached 1, randomization began. Sixty mice were enrolled in this study. All animals were randomly assigned to seven study groups. Randomization was performed based on a randomized block design.

[0243] 4. Measure tumor volume twice weekly in two dimensions with a vernier caliper and calculate the volume in mm using the formula V = (L × W × W) / 2. 3 where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Dosing and measurements of tumor size and body weight were performed in a laminar flow cabinet (see Table 13 and Figure 6).

[0244] 5. Atezolizumab (an anti-PD-L1 antibody also known as Tecentriq or MPDL3280A; see WO2010077634A1) and Antengene-084M (the Fab sequence of MGA271 constructed with murine IgG2a (see U.S. Patent No. 8,802,091B)) were used as controls. [Table 13]

[0245] 2. result As shown in Figure 6, some anti-CD276 antibodies demonstrated potent in vivo efficacy. In particular, 25-C8-D7-C5 mouse IgG2a and Bis-6-D8-E7-A11-reverse mouse IgG2a exhibited higher tumor inhibition than Antengene-084M mouse IgG2a.

[0246] Example 7 Antibody ADC In Vivo Efficacy Testing 1. Study Design and Methods To evaluate the ADC potential of the antibodies, conjugation of the antibodies with VC-MMAE was performed at ChemPartner. A series of VC-MMAE conjugated antibodies was obtained, details of which are shown in Table 14. [Table 14]

[0247] For the Calu-6 test, Balb / c nude mice were inoculated with Calu-6 tumor cells (5 × 10 cells) in 0.1 ml of PBS in the right anterior flank region for tumor development. 6 ) was subcutaneously inoculated. The tumor size was approximately 122 mm 3 Randomization begins when the NIH score reaches 0. Sixty mice were enrolled in this study. The day of randomization is designated as day 0, and dosing begins on day 0.

[0248] 2. result This study investigated the therapeutic efficacy of the test articles in treating subcutaneous human lung cancer Calu-6 in Balb / c nude mice. No obvious weight loss, mortality, or toxic reactions were observed with the designed dosing regimen during the efficacy study. On day 24, MGC018, 10-G6-C4-B2, 16-C6-F7-F5, 15-C8-B5-G7, and 18-F9-D8-G7 at 3 mg / kg as single agents each showed significant antitumor efficacy in the Calu-6 model in Balb / c nude mice. The results are shown in Figure 7 and Table 15. [Table 15]

[0249] Example 8 Antibody T cell activation (MLR assay) test 1. Study design and methods dc induction Monocyte cells were cultured at 5 × 10 in 3 ml of complete medium supplemented with 2 U / ml of dendritic cell culture factors. 6 The monocytes were resuspended in PBS and then cultured in 6-well plates. On day 2, 2 ml / well of fresh complete medium supplemented with 2 U / ml of dendritic cell culture factors was added for an additional 3 days of culture. The monocytes were then differentiated into immature dendritic cells (iDCs). After 48 hours of stimulation with 2 U / ml of dendritic cell maturation factors, the iDCs differentiated into mature dendritic cells.

[0250] Antibody digestion 25 μg of hIgG1 was mixed with 3 μl of 10x Glyco Buffer, and then PBS was added to bring the total reaction volume to 30 μl. 1 μl of IdeZ protease was added, followed by incubation at 37°C for 30 minutes. 10 μl of Protein A / G beads were added to 1 mL of PBS and washed twice. The beads were resuspended in the reaction solution from the previous step.

[0251] The tube was placed on a magnet at room temperature for 1 minute, with the F(ab)2 in the supernatant and the Fc captured by the beads.

[0252] MLR assay T cells were plated at 200,000 cells / well and mDCs at 20,000 cells / well.

[0253] Cells were treated with PBS, IgG1, 25-C8-D7-C5, 30-C7-C11-D4, BMK (MGA271), or their Fabs (final concentration 5 μg / ml) for 24 hours. Supernatants were collected and tested for human IL-2 and IFNγ by ELISA.

[0254] 2. result As shown in Figures 8 and 9, 30-C7-C11-D4 can stimulate T cell activation, but 25-C8-D7-C5 and BMK (MGA271) cannot.

[0255] Example 9 Antibody Humanization and PTM Optimization 9.1 Cell-based affinity testing of humanized antibodies by FACS 10-G6-C4-B2 and 30-C7-C11-D4 were selected for humanization and PTM optimization. The affinity of the humanized candidates and optimized PTM sequences was evaluated by FACS.

[0256] The results are shown in Figures 10 and 11. All sequences showed affinities comparable to the parent antibody.

[0257] 9.2 Protein-based affinity testing with Biacore (ChemPartner) Test conditions Analyte: B7H3. Running buffer: HBS-EP+. Flow rate: 30 μL / min. Capture: antibody, 10 μL / min for 60 s. Injection of serially diluted B7H3. Contact time: 180 s, dissociation time: 400 s. Regeneration: pH=1.5 Gly, 30 μL / min for 30 s. Method: Multiple cycle kinetic / affinity with capture. Machine model: Biacore 8K (GE). Analysis temperature: 25°C.

[0258] The results are shown in Table 16. [Table 16]

[0259] 9.3 In vivo ADC therapeutic potential testing of the humanized 10-G6-C4-B2 antibody The in vivo ADC therapeutic potential of the humanized 10-G6-C4-B2 antibody was evaluated. Antibody conjugates with VC-MMAE were constructed at ChemPartner. A series of VC-MMAE conjugated antibodies were obtained, the details of which are shown in Table 17. [Table 17]

[0260] For the Calu-6 test, each Balb / c nude mouse was inoculated into the right anterior flank region with Calu-6 tumor cells (5 × 10 cells) in 0.1 ml of PBS for tumor development. 6 ) was subcutaneously inoculated. The tumor size was approximately 122 mm 3 Randomization begins when the NIH score reaches 0. Sixty mice were enrolled in this study. The day of randomization is designated as day 0, and dosing begins on day 0.

[0261] The therapeutic efficacy of the test article in treating subcutaneous human lung cancer Calu-6 in Balb / c nude mice was investigated in this study. No obvious weight loss, mortality, or toxic reactions were observed with the designed dosing regimen during the efficacy study.

[0262] The results are shown in Figure 12 and Table 18. At day 26, nearly all of the test articles at 3 mg / kg as single agents each show significant anti-tumor efficacy against the Calu-6 model in Balb / c nude mice. [Table 18]

[0263] Example 10 In vivo efficacy of bispecific antibodies The right hind flank region of each mouse (C57BL / 6) was inoculated with MC-38-hCD276 (B7H3) tumor cells (1 × 10) in 0.1 ml of PBS for tumor development. 6 ) was subcutaneously inoculated. The tumor size was approximately 139 mm 3 Once the NIH reaches 0, randomization begins. 66 mice were enrolled in this study. The day of randomization is designated as day 0, and dosing begins on the day of randomization (day 0).

[0264] As shown in Table 19 and Figure 13, on day 19, Bis-30-C7-C11-D4 at 3 mg / kg and 25-C8-D7-C5-igG2a at 2.25 mg / kg mixed with YN035-IgG2a at 2.25 mg / kg each show significant antitumor efficacy against MC-38-hCD276 (B7H3) murine colon carcinoma in C57BL / 6 mice. [Table 19]

[0265] The right hind flank region of each mouse (C57BL / 6) was inoculated with MC-38-hCD276 (B7H3) tumor cells (1 × 10) in 0.1 ml of PBS for tumor development. 6 ) was subcutaneously inoculated. The tumor size was approximately 100 mm 3 Randomization begins when the NIH score reaches 0. Thirty mice were enrolled in this study. The day of randomization is designated as day 0, and dosing begins on the day of randomization (day 0).

[0266] As shown in Table 20 and FIG. 14, at day 19, Bis-25-C8-D7-C5 at 3 mg / kg and Bis-30-C7-C11-D4 at 3 mg / kg as single agents each demonstrated significant antitumor efficacy against MC-38-hCD276 (B7H3) murine colon carcinoma in C57BL / 6 mice. [Table 20]

[0267] Example 11 In vivo efficacy of bispecific antibodies incorporating PTM optimization A humanized antibody incorporating PTM optimization was constructed and expressed for in vivo efficacy. A bispecific antibody using the B7H3 mAb 30-C7-C11-D4 bearing human IgG1 and a PD-L1 mAb (SHANGHAI ORIGINCELL MEDICAL TECHNOLOGY CO., LTD. See WO2019196309A1, YN035) was also constructed in the IgG(H)scFv format. The N-terminus of the anti-PD-L1 scFv VH followed by (GGGGS)3-VL was linked to the C-terminus of the Fc region of the B7H3-antibody.

[0268] The right hind flank region of each mouse (C57BL / 6) was inoculated with MC-38-hCD276 (B7H3) tumor cells (1 × 10) in 0.1 ml of PBS for tumor development. 6 ) was subcutaneously inoculated. The tumor size was approximately 100 mm 3 Once the NIH reaches 0, randomization begins. 54 mice were enrolled in this study. The day of randomization is designated as day 0, and dosing begins on the day of randomization (day 0).

[0269] As shown in Table 21 and Figure 15, Bis-30-C7-C11-D4_hVH3-hVL2-PTM and Bis-30-C7-C11-D4_hVH4-hVL5-PTM showed comparable tumor growth inhibition to the parental Bis-30-C7-C11-D4. [Table 21]

Claims

1. A multispecific antibody or antigen-binding fragment thereof comprising a first binding domain and a second binding domain, wherein the first binding domain specifically binds to CD276 and the second binding domain specifically binds to a second target other than CD276.

2. The first binding domain is selected from the group consisting of SEQ ID NOs: 1 to 3, 9 to 11, 17 to 19, 25 to 27, 33 to 35, 41 to 43, 49 to 51, 57 to 59, 65 to 67, 73 to 75, 81 to 83, 89 to 91, 97 to 99, 105 to 107, 113 to 115, 121 to 123, 129 to 131, 137 to 139, 145 to 147, 153 to 155, 161 to 163, 169 to 171, 177 to 179, 185 to 187, 193 to 195, 201 to 202 3, 209-211, 217-219, 225-227, 233-235, 241-243, 249-251, 257-259, 265-267, 273-275, 281-283, 289-291, 297-299, 305-307, 313-315, 321-323, 329-331, 337-339, and 374-375, and / or No. 4-6, 12-14, 20-22, 28-30, 36-38, 44-46, 52-54, 60-62, 68-70, 76-78, 84-86, 92-94, 100-102, 108-110, 116-118, 1 24-126, 132-134, 140-142, 148-150, 156-158, 164-166, 172-174, 180-181, 188-190, 196-198, 204-206, 212-214, 22 2. The multispecific antibody or antigen-binding fragment thereof of claim 1, comprising one, two, or three light chain CDR sequences selected from the group consisting of: 0-222, 228-230, 236-238, 244-246, 252-254, 260-262, 268-270, 276-278, 284-286, 292-294, 300-302, 308-310, 316-318, 324-326, 332-334, 340-342, and 376-377.

3. The first binding domain comprises: a) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 1-3; b) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 9-11; c) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 17-19; d) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 25-27; and e) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 33-35; f) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 41-43; and g) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 49-51; h) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 57-59; i) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 65-67; j) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 73-75; k) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 81-83; l) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 89-91; m) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 97-99; n) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 105-107; o) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 113-115; p) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 121-123; q) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 129-131; r) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 137-139; s) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 145-147; t) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 153-155; u) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 161-163; v) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 169-171; w) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 177-179; x) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 185-187; y) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 193-195; z) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 201-203; aa) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 209-211; bb) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 217-219; cc) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 225-227; dd) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 233-235; ee) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 241-243; ff) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 249-251; gg) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 257-259; hh) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 265-267; ii) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 273-275; and jj) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 281-283; kk) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 289-291; ll) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 297-299; mm) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 305-307; nn) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 313-315; oo) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 321-323; pp) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 329-331; qq) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 337-339; rr) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 9, 374, and 375; 2. The multispecific antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region selected from the group consisting of:

4. The first binding domain comprises: a) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 4-6; b) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 12-14; and c) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 20-22; and d) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 28-30; and e) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 36-38; and f) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 44-46; and g) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 52-54; h) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 60-62; and i) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 68-70; j) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 76-78; k) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 84-86; l) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 92-94; and m) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 100-102; n) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 108-110; o) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 116-118; p) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 124-126; q) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 132-134; r) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 140-142; s) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 148-150; t) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 156-158; u) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 164-166; v) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 172-174; and w) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 180-181; x) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 188-190; and y) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 196-198; z) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 204-206; and aa) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 212-214; bb) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 220-222; cc) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 228-230; and dd) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 236-238; ee) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 244-246; ff) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 252-254; gg) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 260-262; hh) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 268-270; ii) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 276-278; and jj) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 284-286; kk) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 292-294; ll) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 300-302; mm) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 308-310; nn) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 316-318; oo) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 324-326; pp) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 332-334; qq) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 340-342; rr) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 376, 13, and 14; ss) a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NOs: 377, 45, and 46; 10. The multispecific antibody or antigen-binding fragment thereof of claim 1, comprising a light chain variable region selected from the group consisting of:

5. The first binding domain comprises: a) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; b) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; c) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; d) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; e) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38; f) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46; g) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54; h) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:62; i) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70; j) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; k) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86; l) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:91, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:92, SEQ ID NO:93, and SEQ ID NO:94; m) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102; n) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110; o) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118; p) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126; q) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 134; r) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142; s) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150; t) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 153, SEQ ID NO: 154, and SEQ ID NO: 155, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158; u) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166; v) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174; w) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 179, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 180, SEQ ID NO: 181, and SEQ ID NO: 182; x) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 187, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 189, and SEQ ID NO: 190; y) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 198; z) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:201, SEQ ID NO:202, and SEQ ID NO:203, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:204, SEQ ID NO:205, and SEQ ID NO:206; aa) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 209, SEQ ID NO: 210, and SEQ ID NO: 211, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 214; bb) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:217, SEQ ID NO:218, and SEQ ID NO:219, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:220, SEQ ID NO:221, and SEQ ID NO:222; cc) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:225, SEQ ID NO:226, and SEQ ID NO:227, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:228, SEQ ID NO:229, and SEQ ID NO:230; dd) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 236, SEQ ID NO: 237, and SEQ ID NO: 238; ee) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:241, SEQ ID NO:242, and SEQ ID NO:243, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:244, SEQ ID NO:245, and SEQ ID NO:246; ff) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 249, SEQ ID NO: 250, and SEQ ID NO: 251, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 252, SEQ ID NO: 253, and SEQ ID NO: 254; gg) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:257, SEQ ID NO:258, and SEQ ID NO:259, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:260, SEQ ID NO:261, and SEQ ID NO:262; hh) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:265, SEQ ID NO:266, and SEQ ID NO:267, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:268, SEQ ID NO:269, and SEQ ID NO:270; ii) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:273, SEQ ID NO:274, and SEQ ID NO:275, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:276, SEQ ID NO:277, and SEQ ID NO:278; jj) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:281, SEQ ID NO:282, and SEQ ID NO:283, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:284, SEQ ID NO:285, and SEQ ID NO:286; kk) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:289, SEQ ID NO:290, and SEQ ID NO:291, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:292, SEQ ID NO:293, and SEQ ID NO:294; ll) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:297, SEQ ID NO:298, and SEQ ID NO:299, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:300, SEQ ID NO:301, and SEQ ID NO:302; mm) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 305, SEQ ID NO: 306, and SEQ ID NO: 307, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310; nn) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 313, SEQ ID NO: 314, and SEQ ID NO: 315, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 316, SEQ ID NO: 317, and SEQ ID NO: 318; oo) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 321, SEQ ID NO: 322, and SEQ ID NO: 323, and a light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 324, SEQ ID NO: 325, and SEQ ID NO: 326; pp) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 329, SEQ ID NO: 330, and SEQ ID NO: 331, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO: 332, SEQ ID NO: 333, and SEQ ID NO: 334; qq) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:337, SEQ ID NO:338, and SEQ ID NO:339, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:340, SEQ ID NO:341, and SEQ ID NO:342; rr) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:9, SEQ ID NO:374, and SEQ ID NO:375, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:376, SEQ ID NO:13, and SEQ ID NO:14; or ss) a heavy chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43, and a kappa light chain variable region comprising one, two, or three CDR sequences selected from SEQ ID NO:377, SEQ ID NO:45, and SEQ ID NO:46; 10. The multispecific antibody or antigen-binding fragment thereof of claim 1 , comprising:

6. The first binding domain is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:119, SEQ ID NO:127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:199, SEQ ID NO:207, SEQ ID NO:215, SEQ ID NO:22 3. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a heavy chain variable region selected from the group consisting of SEQ ID NO:3, SEQ ID NO:231, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:255, SEQ ID NO:263, SEQ ID NO:271, SEQ ID NO:279, SEQ ID NO:287, SEQ ID NO:295, SEQ ID NO:303, SEQ ID NO:311, SEQ ID NO:319, SEQ ID NO:327, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:347, and SEQ ID NO:349 and homologous sequences with at least 80% sequence identity thereof.

7. The first binding domain is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:104, SEQ ID NO:112, SEQ ID NO:120, SEQ ID NO:128, SEQ ID NO:136, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:1756, SEQ ID NO:184, SEQ ID NO:192, SEQ ID NO:200, SEQ ID NO:208, SEQ ID NO:216, SEQ ID NO:22 4, SEQ ID NO:232, SEQ ID NO:240, SEQ ID NO:248, SEQ ID NO:256, SEQ ID NO:264, SEQ ID NO:272, SEQ ID NO:280, SEQ ID NO:288, SEQ ID NO:296, SEQ ID NO:304, SEQ ID NO:312, SEQ ID NO:320, SEQ ID NO:328, SEQ ID NO:336, SEQ ID NO:344, SEQ ID NO:348, and SEQ ID NO:350 and homologous sequences with at least 80% sequence identity thereof.

8. The first binding domain is selected from the group consisting of SEQ ID NO:378 (EVQLVESGGGLXQPGXSLRLSCXTSGFTLSDYYMSWVRQXPGKGLEWVXFMRNKANXYTTEYSASVRGRFTISRDTSKSXIYLQMNSLXXEDTAVYYCVRDRXGRPFAYWGQGTLVTVSS), wherein X at position i (i=12, 16, 23, 40, 49, 57, 80, 89, 90 and 103) of SEQ ID NO:378 is Ｈｉ It is called X Ｈ１２ is V or I, and X Ｈ１６ is G or R, and X Ｈ２３ is A or T, and X Ｈ４０ is A or P, and X Ｈ４９ is G or S, and X Ｈ５７ is A or G, and X Ｈ８０ is I or T, and X Ｈ８９ is R or K, and X Ｈ９０ is A or T, and X Ｈ１０３ is D or E; and SEQ ID NO:379 (DIXMTQSPXSLXXXGXXXXIXCKSSQSLLNXINQKNFLTWYXQKPGXXPXLLIYWASTRESGVPXRFSGSGSGTDFTLXISXXXXEDLXXYYCQNDYTYPLTFGQGTKLEIK), wherein the X at position i (i=3, 9, 12, 13, 14, 15, 17, 18, 19, 20, 22, 32, 43, 48, 49, 51, 66, 80, 83, 84, 85, 86, 90, and 91) of SEQ ID NO:379 is X Ｌｉ It is called X Ｌ３ is V or Q, and X Ｌ９ is D, L or S, and X Ｌ１２ is A, S or P, and X Ｌ１３ is A or V, and X Ｌ１４ is S or T, and X Ｌ１５ is L, V or P, and X Ｌ１７ is D or E, and X Ｌ１８ is R or P, and X Ｌ１９ is A or V, and X Ｌ２０ is S or T, and X Ｌ２２ is N, T or S, and X Ｌ３２ is A or S, and X Ｌ４３ is Q or L, and X Ｌ４８ is Q or K, and X Ｌ４９ is A, P or S, and X Ｌ５１ is K or Q, and X Ｌ６６ is S or D, and X Ｌ８０ is K or T, and X Ｌ８３ is R or S, and X Ｌ８４ is L or V, and X Ｌ８５ is Q or E, and X Ｌ８６ is A or P, and X Ｌ９０ is A or G, and X Ｌ９１ and a light chain variable region comprising:

9. The first binding domain has the structure of SEQ ID NO:380 (QVQLQESGPGLVKPSXTLSLTCXVXGYSITSDYAWNWIRQXPGKGLEWIGYISHSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSLGRRWYFDVWGQGTTVTVSS), wherein X at position i (i=16, 23, 25 and 41) of SEQ ID NO:380 is Ｈｉ It is called X Ｈ１６ is E or Q, and X Ｈ２３ is A or T, and X Ｈ２５ is S or Y, and X Ｈ４１ is H or P; and SEQ ID NO:381 (DIXMTQSPXSLXXXGXXXXIXCKSSQSLLXSSTQKNYLAWYXQKPGXXPXLLIYFASTRDSGVPXRFSGSGSGTDFTLXISXXXXEDLXXYFCQQHYIIPFFTFGQGTKLEIK), wherein the X at position i (i=3, 9, 12, 13, 14, 15, 17, 18, 19, 20, 22, 31, 43, 48, 49, 51, 66, 80, 83, 84, 85, 86, 90, and 91) of SEQ ID NO:381 is X Ｌｉ It is called X Ｌ３ is V or Q, X Ｌ９ D, L or S, X Ｌ１２ is A, S or P, X Ｌ１３ is A or V, X Ｌ１４ is S or T, X Ｌ１５ is L, V or P, X Ｌ１７ Is D or E, X Ｌ１８ is R or P, X Ｌ１９ is A or V, X Ｌ２０ is S or T, X Ｌ２２ is N, T or S, X Ｌ３１ is N or Q, X Ｌ４３ is Q or L, X Ｌ４８ is Q or K, X Ｌ４９ A, P or S, X Ｌ５１ is K or Q, X Ｌ６６ is S or D, X Ｌ８０ is K or T, X Ｌ８３ is R or S, X Ｌ８４ is L or V, X Ｌ８５ Is Q or E, X Ｌ８６ is A or P, X Ｌ９０ A or G, X Ｌ９１ and a light chain variable region comprising:

10. The first binding domain comprises: (i) a heavy chain variable region comprising one, two, or three CDR sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10, or 374 (MRNKANAYTT) and SEQ ID NO: 11 or 375 (VRDREGRPFAY), respectively, and a kappa light chain variable region comprising one, two, or three CDR sequences set forth in SEQ ID NO: 12 or 376 (QSLLNAINQKNF), SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or (ii) a heavy chain variable region comprising one, two, or three CDR sequences set forth in SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively, and a kappa light chain variable region comprising one, two, or three CDR sequences set forth in SEQ ID NO: 44 or 377 (QSLLQSSTQKNY), SEQ ID NO: 45, and SEQ ID NO: 46, respectively; 10. The multispecific antibody or antigen-binding fragment thereof of claim 9, comprising:

11. 10. The multispecific antibody or antigen-binding fragment thereof of claim 9, wherein the first binding domain comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 351, 353, 355, 357, 358, 360, 362, 364, 365, 367, and 370, and a light chain variable region comprising a sequence selected from SEQ ID NOs: 352, 354, 356, 359, 361, 363, 366, 368, 369, 371, 372, and 373.

12. The first binding domain comprises: a) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8; b) a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 16; c) a heavy chain variable region comprising SEQ ID NO: 23 and a light chain variable region comprising SEQ ID NO: 24; d) a heavy chain variable region comprising SEQ ID NO: 31 and a light chain variable region comprising SEQ ID NO: 32; e) a heavy chain variable region comprising SEQ ID NO: 39 and a light chain variable region comprising SEQ ID NO: 40; f) a heavy chain variable region comprising SEQ ID NO: 47 and a light chain variable region comprising SEQ ID NO: 48; g) a heavy chain variable region comprising SEQ ID NO: 55 and a light chain variable region comprising SEQ ID NO: 56; h) a heavy chain variable region comprising SEQ ID NO: 63 and a light chain variable region comprising SEQ ID NO: 64; i) a heavy chain variable region comprising SEQ ID NO: 71 and a light chain variable region comprising SEQ ID NO: 72; j) a heavy chain variable region comprising SEQ ID NO: 79 and a light chain variable region comprising SEQ ID NO: 80; k) a heavy chain variable region comprising SEQ ID NO: 87 and a light chain variable region comprising SEQ ID NO: 88; l) a heavy chain variable region comprising SEQ ID NO: 95 and a light chain variable region comprising SEQ ID NO: 96; m) a heavy chain variable region comprising SEQ ID NO: 103 and a light chain variable region comprising SEQ ID NO: 104; n) a heavy chain variable region comprising SEQ ID NO: 111 and a light chain variable region comprising SEQ ID NO: 112; o) a heavy chain variable region comprising SEQ ID NO: 119 and a light chain variable region comprising SEQ ID NO: 120; p) a heavy chain variable region comprising SEQ ID NO: 127 and a light chain variable region comprising SEQ ID NO: 128; q) a heavy chain variable region comprising SEQ ID NO: 135 and a light chain variable region comprising SEQ ID NO: 136; r) a heavy chain variable region comprising SEQ ID NO: 143 and a light chain variable region comprising SEQ ID NO: 144; s) a heavy chain variable region comprising SEQ ID NO: 151 and a light chain variable region comprising SEQ ID NO: 152; t) a heavy chain variable region comprising SEQ ID NO: 159 and a light chain variable region comprising SEQ ID NO: 160; u) a heavy chain variable region comprising SEQ ID NO: 167 and a light chain variable region comprising SEQ ID NO: 168; v) a heavy chain variable region comprising SEQ ID NO: 175 and a light chain variable region comprising SEQ ID NO: 176; w) a heavy chain variable region comprising SEQ ID NO: 183 and a light chain variable region comprising SEQ ID NO: 184; x) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 192; y) a heavy chain variable region comprising SEQ ID NO: 199 and a light chain variable region comprising SEQ ID NO: 200; z) a heavy chain variable region comprising SEQ ID NO: 207 and a light chain variable region comprising SEQ ID NO: 208; aa) a heavy chain variable region comprising SEQ ID NO: 215 and a light chain variable region comprising SEQ ID NO: 216; bb) a heavy chain variable region comprising SEQ ID NO: 223 and a light chain variable region comprising SEQ ID NO: 224; cc) a heavy chain variable region comprising SEQ ID NO: 231 and a light chain variable region comprising SEQ ID NO: 232; dd) a heavy chain variable region comprising SEQ ID NO: 239 and a light chain variable region comprising SEQ ID NO: 240; ee) a heavy chain variable region comprising SEQ ID NO: 247 and a light chain variable region comprising SEQ ID NO: 248; ff) a heavy chain variable region comprising SEQ ID NO: 255 and a light chain variable region comprising SEQ ID NO: 256; gg) a heavy chain variable region comprising SEQ ID NO: 263 and a light chain variable region comprising SEQ ID NO: 264; hh) a heavy chain variable region comprising SEQ ID NO: 271 and a light chain variable region comprising SEQ ID NO: 272; ii) a heavy chain variable region comprising SEQ ID NO: 279 and a light chain variable region comprising SEQ ID NO: 280; jj) a heavy chain variable region comprising SEQ ID NO: 287 and a light chain variable region comprising SEQ ID NO: 288; kk) a heavy chain variable region comprising SEQ ID NO: 295 and a light chain variable region comprising SEQ ID NO: 296; ll) a heavy chain variable region comprising SEQ ID NO: 303 and a light chain variable region comprising SEQ ID NO: 304; mm) a heavy chain variable region comprising SEQ ID NO: 311 and a light chain variable region comprising SEQ ID NO: 312; nn) a heavy chain variable region comprising SEQ ID NO: 319 and a light chain variable region comprising SEQ ID NO: 320; oo) a heavy chain variable region comprising SEQ ID NO: 327 and a light chain variable region comprising SEQ ID NO: 328; pp) a heavy chain variable region comprising SEQ ID NO: 335 and a light chain variable region comprising SEQ ID NO: 336; qq) a heavy chain variable region comprising SEQ ID NO: 343 and a light chain variable region comprising SEQ ID NO: 344; rr) a heavy chain variable region comprising SEQ ID NO: 347 and a light chain variable region comprising SEQ ID NO: 348; or ss) a heavy chain variable region comprising SEQ ID NO: 349 and a light chain variable region comprising SEQ ID NO: 350; 10. The multispecific antibody or antigen-binding fragment thereof of claim 1 , comprising:

13. 10. The multispecific antibody or antigen-binding fragment thereof of claim 1 , wherein the first binding domain further comprises one or more amino acid residue substitutions or modifications while retaining specific binding affinity to human CD276.

14. 14. The multispecific antibody or antigen-binding fragment thereof of claim 13, wherein the substitutions are in one or more CDR sequences and / or in one or more of the VH or VL sequences but not in any of the CDR sequences.

15. 10. The multispecific antibody or antigen-binding fragment thereof of claim 1, further comprising an activating receptor binding domain, optionally wherein the activating receptor binding domain is a human Ig constant region or a human IgG constant region, and optionally wherein the first binding domain is an intact antibody.

16. 16. The multispecific antibody or antigen-binding fragment thereof of claim 15, wherein the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

17. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the second target is PD-L1.

18. 3. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the second binding domain comprises one, two, or three heavy chain complementarity determining region (CDR) sequences selected from the group consisting of SEQ ID NOs: 384 to 386 and / or one, two, or three light chain CDR sequences selected from the group consisting of SEQ ID NOs: 387 to 389.

19. 3. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable region comprising the three CDR sequences set forth in SEQ ID NOs: 384, 385 and 386, respectively, and a light chain variable region comprising the three CDR sequences set forth in SEQ ID NOs: 387, 388 and 389, respectively.

20. 3. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable region set forth in SEQ ID NO: 382 and / or a light chain variable region set forth in SEQ ID NO:

383.

21. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the second binding domain further comprises one or more amino acid residue substitutions or modifications while retaining specific binding affinity to human PD-L1.

22. 17. The multispecific antibody or antigen-binding fragment thereof of claim 16, wherein the substitutions are in one or more CDR sequences and / or in one or more of the VH or VL sequences but not in any of the CDR sequences.

23. The multispecific antibody or antigen-binding fragment thereof of claim 16, wherein the second binding domain comprises a VH-linker-VL scFv structure.

24. The linker is (GGGGS) ３ 390. The multispecific antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising the sequence: (SEQ ID NO: 390).

25. 17. The multispecific antibody or antigen-binding fragment thereof of claim 16, wherein the N-terminus of the second binding domain is operably linked to the C-terminus of the activating receptor binding domain.

26. 10. The multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a bispecific or trispecific antibody.

27. 10. The multispecific antibody or antigen-binding fragment thereof of any one of the preceding claims, linked to one or more conjugates, optionally covalently bound directly or via a linker.

28. 26. The multispecific antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or another anti-cancer agent.

29. 10. A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier.

30. An isolated polynucleotide encoding the multispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 27.

31. 32. A vector comprising the isolated polynucleotide of claim 31.

32. A host cell comprising the vector of claim 32.

33. 38. A method for expressing the multispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 27, comprising culturing the host cell of claim 33 under conditions in which the vector of claim 32 is expressed.

34. 31. A method of treating a disease or condition in a subject that is benefited from modulation of CD276 activity and / or PD-L1, comprising administering to said subject a therapeutically effective amount of the multispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 27 or the pharmaceutical composition of any one of claims 28 to 30.

35. The method of claim 35, wherein the disease or condition is a CD276- and / or PD-L1-associated disease or condition.

36. 36. The method of claim 35, wherein the disease or condition is cancer, an adaptive immune disorder, an autoimmune disease, an inflammatory disease, or an infectious disease.

37. The cancers include adrenal gland tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumors, breast cancer, carotid globe tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplasia ossificans, fibrous dysplasia, gallbladder or bile duct cancer, gastric cancer cancer), gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, pancreatic islet tumors, Kaposi's sarcoma, kidney cancer, leukemia, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumors, childhood cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare blood disorders, metastatic kidney cancer, rhabdomyosarcoma-like tumors, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer (stomach cancer) 38. The method of claim 37, wherein the cancer is selected from the group consisting of thyroid cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer, and optionally, the cancer is chemotherapy-resistant.

38. 38. The method of claim 37, wherein the disease or condition is a hematological cancer selected from B-cell lymphoma, and optionally Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma (MZL), mantle cell lymphoma (MCL), Richter's syndrome, Burkitt's lymphoma, or follicular lymphoma.

39. 36. The method of claim 35, wherein the subject is a human.

40. 36. The method of claim 35, comprising administering to the subject a therapeutically effective amount of one or more therapeutic agents.

41. 36. The method of claim 35, wherein the therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, a hormonal therapeutic agent, a toxin, or an immunotherapeutic agent.

42. 43. The method of any one of claims 35 to 42, wherein the administration is oral, nasal, intravenous, subcutaneous, sublingual or intramuscular.

43. The method of claims 35 to 43, wherein the method further comprises the administration of one or more additional cancer treatments selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, hormone therapy, and surgery.

44. 28. Use of a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 in the manufacture of a medicament for treating a CD276 and / or PD-L1 associated disease or condition in a subject.

45. 28. Use of the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 27 in a method for treating a CD276- and / or PD-L1-associated disease or condition in a subject.

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