WO2024099295A1 - Novel multi-specific antibodies and uses thereof - Google Patents

Novel multi-specific antibodies and uses thereof Download PDF

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Publication number
WO2024099295A1
WO2024099295A1 PCT/CN2023/130106 CN2023130106W WO2024099295A1 WO 2024099295 A1 WO2024099295 A1 WO 2024099295A1 CN 2023130106 W CN2023130106 W CN 2023130106W WO 2024099295 A1 WO2024099295 A1 WO 2024099295A1
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seq
variable region
chain variable
cdr sequences
sequences selected
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PCT/CN2023/130106
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French (fr)
Inventor
Hui YUWEN
Bing HOU
Tengteng LI
Peng Chen
Min Deng
Bo Shan
Jay Mei
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Antengene (Hangzhou) Biologics Co., Ltd.
Antengene Biologics Limited
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Publication of WO2024099295A1 publication Critical patent/WO2024099295A1/en

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  • the present disclosure generally relates to novel multi-specific antibodies targeting CD276 and one or more additional targets.
  • B7-H3 (CD276, UniProt IDs for human amino acid sequence: Q5ZPR3 and mouse amino acid sequence: Q8VE98) is an important newly found immune checkpoint member of the B7 and CD28 families, which is a type I transmembrane co-stimulatory molecule, existing in two isoforms determined by its extracellular domain.
  • 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 has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. However, B7-H3 protein is expressed at high frequency on many different cancer types (60%of all cancers) ("B7-H3: an attractive target for antibody-based immunotherapy” . Clinical Cancer Research: clincanres. 2584.2020) .
  • B7-H3 has been controversial. It was classified as either a co-stimulatory molecule for T cell activation that inhibits tumor antigen-specific immune responses, or the non-immunological role such as promoting migration, tumor growth, invasion, metastasis, malignant stage, recurrence rate, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism.
  • the receptor for B7-H3 has been reported to be triggering receptor expressed on myeloid cell (TREM) -like transcript 2 (TLT-2, or TREML2) , which binds B7-H3 and costimulates activation of CD8 T cells in particular.
  • B7-H3 is also reported an inhibitor for NK cells and osteoblastic cells by ligating unknown receptor (s) . (The contrasting role of B7-H3, PNAS July 29, 2008, 105 (30) 10277-10278) .
  • 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 observed tumor growth suppression in vitro and in vivo.
  • CD276 is also reported to be expressed in hematological 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 can also be a potential target for treating hematological cancers.
  • an antibody means one antibody or more than one antibody.
  • the present disclosure provides novel anti-CD276 antibody molecules, amino acid and nucleotide sequences thereof, and uses thereof.
  • the present disclosure provides an multi-specific antibody or an 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.
  • the first binding domain comprises 1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, 41-43, 49-51, 57-59, 65-67, 73-75, 81-83, 89-91, 97-99, 105-107, 113-115, 121-123, 129-131, 137-139, 145-147, 153-155, 161-163, 169-171, 177-179, 185-187, 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, and/or 1, 2, or 3 light chain CDR sequences selected from the group consist
  • the first binding domain comprises a heavy chain variable region selected from the group consisting of:
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 1-3;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9-11;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 17-19;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 25-27;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 41-43;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 49-51;
  • h a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 57-59;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 65-67;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 73-75;
  • k a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 81-83;
  • m a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 97-99;
  • n a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 105-107;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 113-115;
  • p a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 121-123;
  • r a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 137-139;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 145-147;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 161-163;
  • v a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 169-171;
  • w a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 177-179;
  • x a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 185-187;
  • y a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 193-195;
  • z a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 201-203;
  • aa a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 209-211;
  • bb a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 217-219;
  • cc a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 225-227;
  • dd a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 233-235;
  • ee a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 241-243;
  • ff a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 249-251;
  • gg a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 257-259;
  • hh a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 265-267;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 273-275;
  • jj a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 281-283;
  • kk a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 289-291;
  • ll a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 297-299;
  • mm a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 305-307;
  • nn a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 313-315;
  • oo a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 321-323;
  • pp) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 329-331;
  • qq a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 337-339;
  • rr a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9, 374 and 375.
  • the first binding domain provided herein comprises a light chain variable region selected from the group consisting of:
  • a) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 4-6;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20-22;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 28-30;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 52-54;
  • h a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 60-62;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 68-70;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 76-78;
  • k a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 84-86;
  • m a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 100-102, 108-110;
  • n a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 108-110;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 116-118;
  • p a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 124-126;
  • r a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 140-142;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 164-166;
  • v) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 172-174;
  • w a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 180-181;
  • x a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 188-190;
  • y a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 196-198;
  • z a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 204-206;
  • aa a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 212-214;
  • bb a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 220-222;
  • cc a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 228-230;
  • dd a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 236-238;
  • ee a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 244-246;
  • ff a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 252-254;
  • gg a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 260-262;
  • hh a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 268-270, ;
  • a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 276-278;
  • jj a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 284-286;
  • kk a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 292-294;
  • ll a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 300-302;
  • mm a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 308-310;
  • nn a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 316-318;
  • oo a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 324-326;
  • pp) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 332-334;
  • qq a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 340-342;
  • rr a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 376, 13 and 14;
  • ss a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 377, 45 and 46.
  • the first binding domain comprises:
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62;
  • a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70;
  • a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78;
  • k a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86;
  • m a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102;
  • n a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110;
  • p a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150;
  • a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166;
  • w a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 179; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 180, SEQ ID NO: 181, and SEQ ID NO: 182;
  • x a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 187; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 189, and SEQ ID NO: 190;
  • y a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198;
  • z a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 204, SEQ ID NO: 205, and SEQ ID NO: 206;
  • aa a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 209, SEQ ID NO: 210, and SEQ ID NO: 211; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 214;
  • bb a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222;
  • cc a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 225, SEQ ID NO: 226, and SEQ ID NO: 227; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 228, SEQ ID NO: 229, and SEQ ID NO: 230;
  • dd a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 236, SEQ ID NO: 237, and SEQ ID NO: 238;
  • ee a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 244, SEQ ID NO: 245, and SEQ ID NO: 246;
  • ff a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 252, SEQ ID NO: 253, and SEQ ID NO: 254;
  • gg a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 260, SEQ ID NO: 261, and SEQ ID NO: 262;
  • hh a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 265, SEQ ID NO: 266, and SEQ ID NO: 267; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 268, SEQ ID NO: 269, and SEQ ID NO: 270;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 273, SEQ ID NO: 274, and SEQ ID NO: 275; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278;
  • jj a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 281, SEQ ID NO: 282, and SEQ ID NO: 283; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 284, SEQ ID NO: 285 and SEQ ID NO: 286;
  • kk a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 289, SEQ ID NO: 290, and SEQ ID NO: 291; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 292, SEQ ID NO: 293, and SEQ ID NO: 294;
  • ll a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 297, SEQ ID NO: 298, and SEQ ID NO: 299; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 300, SEQ ID NO: 301, and SEQ ID NO: 302;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 305, SEQ ID NO: 306, and SEQ ID NO: 307; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310;
  • nn a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 313, SEQ ID NO: 314, and SEQ ID NO: 315; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 316, SEQ ID NO: 317, and SEQ ID NO: 318;
  • a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 321, SEQ ID NO: 322, and SEQ ID NO: 323; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 324, SEQ ID NO: 325, and SEQ ID NO: 326;
  • qq a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 377, SEQ ID NO: 45, and SEQ ID NO: 46.
  • the first binding domain comprises a heavy chain variable region 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:
  • the first binding domain comprises a light chain variable region 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: 232, SEQ ID NO: 240, SEQ ID NO: 248, SEQEQ ID NO:
  • the first binding domain comprises: (i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences shown 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 1, 2, or 3 CDR sequences shown 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 1, 2, or 3 CDR sequences shown as SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences shown as SEQ ID NO: 44 or 377 (QSLLQSSTQKNY) , SEQ ID NO: 45, and SEQ ID NO: 46, respectively.
  • the first binding domain comprises a heavy chain variable region comprising a sequence selected from SEQ ID NO: 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 NO: 352, 354, 356, 359, 361, 363, 366, 368, 369, 371, 372 and 373.
  • the first binding domain comprises:
  • aa a heavy chain variable region comprising SEQ ID NO: 215 and a light chain variable region comprising SEQ ID NO: 216;
  • 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;
  • nn a heavy chain variable region comprising SEQ ID NO: 319 and a light chain variable region comprising SEQ ID NO: 320;
  • ss a heavy chain variable region comprising SEQ ID NO: 349 and a light chain variable region comprising SEQ ID NO: 350.
  • the first binding domain comprises:
  • the first binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to human CD276.
  • the substitution is 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.
  • the multi-specific antibody or antigen-binding fragment thereof further comprises an activating receptor binding domain, optionally a constant region of human Ig, or optionally a constant region of human IgG.
  • the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4.
  • the second target is PD-L1.
  • the second binding domain comprises 1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 384-386, and/or 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 387-389.
  • CDR heavy chain complementarity determining region
  • the second binding domain comprises a heavy chain variable region comprising three CDR sequences set forth as SEQ ID NOs: 384, 385 and 386, respectively; and a light chain variable region comprising three CDR sequences set forth as SEQ ID NOs: 387, 388 and 389, respectively.
  • the second binding domain comprises a heavy chain variable set forth as SEQ ID NO: 382, and/or a light chain variable region set forth as SEQ ID NO: 383.
  • the second binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to human PD-L1.
  • the substitution is 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.
  • the second binding domain comprising an scFv structure of VH-linker-VL.
  • the linker comprises a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
  • the N-termination of the second binding domain is operably linked to the C-termination of the activating receptor binding domain.
  • the multi-specific antibody or antigen-binding fragment thereof provided herein is a bi-specific or tri-specific antibody.
  • the multi-specific antibody or antigen-binding fragment is linked to one or more conjugates.
  • the conjugate is covalently attached either directly or via a linker.
  • the conjugate comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
  • the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof provided herein.
  • the present disclosure provides a vector comprising the isolated polynucleotide provided herein.
  • the present disclosure provides a host cell comprising the vector provided herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the multi-specific antibody or antigen-binding fragment thereof provided herein or the polynucleotide encoding the multi-specific antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of expressing the multi-specific antibody or antigen-binding fragment thereof provided herein, comprising culturing the host cell provided herein under the condition at which the vector provided herein is expressed.
  • the present disclosure provides a method of treating a disease or condition in a subject that would benefit from modulation of CD276 activity, comprising administering to the subject a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof provided herein or the pharmaceutical composition provided herein.
  • the disease or condition is a CD276 related disease or condition.
  • the disease or condition is cancer, adaptive immune disease, autoimmune disease, inflammatory disease, or infectious disease.
  • the cancer is adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck
  • the disease or condition is hematological cancer selected from B-cell lymphomas, such as 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.
  • B-cell lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) , acute lymphocytic leukemia (ALL) , acute myeloid leukemia (AML)
  • the subject is human.
  • the method provided herein comprises administering to the subject a therapeutically effective amount of one or more therapeutic agent.
  • said therapeutic agent is a chemotherapeutic agent, a radiation therapeutic agent, a hormonal therapeutic agent, a toxin or an immunotherapeutic agent.
  • the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
  • said method further comprises administration of one or more additional cancer therapies selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, hormonal therapy, and surgery.
  • the present disclosure provides a method of modulating CD276 activity in a CD276-expressing cell, comprising exposing the CD276-expressing cell to the antibody or antigen-binding fragment thereof provided herein.
  • the present disclosure provides a method of modulating PD-1/PD-L1 pathway activity in a PD-L1-expressing cell, comprising exposing the PD-L1-expressing cell to the multi-specific antibody or antigen-binding fragment thereof provided herein.
  • the present disclosure provides use of the multi-specific antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
  • the medicament further comprises a second therapeutic agent.
  • the second therapeutic agent is a chemotherapeutic agent, a radiation therapeutic agent, a hormonal therapeutic agent, a toxin or an immunotherapeutic agent.
  • Figure 1 shows the binding affinity of the anti-CD276 antibodies provided herein on SKOV3 cells as measured by FACs analysis.
  • Figure 2A-2E show binding of 6-D8-E7-A11 to several cancer cell lines that express B7H3 as measured by FACS analysis.
  • Figure 3A-3C show ADCC effect of the anti-CD276 antibodies provided herein on SKOV3 cells.
  • Figure 4A and 4B show CDC effect of the anti-CD276 antibodies provided herein on CHO-S-hCD276 cells.
  • Figure 5A-5E show indirect ADC cytotoxicity effect of the anti-CD276 antibodies provided herein on SKOV3 cells.
  • Figure 6 shows in vivo efficacy of the anti-CD276 antibodies provided herein in inhibiting the tumor growth in the mouse model inoculated with MC-38-hCD276 (B7H3) tumor cells.
  • Figure 7 shows effects on tumor growth in subcutaneous calu-6 model in balb/c nude mice (mean ⁇ sem) .
  • Figure 8 shows IL2 release by T cell activation in MLR assay.
  • Figure 9 shows IFN ⁇ release by T cell activation in MLR assay.
  • Figure 10 shows the binding affinity on SKOV3 of humanized antibodies derived from 30-C7-C11-D4.
  • Figure 11 shows binding affinity on SKOV3 of humanized antibodies derived from 10-G6-C4-B2.
  • Figure 12 shows effects on tumor growth in subcutaneous Calu-6 model in Balb/c nude mice (Mean ⁇ SEM) .
  • Figure 13 shows effects of test articles on tumor growth in subcutaneous MC-38-hCD276 (B7H3) model in C57BL/6 Mice (Mean ⁇ SEM) .
  • Figure 14 shows effects of test articles on tumor growth in subcutaneous MC-38-hCD276 (B7H3) Model in C57BL/6 Mice (Mean ⁇ SEM) .
  • Figure 15 shows effects of test articles on tumor growth in subcutaneous MC-38-hCD276 (B7H3) Model in C57BL/6 Mice (Mean ⁇ SEM) .
  • antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, or monovalent antibody that binds to a specific antigen.
  • a native intact antibody comprises two heavy (H) chains and two light (L) chains.
  • Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (V H ) and a first, second, and third constant region (C H1 , C H2 , C H3 , respectively) ;
  • mammalian light chains are classified as ⁇ or ⁇ , while each light chain consists of a variable region (V L ) and a constant region.
  • the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
  • Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions 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 the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, and HCDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding domains disclosed herein may be defined or identified by the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., 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, A.M., J. Mol. Biol., 196, 901 (1987) ; N. R. Whitelegg et al, Protein Engineering, v13(12) , 819-824 (2000) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83
  • the constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • IgG1 gamma1 heavy chain
  • IgG2 gamma2 heavy chain
  • IgG3 gamma3 heavy chain
  • IgG4 gamma4 heavy chain
  • IgA1 alpha1 heavy chain
  • IgA2 alpha2 heavy chain
  • antibody molecule refers to an antigen-binding protein or polypeptide comprising at least one antibody fragment (such as CDR, and/or variable region sequence) .
  • An antibody molecule includes, for example, a monoclonal antibody, an antibody fragment or domain, a fusion protein comprising an antibody fragment or domain, a polypeptide complex comprising an antibody fragment or domain, and so on.
  • antigen-binding domain e.g. CD276-binding domain or PD-L1 binding domain
  • 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.
  • antigen-binding domain examples include, without limitation, a Fab, a Fab', a F (ab') 2 , an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a single-chain antibody molecule (scFv) , a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
  • An antigen-binding domain is capable of binding to the same antigen to which the parent antibody binds.
  • an antigen-binding domain may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • Fab with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F (ab') 2 refers to a dimer of Fab’ .
  • a “fragment difficult (Fd) ” with regard to an antibody refers to the amino-terminal half of the heavy chain fragment that can be combined with the light chain to form Fab.
  • Fd fragment may consists of the VH and CH1 domains
  • Fv with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • a number of Fv designs have been provided, including dsFvs, in which the association between the two domains is enhanced by an introduced disulphide bond; and scFvs can be formed using a peptide linker to bind the two domains together as a single polypeptide.
  • Fvs constructs containing a variable domain of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced.
  • 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 one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .
  • a “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • a “ (dsFv) 2 ” or “ (dsFv-dsFv') ” comprises three peptide chains: two V H moieties linked by a peptide linker (e.g., a long flexible linker) and bound to two V L moieties, respectively, via disulfide bridges.
  • dsFv-dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
  • Fc with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding.
  • the Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • “Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two V H domains and no 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 Camelidae (camels, dromedaries, and llamas) .
  • VHH domain The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .
  • a “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
  • 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.
  • two or more V H domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody.
  • the two V H domains of a bivalent domain antibody may target the same or different antigens.
  • chimeric means an antibody or antigen-binding domain, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
  • a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse.
  • the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
  • humanized means that the antibody or antigen-binding domain comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
  • operably link refers to a juxtaposition, with or without a spacer or a linker or an intervening sequence, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner.
  • polypeptide sequences When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function.
  • an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity.
  • an antigen-binding domain can be operably linked to another antigen-binding domain with an intervening sequence there between, and such intervening sequence can be a spacer or can comprise a much longer sequence such as a constant region of an antibody.
  • the term may also be used with respect to polynucleotides.
  • a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.
  • fusion refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence which does not exist naturally.
  • a fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
  • an “antigen” as used herein refers to a compound, composition, peptide, polypeptide, protein or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma) , or injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody) , including those induced by heterologous antigens.
  • CD276 protein or “B7-H3 protein” as used herein is intended to encompass any form of CD276, for example, 1) native unprocessed CD276 molecule, “full-length” CD276 chain or naturally occurring variants of CD276, including, for example, splice variants or allelic variants; 2) any form of CD276 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CD276 subunit generated through recombinant method.
  • a fragment e.g., a truncated form, an extracellular/transmembrane domain
  • a modified form e.g. a mutated form, a glycosylated/PEGylated,
  • anti-CD276 antibody refers to an antibody or antigen-binding domain that is capable of specifically binding to CD276 (e.g. human, monkey or mouse CD276) .
  • PD-L1 refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192: 1027) .
  • Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3.
  • PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells.
  • PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells.
  • the binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation.
  • PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.
  • binding or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
  • the antibody molecules or antigen-binding domains provided herein specifically bind to CD276 and/or PD-L1 with a binding affinity (K D ) of ⁇ 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) .
  • K D binding affinity
  • K D used herein refers to the ratio of the dissociation rate to the association rate (k off /k on ) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method.
  • the K D value can be appropriately determined by using flow cytometry.
  • Binding affinity to CD276 and/or PD-L1 can also be represented by “half maximal effective concentration” (EC 50 ) value, which refers to the concentration of an antibody where 50%of its maximal effect (e.g., binding or inhibition etc. ) is observed.
  • the EC 50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays.
  • the antibodies and the fragments thereof provided herein specifically bind to CD276 and/or PD-L1 at an EC 50 (i.e.
  • 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.
  • 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 may be greater than 85%, or greater than 90%.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitope) .
  • Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on the protein and the small segments of the contiguous amino acids can be digested from an antigen binding with major histocompatibility complex (MHC) molecules or retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen.
  • an antibody or antigen-binding domain blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding domain may be considered to bind the same/closely related epitope as the reference antibody.
  • a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
  • conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) .
  • conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
  • homolog and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.
  • Percent (%) sequence identity with respect to 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 to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F.
  • effector functions refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex, Fc receptor and effector cell (e.g., macrophage) .
  • exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and antibody-dependent cellular phagocytosis (ADCP) induced by binding of Fc region of an antibody to phagocytosis.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
  • subject or “individual” or “animal” or “patient” as used herein refers to human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease or disorder.
  • Mammalian subjects include humans, monkeys, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
  • vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
  • a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
  • vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
  • a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
  • a vector can be an expression vector or a cloning vector.
  • host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
  • CD276-related disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of CD276.
  • the CD276 related condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease.
  • a “PD-L1-related” disease or condition as used herein refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased expression or activities of PD-L1.
  • the PD-L1 related disease or condition is accompanied with a suppressed immune system.
  • the PD-L1 related disease or condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease.
  • Cancer refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia.
  • solid tumor refers to a solid mass of neoplastic and/or malignant cells.
  • cancer or tumors include adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer,
  • the hematological malignancies includes B-cell lymphomas, 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.
  • B-cell lymphomas optionally Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) , acute lymphocytic leukemia (ALL) , acute myeloid leukemia (AML) , chronic lymph
  • 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 a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.
  • the cancer is chemoresistant.
  • chemoresistant cancer refers to a type of cancer that are not responsive to the effects of chemotherapy. For example, a cancer that has been responding to a chemotherapy or a combination of different chemotherapies suddenly begins to grow can be referred to as a chemoresistant cancer.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • the present disclosure provides a multi-specific antibody or antigen-binding fragment thereof comprises multiple antigen binding domains and an activating receptor binding domain.
  • the multiple antigen binding domains comprise at least a first binding domain targeting a first antigen and a second binding domain targeting a second antigen, wherein one of the first antigen and the second antigen is an immune checkpoint molecule or a tumor antigen, while the other one is one or more additional targets.
  • the one or more additional targets comprise a target which is another immune checkpoint molecule (e.g., PD-L1) .
  • the one or more additional targets comprise targets relating to immune cell (e.g., T cells or B cells) function.
  • the one or more additional targets comprises a target selected from the group consisting of CD40, CD3, CD28, etc.
  • 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.
  • the CD276 binding domain comprises one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CD276 antibody 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-D7-C5, 4-D5-B9-B11, 10-B9-D10-A12, 15-G1-D1-E3, 8-B4-F5-E11, 6-F3-G
  • the first binding domain is capable of specifically binding to CD276.
  • the CD276 are derived from human, monkey or mouse.
  • the CD276 is a recombinant CD276 or a CD276 expressed on a cell surface.
  • All of these 43 anti-CD276 antibodies thereof provided herein are mouse monoclonal antibodies.
  • Table 1 shows the CDR sequences of these 43 anti-CD276 antibodies according to IMGT numbering system.
  • the heavy chain and light chain variable region sequences are also provided below.
  • CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable.
  • the CD276 binding domain comprises a heavy chain CDR3 sequence of one of 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-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
  • 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.
  • Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen.
  • the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81) .
  • the diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) as well as desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263: 551-67) .
  • the CD276 binding domain comprise suitable framework region (FR) sequences, as long as the antibodies and/or antigen-binding fragments thereof can specifically bind to CD276.
  • FR framework region
  • the CDR sequences provided in Table 1 are obtained from mouse antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
  • sequences of the CD276 binding domain are PTM optimized.
  • PTM optimization refers to post-translation modification, aiming at avoiding potential aggregation, activity loss or other risk.
  • Exemplary PTM optimized CD276 binding domain includes the antigen-binding domain of 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, wherein all the CDR regions are underlined.
  • the CD276 binding domain is humanized.
  • a humanized antibody or antigen-binding fragment is desirable in its reduced immunogenicity in human.
  • a humanized antibody is chimeric in its variable regions, as non-human CDR sequences are grafted to human or substantially human FR sequences.
  • Humanization of an antibody or antigen-binding fragment can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536) .
  • Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art.
  • “best-fit” approach can be used, where 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 the human scaffold for grafting the non-human CDR sequences (see, for example, Sims et al, (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mot. Biol. 196: 901) .
  • a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623) .
  • the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences except for the CDR sequences which are non-human.
  • the variable region FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences.
  • the human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
  • Table 4 below shows the heavy chain and light chain variable region amino acid sequences of humanized antibodies for 10-G6-C4-B2 and 30-C7-C11-D4, which are designated as hVH2-hVL1-10, hVH3-hVL3-10, hVH4-hVL1-10, hVH4-hVL2-10, hVH5-hVL1-10, hVH5-hVL3-10, 10-G6-C4-B2_hVH2-VL1_PTM, 10-G6-C4-B2_hVH3-VL3_PTM, 10-G6-C4-B2_hVH4-VL1_PTM, 10-G6-C4-B2_hVH4-VL2_PTM, 10-G6-C4-B2_hVH5-VL1_PTM, 10-G6-C4-B2_hVH5-VL3_PTM, hVH1-hV
  • the humanized CD276 binding domain is composed of substantially all human sequences except for the CDR sequences which are non-human.
  • the variable region FRs, and constant regions if present are entirely or substantially from human immunoglobulin sequences.
  • the human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
  • the humanized CD276 domain comprises human heavy chain HFR1-4, and/or light chain LFR1-4.
  • the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived.
  • one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or its fragment closely approximate the non-human parent antibody structure, so as to optimize binding characteristics (for example, increase binding affinity) .
  • the humanized antibody or antigen-binding fragment thereof provided herein comprises 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 the FR sequences of a heavy or a light chain variable domain.
  • such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.
  • one or more amino acids of the human FR sequences are randomly mutated to increase binding affinity.
  • one or more amino acids of the human FR sequences are back mutated to the corresponding amino acid (s) of the parent non-human antibody so as to increase binding affinity.
  • variable regions of the PD-L1 binding domain comprised in the multi-specific antibody or antigen-binding fragments thereof disclosed herein can be any of those PD-L1 antibody heavy chain variable regions and light chain variable regions known in the art.
  • the PD-L1 binding domain comprises the heavy chain variable region and the light chain variable region from YN035 recited in WO2019196309A1.
  • the multi-specific antibody or antigen-binding fragments thereof further comprises an activating receptor-binding domain.
  • activating receptor refers to receptors (e.g., Fc ⁇ R) expressed on an immune effector cell (e.g., a phagocytic cell, such as a macrophage) , which upon activation by binding to, for example, Fc domain, mediates at least one effector functions of the immune effector cell (e.g., a phagocytic cell, such as a macrophage) or pro-inflammatory response.
  • the immune effector cell provided herein co-expresses CD276 and one or more additional targets (e.g., PD-L1) .
  • the activating receptor is an Fc ⁇ R
  • the activating receptor-binding domain is an Fc domain.
  • Fc domain can activate Fc receptors (FcRs) on macrophages to drive a phosphorylation cascade propagated by the receptors’ immunoreceptor tyrosine-based activation motifs (ITAMs) .
  • 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 featured with a conserved amino acid sequence motif, which consists of paired YXXL/I motifs (wherein Y, L and I refer to Tyrosine, Lysine and Isoleucine respectively) and separated by a defined interval of six to eight amino acids. Phosphorylation of residues within the ITAM recruits several signaling molecules for phagocytosis activation. Accordingly, the activating receptor can be any receptor expressed on an immune effector cell that can be bound and activated to induce phagocytosis via an ITAM-comprising intracellular phagocytosis signaling domain.
  • the activating receptor is a receptor involved in a different phagocytic signaling or mechanism, such as, Akt mediated signaling cascade (via CD19, CD28, CSFR or PDGFR receptor) , clustering of a group of receptors on an immune effector cell (e.g., macrophage) that potentiates phagocytosis (via, for example, integrins or selectins) , or antigen mediated cytotoxicity (via FcDR1 (CD89) receptor or CD206) .
  • Akt mediated signaling cascade via CD19, CD28, CSFR or PDGFR receptor
  • an immune effector cell e.g., macrophage
  • FcDR1 (CD89) receptor or CD206 antigen mediated cytotoxicity
  • activating receptors that are relevant to effector functions can be fragment crystallizable ⁇ receptors (Fc ⁇ Rs) , TREM2, lectin, 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, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, dectin 1, RAGE (SR-E1) , LRPl, LRP2, ASGP, SR-PSOX, CXCL16, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg (L) , and CD169 receptor or
  • Fc ⁇ Rs fragment crystalliz
  • the activating receptor that can generate pro-inflammatory signals upon activation include without limitation, PI3K, Fc ⁇ Rl, Fc ⁇ R2A, Fc ⁇ R2B2, Fc ⁇ R2C, Fc ⁇ R3A, BAH. Tyro3, Axl, Traf6, Syk, MyD88, Zap70, Fc ⁇ Rl, Fc ⁇ Rl, BAFF-R, DAP 12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.
  • activating receptor-binding domain refers to domain (e.g., a portion of an antibody) that is capable of specifically binding to an activating receptor on an immune effector cell and such binding leads to activation of the receptor as well as downstream signaling thereof (e.g., effector function or pro-inflammatory response of the immune cell) .
  • the activating receptor-binding domain comprises an Fc domain of an antibody or a variant thereof.
  • the Fc domain can be derived from IgG1 or IgG4.
  • the activating receptor-binding domain of the multi-specific molecules provided herein binds and activates activating receptor selected from the group consisting of fragment crystallizable ⁇ receptors (Fc ⁇ Rs) , TREM2, lectin, 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, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, dectin 1, RAGE (SR-E1) , LRPl, LRP2, ASGP, SR-PSOX, CXCL16, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CCR2, CX3CR1, CSF1R, Tie2,
  • the activating receptor-binding domain of the multi-specific molecule provided herein comprises a Fc domain or a variant thereof, which activates Fc receptors (FcRs) on macrophages, such as Fc ⁇ RII.
  • FcRs Fc receptors
  • the activating receptor-binding domain comprises a heavy chain and/or a light chain constant region.
  • the heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions.
  • the heavy chain constant region comprises an Fc region.
  • the light chain constant region comprises C ⁇ or C ⁇ .
  • 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 human IgG1, IgG2, IgG3, or IgG4.
  • Ig immunoglobulin
  • the activating receptor-binding domain is derived from human IgG1, IgG2, IgG3, or IgG4.
  • Human IgG isotypes (the subclasses of mature gamma globulin class G antibodies; IgG1, IgG2, IgG3 and IgG4) exhibit differential capacity 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 engagement of such effector functions is based on selectivity for Fc receptors on distinct immune cells and the ability to bind C1q thereby activating the assembly of a membrane attack complex (MAC) .
  • MAC membrane attack complex
  • Fc ⁇ receptors which include Fc ⁇ RI, Fc ⁇ RIIa/b/c, and Fc ⁇ RIIIa/b is high for IgG1 and IgG3.
  • Fc ⁇ affinity for IgG2 is considerably lower with the exception of Fc ⁇ RIIa H131 polymorphism and IgG4 only has measurable affinity for Fc ⁇ RI.
  • the activating receptor-binding domain is derived from human IgG1 isotype, which could induce ADCC, CDC or ADCP, or a constant region of IgG4 or IgG2 isotype, which has reduced or depleted effector function.
  • Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing tumor antigen (e.g., CD276) .
  • Effector functions can be evaluated using various assays such as Fc receptor binding assay, C1q binding assay, and cell lysis assay.
  • the activating receptor-binding domain is derived from a constant region of mouse IgG2 isotype, which could induce ADCC, CDC or ADCP.
  • the multi-specific antibody or antigen-binding fragments thereof provided herein is a recombinant protein comprising multiple antigen binding domains described throughout the specification, wherein each of the multiple antigen binding domains has specific binding affinity to each targeted antigen, and is connected with each other by one or more linkers.
  • the one or more linkers may have cognate peptides that exhibit complementary binding with each other.
  • the first binding domain of the multi-specific antibody or antigen-binding fragments thereof provided herein is fused with the first of a pair of cognate peptides, while the second binding domain thereof is fused with the second of the pairs of cognate peptides; as such, the first binding domain and the second binding domain can be connected by the pair of cognate peptides through the complementary binding between each of the pair of cognate peptides.
  • the pair of cognate peptides comprises two heavy chains of an antibody or any complementary portion thereof, a pair of light chain and heavy chain complementary to each other of an antibody or any complementary portion thereof, leucine zipper domains that exhibit complementary binding with each other (e.g., the zipper sequences within the binding regions of c-Fos and c-June proteins) , or synthetic peptides designed to specifically bind to each other via synthetic connectors.
  • the multiple antigen binding domains (e.g., the first binding domain and the second binding domain) and the activating receptor binding domain of the multi-specific antibody or antigen-binding fragments thereof provided herein can also be connected via chemical binding, such as crosslinking (e.g., BS2G crosslinker (Bis [Sulfosuccmimidyl] glutarate) , BS3 crosslinker (Bis [sulfosuccinimidyl] suberate) , Sulfo-DSS, DST crosslinker (Disuccinimidyl tartrate) , BMPS (N- (B-Maleimidopropyloxy) succinimide ester; MBS crosslinker (mMaleimidobenzoyl-N-hydroxysuccinimide ester) ; or PDPH crosslinker (3- [2-Pyridyldithio] propionylhydrazide) ) .
  • crosslinking e.g., BS
  • the first binding domain (e.g., CD276 binding domain or PD-L1 binding domain) is linked to N-termination of the activating receptor-binding domain (e.g., Fc domain) .
  • the first binding antibody domain (e.g., CD276 binding domain or PD-L1 binding domain) comprises a Fab domain, optionally, the Fab domain comprises a heavy chain linked to one of the N-termination of the activating receptor-binding domain (e.g., Fc domain) .
  • both of the first binding domain and the second binding domain comprise a Fab domain
  • each of the Fab domains comprises a heavy chain linked to each N-termination of the activating receptor-binding domain (e.g., Fc domain) , respectively.
  • the second binding domain (e.g., CD276 binding domain or PD-L1 binding domain) is linked to the activating receptor-binding domain (e.g., Fc domain) or to the first binding domain.
  • the second binding domain comprises an scFv structure.
  • the second binding domain is linked to the C-termination of light chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) . In certain embodiments, the second binding domain is linked to the N-termination of light chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) . In certain embodiments, the second binding domain is linked to the C-termination of heavy chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) . In certain embodiments, the second binding domain is linked to the N-termination of heavy chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) .
  • linked to refers to covalent or non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds) between two components.
  • the second binding domain is linked to C-termination of the activating receptor-binding domain (e.g., Fc domain) .
  • the first binding domain e.g., the first binding domain comprising a Fab domain
  • the second binding domain is linked to the C-termination of activating receptor-binding domain.
  • the first binding domain e.g., CD276 binding domain or PD-L1 binding domain
  • the activating receptor binding domain e.g., Fc domain
  • the second binding domain comprises an scFv structure from N-termination to C-termination comprising a second heavy chain variable region, a first linker and a second light chain variable region.
  • the first linker has a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
  • the N-termination of the second binding domain is linked to the C-termination of the activating receptor binding domain via a second linker.
  • the second linker has a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
  • the multi-specific antibody or antigen-binding fragments thereof disclosed herein comprises two second binding domains, each of which is operably linked to each C-termination of the activation receptor binding domain, respectively.
  • the multi-specific antibody or antigen-binding fragments thereof comprises four chains, which from N-termination to C-termination have the structures as following:
  • Chains 1 and 4 VL1-CL;
  • Chains 2 and 3 VH1-CH1-hing region-CH2-CH3-linker 2-VH2-linker 1-VL2;
  • VH1 stands for the heavy chain variable region of the first binding domain
  • VL1 stands for the light chain variable region of the first binding domain
  • VH2 stands for the heavy chain variable region of the second binding domain
  • VL2 stands for the light chain variable region of the second binding domain
  • linker 1 stands for the first linker
  • linker 2 stands for the second linker.
  • the first binding domain is a CD276 binding domain and the second binding domain is a PD-L1 binding domain. In some other certain embodiments, the first binding domain is a PD-L1 binding domain and the second binding domain is a CD276 binding domain.
  • the present disclosure also encompasses various variants of the multi-specific antibodies and/or antigen-binding fragments thereof provided herein.
  • the present disclosure encompasses various types of variants of an exemplary antibody provided herein, i.e., the antibody with 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.
  • the antibody variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, 3 or 5, one or more FR sequences, the heavy or light chain variable region sequences provided in Table 2, 4 or 6, and/or the activating receptor binding domain (e.g. Fc region) .
  • Such variants retain specific binding affinity to CD276 and/or PD-L1 of their parent antibodies, but have one or more desirable properties conferred by the modification (s) or substitution (s) .
  • the antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function (s) , improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues) .
  • the parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) .
  • target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution.
  • the potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc. ) .
  • Affinity variant may contain modifications or substitutions in one or more CDR sequences as provided in Table 1, 3 or 5, one or more FR sequences, or the heavy or light chain variable region sequences in provided in Table 2, 3, 4 or 6.
  • the affinity variants retain specific binding affinity to CD276 and/or PD-L1 of the parent antibody, or even have improved CD276 and/or PD-L1 specific binding affinity over the parent antibody.
  • at least one (or all) of the substitution (s) in the CDR sequences, FR sequences, or variable region sequences comprises a conservative substitution.
  • one or more amino acid residues may be substituted yet the resulting antibody or antigen-binding fragment still retain the binding affinity to CD276 and/or PD-L1, or even have an improved binding affinity.
  • Various methods known in the art can be used to achieve this purpose.
  • a library of antibody variants such as Fab or scFv variants
  • phage display technology can be generated and expressed with phage display technology, and then screened for the binding affinity to CD276 and/or PD-L1.
  • computer software can be used to virtually simulate the binding of the antibodies to CD276 and PD-L1, and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding.
  • the humanized antibody or antigen-binding fragment provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences.
  • an affinity variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences in total.
  • the multi-specific antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1 or 5, and in the meantime retain the binding affinity to CD276 and/or PD-L1 at a level similar to or even higher than its parent antibody.
  • the multi-specific 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 that (or those) listed in Table 2, 4 or 6, and in the meantime retain the binding affinity to CD276 and/or PD-L1 at a level similar to or even higher than its parent antibody.
  • a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a sequence selected from that (or those) listed in Table 1.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) .
  • the multi-specific antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment thereof.
  • the multi-specific antibody or antigen binding fragment thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, 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-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
  • the multi-specific antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
  • a free cysteine residue is one which is not part of a disulfide bridge.
  • a cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl.
  • Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
  • the multi-specific antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.
  • the multi-specific antibodies or antigen-binding fragments comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) .
  • FcRn neonatal Fc receptor
  • Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell.
  • Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al, Structure, 6 (1) : 63-73, 1998; Kontermann, R.
  • the multi-specific antibodies or antigen-binding fragments comprise one or more amino acid substitution (s) that alters the antibody-dependent cellular cytotoxicity (ADCC) .
  • Certain amino acid residues at CH2 domain of the Fc region can be substituted to provide for enhanced ADCC activity.
  • carbohydrate structures on the antibody can be changed to enhance ADCC activity.
  • the multi-specific antibodies or antigen-binding fragments comprise one or more amino acid substitution (s) that alters Complement Dependent Cytotoxicity (CDC) , for example, by improving or diminishing C1q binding and/or CDC (see, for example, 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 concerning other examples of Fc region variants.
  • CDC Complement Dependent Cytotoxicity
  • the multi-specific antibodies or antigen-binding fragments thereof comprise one or more amino acid substitution (s) in the interface of the Fc region to facilitate and/or promote heterodimerization.
  • modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex.
  • the multi-specific antibodies and antigen-binding fragments thereof is linked to one or more conjugates, optionally, wherein the conjugate is covalently attached either directly or via a linker.
  • a conjugate is a non-proteinaceous moiety that can be attached to the 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, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) .
  • conjugates may be linked to the multi-specific antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
  • the conjugate comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
  • the multi-specific antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates.
  • a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate.
  • the multi-specific antibodies may be linked to a conjugate indirectly, or through another conjugate.
  • the multi-specific antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin.
  • the conjugate can be a toxin (e.g., a chemotherapeutic agent) , a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) .
  • a “toxin” can be any agent that is detrimental to cells or that can damage or kill cells.
  • toxin include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioe
  • detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or ⁇ -D-galactosidase) , radioisotopes (e.g.
  • the conjugate can be a pharmacokinetic modifying moiety which helps increase half-life of the antibody.
  • Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like.
  • 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 if more than one polymer are attached, they can be the same or different molecules.
  • the conjugate can be a purification moiety such as a magnetic bead.
  • the multi-specific antibodies and/or antigen-binding fragments thereof provided herein is used for a base for a conjugate.
  • the present disclosure provides isolated polynucleotides that encode the multi-specific antibodies and antigen-binding fragments thereof.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
  • the encoding DNA may also be obtained by synthetic methods.
  • the isolated polynucleotide that encodes the multi-specific antibodies and antigen-binding fragments thereof can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • the vector system includes mammalian, bacterial, yeast systems, etc, and comprises plasmids 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, and other laboratorial and commercially available vectors.
  • Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated vectors
  • Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for multi-specific antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424) , K. bulgaricus (ATCC 16,045) , K. wickeramii (ATCC 24,178) , K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226)
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Neurospora crassa
  • Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (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.
  • mice 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 liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .
  • Host cells are transformed with the above-described expression or cloning vectors for multi-specific antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the antibody may be produced by homologous recombination known in the art.
  • the host cells used to produce the multi-specific antibodies or antigen-binding fragments provided herein may 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 the host cells.
  • any of these media may be supplemented as necessary 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 GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually 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 that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the multi-specific antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the multi-specific antibodies and antigen-binding fragments thereof prepared from the 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 the preferred purification technique.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) .
  • Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) .
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bakerbond ABX. TM. resin J.T. Baker, Phillipsburg, N.J. ) is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
  • the present disclosure further provides pharmaceutical compositions comprising the multi-specific antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • Suitable components 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 hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
  • compositions comprising an antibody or antigen-binding fragment and conjugates as provided herein decreases oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain embodiments compositions are provided that comprise one or more multi-specific antibodies or antigen-binding fragments thereof as disclosed herein and one or more antioxidants such as methionine.
  • pharmaceutical 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, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at 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 carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, 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 buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
  • a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents.
  • the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial can contain a single dosage or multiple dosages of the anti-CD276 antibody or antigen-binding fragment thereof or composition thereof.
  • Overfilling vials with a small amount above that needed for a dose or set of doses e.g., about 10% is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
  • the present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof as provided herein to a subject in need thereof, thereby treating or preventing a CD276 and/or PD-L1 related disease or condition.
  • the CD276-related disease or condition is cancer, autoimmune disease, inflammatory disease, adaptive immune disease or infectious disease.
  • cancer examples include but are not limited to, non-small cell lung cancer (squamous/nonsquamous) , small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast 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, myelomas, mycoses fungoids, merkel cell cancer, hepatocellular carcinoma (HCC) , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyos
  • the cancer is adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck
  • the disease or condition is hematological cancer chosen from B-cell lymphomas.
  • B-cell lymphomas includes 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.
  • NHL non-Hodgkin's lymphoma
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • Autoimmune diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is 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) , Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos'disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglob
  • Inflammatory disorders include, for example, chronic and acute inflammatory disorders.
  • inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.
  • the CD276 associated conditions are inflammatory diseases such as systemic lupus erythematosus (SLE) , intestinal mucosal inflammation, wasting disease associated with colitis, multiple sclerosis, viral infections, rheumatoid arthritis, osteoarthritis, Cohn's disease, and inflammatory bowel disease, psoriasis, systemic scleroderma, autoimmune diabetes and the like.
  • SLE systemic lupus erythematosus
  • Infectious disease include, but are not limited to, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis
  • the subject is human.
  • methods are provided to treat a disease or condition in a subject that would benefit from modulation of CD276 activity, comprising administering a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof as provided herein to a subject in need thereof.
  • methods are provided to treat a disease or condition in a subject that would benefit from modulation of PD-1/PD-L1 pathway activity, comprising administering a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof as provided herein to a subject in need thereof.
  • the term “disease or condition” as used herein can be used interchangeably with the term “CD276 and/or PD-L1 related disease or condition” .
  • an antibody or antigen-binding fragment thereof as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
  • an multi-specific antibody or antigen-binding fragment thereof as provided herein may be administered at a therapeutically effective dosage 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) .
  • the antibody or antigen-binding fragment thereof is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage 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.
  • the administration dosage may change over the course of treatment. For example, in certain embodiments, the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single dose may be administered, or several divided doses may be administered over time.
  • the multi-specific antibodies and antigen-binding fragments thereof disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
  • parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
  • non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
  • the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents. In some embodiments, the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered alone or in combination with a second therapeutic agent. For example, the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with a second therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug.
  • a second therapeutic agent for example, a chemotherapeutic agent or an anti-cancer drug.
  • the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with an antagonist of one or more immunoinhibitory molecule, e.g., CD24, CD47, SIRP ⁇ , PD-L1, or the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M) .
  • an antagonist of one or more immunoinhibitory molecule e.g., CD24, CD47, SIRP ⁇ , PD-L1, or the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M) .
  • the term “antagonist” as used herein comprises can refer to any small molecule, small or micro RNAs, or antibodies or antigen-binding fragments thereof that blocks or inhibits binding of CD24, CD47, SIRP ⁇ , PD-L1 or B2M to their respective binding partners so as to prevent elicit of immunoinhibitory signals.
  • a multi-specific antibody or antigen-binding fragment thereof as 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, and in certain of these embodiments the multi-specific antibody or antigen-binding fragment thereof and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition.
  • an antibody or antigen-binding fragment thereof administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent.
  • An antibody or antigen-binding fragment thereof administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment thereof and a second agent are administered via different routes.
  • additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians’ Desk Reference, 57 th Edition; Medical Economics Company; ISBN: 1563634457; 57 th Edition (November 2002) ) or protocols well known in the art.
  • the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
  • the present disclosure also provides a method of modulating CD276 activity in a CD276-expressing cell, comprising exposing the CD276-expressing cell to the multi-specific antibody or antigen-binding fragment thereof provided herein.
  • the present disclosure also provides a method of modulating PD-1/PD-L1 pathway activity in a PD-L1-expressing cell, comprising exposing the PD-L1-expressing cell to the multi-specific antibody or antigen-binding fragment thereof provided herein.
  • the present disclosure also provides use of the multi-specific antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
  • CHO-Scells 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 immunogen.
  • Recombinant human CD276 protein (SEQ ID NO: 346) : the recombinant human CD276 protein was prepared by digesting the human CD276 protein with enterokinase, and the extracellular domain of human CD276 was fused with 6xHis tag and DDDDK (SEQ ID NO: 345) .
  • Recombinant human CD276 protein (SEQ ID NO: 346) :
  • mice Balb/c and SJL mice were immunized as shown below. The primary immunization was followed by several boosts until animals developed satisfactory antiserum titers suitable for hybridoma development.
  • Screening-Test bleeds were performed and evaluated by testing using FACS on CHO-Scell line stably over-expressing human and/or mouse CD276 (CHO-S-hCD276 and/or CHO-S-mCD276) .
  • Screening-Test bleeds were performed and evaluated by testing using Elisa with extra-cellular domain of recombinant human CD276 protein.
  • Fusion-Splenocyte fusions were performed on the mice which responded the best to the immunizations as determined by test bleed FACS.
  • the lymphocytes from spleens and lymph nodes were fused to a Sp2/0 cell line using an optimized electrofusion protocol. Multiple fusions were performed to ensure success of the cell fusion.
  • fusion was plated (2 x 10 4 to 10 5 per well) into a stack of 96-well plates. Plates were monitored for growth and fed weekly. Wells with cell growth were screened by primary screening assays in 10-14 days with FACS and/or other feasible assays such as Elisa. Multiple fusions for each targeting antigen were performed and screened. The positive parental clones which showed positive binding with CHO-S-CD276 and positive Elisa signal from primary screening were expanded into 24-well plates for secondary screening.
  • Hybridomas of interest were chosen to proceed to subcloning.
  • Sequences of 31 mouse antibodies from Table 5 were selected to generate and produce human IgG1 chimeric antibodies.
  • the binding affinity of these antibodies and bench mark antibody, Enoblituzumab see US8802091, MGA271, specifically with a construct designated as hBRCA84D-2) and MGC018 (an antibody-drug-conjugate, in which the 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 in both dividing and non-dividing cells, thereby causing cell death) with human patient derived ovarian cancer cell line, SKOV3, was determined by FACS analysis.
  • DUBA prodrug seco-DUocarmycin hydroxyBenzamide Azaindole
  • step 4 Washed the cells twice by using the condition in step 2. Resuspended the cells with 100 ⁇ l/well diluted 2 nd antibody, incubated at 4°C for 1 hour in the dark.
  • step 5 Washed the cells twice by using the condition in step 2. Resuspended the cells with 100 ⁇ l/well FACS buffer. Kept the cells in dark for FACS analysis.
  • the binding affinity of the selected antibodies on SKOV3 are higher, lower or comparable with bench mark antibody Enoblituzumab (see Table 8 and Figure 1) .
  • Table 9 shows the binding affinity of the chimeric antibodies on hB7H3 by Biacore (ChemPartner)
  • B7H3 expression were detected by FACS with 6-D8-E7-A11. High expression level of B7H3 on several cancer cell lines, such as BxPC3 (Pancreatic) , MCF7 (Breast) , Dentroit562 (Head and neck) , RKO (Colon) and SUN620 (Gastric) , were found (see Figure 2) .
  • SKOV3 cells ahuman ovarian cancer cell line
  • Some of the antibodies showed lower or comparable EC 50 compared with bench mark antibody, Enoblituzumab (Table 10 and figure 3) , indicating that they are more potent in mediating ADCC effect on SKOV3 cells than Enoblituzumab (MGA271) .
  • CHO-S-hCD276 cells were resuspended in cell culture medium at 4E5 cells/mL and were then added into a 96-well opaque wall plate at 50 ⁇ L/well.
  • Anti-CD276 antibodies were diluted with complete F-12K medium and added to the 96-well opaque wall plate at 50 ⁇ L/well.
  • Human serum complement was diluted with cell culture medium and was added to the same plate at 50 ⁇ L/well. The mixture was incubated for 2 hours in a CO 2 incubator at 37°C. CellTiter-Glo reagent for determining the cell cytotoxicity was added at 50 ⁇ L/well and the mixture was incubated for 10 mins at R.T. Luminescence signal of viable cells on a microplate reader was recorded.
  • Fab-ZAP is a chemical conjugate of goat anti-human monovalent antibody (asecondary antibody) and the ribosome-inactivating protein, saporin. Fab-ZAP is used to determine the internalization ability of antibodies.
  • 80 ⁇ L SKOV-3 cells were plated at 2000 cells/well in a 96-well plate and incubated overnight at 37°C.
  • Anti-CD276 antibodies were then added at 40 ⁇ L/well.
  • Fab-ZAP human dilution were added at 40 ⁇ L/well and incubated for 96 hours in a CO 2 incubator at 37°C.
  • CellTiter-Glo reagent for determining the cell cytotoxicity were added at 100 ⁇ L/well and incubated for 10 mins at R.T. Luminescence signal of viable cells on a microplate reader were recorded.
  • All the anti-CD276 antibodies showed potent indirect ADC effect on SKOV3 cells, with lower or comparable IC 50 compared with bench mark antibody, MGC018 (Table 12, Figure 5) , indicating that they are potential candidates for making ADCs.
  • EXAMPLE 6 Antibody in vivo efficacy in the treatment of subcutaneous MC-38-hCD276 murine colon carcinoma in female C57BL/6 mice
  • bi-specific antibodies follows the following pattern.
  • the CD276 binding domain i.e., the binding domain derived from 25-C8-D7-C5
  • the PD-L1 binding domain is the second binding domain
  • the CD276 binding domain i.e., the binding domain derived from 25-C8-D7-C5
  • the CD276 binding domain is the second binding domain
  • the PD-L1 binding domain is the first binding domain.
  • the MC-38-hCD276 (B7H3) tumor cells were maintained in vitro with DMEM medium supplemented with 10%fetal bovine serum at 37°C in an atmosphere of 5%CO 2 in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation.
  • mice Each mouse was inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 10 ⁇ 6) in 0.1 ml of PBS for tumor development. The date of randomization was denoted as day 0, and dosing starts from day 0.
  • the randomization will start when the mean tumor size reaches approximately 50-60 mm 3 .60 mice were enrolled in this study. All animals were randomly allocated to 7 study groups. Randomization was performed based on randomized block design.
  • Atezolizumab an anti-PD-L1 antibody, also named Tecentriq or MPDL3280A, see WO2010077634A1
  • Antengene-084M MAA271’s Fab sequence (see US8802091B) constructed with mouse IgG2a) were used as control.
  • Each Balb/c nude mouse for Calu-6 study was inoculated subcutaneously in the right front flank region with Calu-6 tumor cells (5 x 10 6 ) in 0.1 ml of PBS for tumor development. The randomization starts when the mean tumor size reaches approximately 122 mm 3 . 60 mice were enrolled in the study. The date of randomization was denoted as day 0, dosing starts from day 0.
  • a Mean ⁇ SEM
  • b TGI, T/C and p values were compared with group 1 tumor volume on day 24 by using Dunnett’s tests.
  • Monocyte cells were re-suspended at 5 x 10 6 in 3 ml complete medium supplemented with 2 U/ml of dendritic cell culture factor and then culture cells in 6-well plate. At the second day, 2 ml/well of fresh complete medium supplemented with 2 U/ml of dendritic cell culture factor were added for another three days culture. Then monocytes differentiated into immature dendritic cells (iDC) . After stimulated 48 hours with 2 U/ml of dendritic cell mature factor, iDCs would differentiate into mature dendritic cells.
  • iDC immature dendritic cells
  • the tube was put on a magnet for 1 min RT, the F (ab) 2 is in supernatant and the Fc was captured by beads.
  • 30-C7-C11-D4 can activate T cell activation while 25-C8-D7-C5 and BMK (MGA271) cannot.
  • 10-G6-C4-B2 and 30-C7-C11-D4 were chosen to perform humanization and PTM optimization.
  • the affinity of humanized candidates and optimized PTM sequences were evaluated by FACS.
  • Analyte B7H3.
  • Running buffer HBS-EP+.
  • Flow Rate 30 ⁇ L/min.
  • Capture Abs, 10 ⁇ L/min for 60 s.
  • Method Multiple cycle kinetics/affinity using capture.
  • Machine Model Biacore 8K (GE) . Analysis Temperature: 25°C.
  • Each Balb/c nude mouse for Calu-6 study was inoculated subcutaneously in the right front flank region with Calu-6 tumor cells (5 x 10 6 ) in 0.1 ml of PBS for tumor development.
  • the randomization starts when the mean tumor size reaches approximately 122 mm 3 .60 mice were enrolled in the study.
  • the date of randomization was denoted as day 0, dosing starts from day 0.
  • mice C57BL/6 were inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 10 6 ) in 0.1 ml of PBS for tumor development.
  • the randomization starts when the mean tumor size reaches approximately 139 mm 3 .66 mice were enrolled in the study.
  • the date of randomization was denoted as day 0, dosing starts from the day of randomization (day 0) .
  • mice C57BL/6 were inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 10 6 ) in 0.1 ml of PBS for tumor development.
  • the randomization starts when the mean tumor size reaches approximately 100 mm 3 .30 mice were enrolled in the study.
  • the date of randomization was denoted as day 0, dosing starts from the day of randomization (day 0) .
  • the humanized antibodies incorporated PTM optimization were constructed and expressed for the in vivo efficacy.
  • a bispecific antibody using B7H3 mAb 30-C7-C11-D4 and PD-L1 mAb (SHANGHAI ORIGINCELL MEDICAL TECHNOLOGY CO., LTD; see WO2019196309A1, YN035) with human IgG1 was also constructed in the format of IgG (H) scFV.
  • the N terminal of the anti-PD-L1 scFv VH followed by (GGGGS) 3-VL was linked to the C terminal of the Fc region of the B7H3-antibody.
  • mice C57BL/6 were inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 10 6 ) in 0.1 ml of PBS for tumor development.
  • the randomization starts when the mean tumor size reaches approximately 100 mm 3 .54 mice were enrolled in the study.
  • the date of randomization was denoted as day 0, dosing starts from the day of randomization (day 0) .
  • Bis-30-C7-C11-D4_hVH3-hVL2-PTM and Bis-30-C7-C11-D4_hVH4-hVL5-PTM showed comparable tumor growth inhibition with parental Bis-30-C7-C11-D4, as shown in Table 21 and Figure 15.

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Abstract

Provided are multi-specific molecules specific for CD276 and PD-L1, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the uses thereof.

Description

NOVEL MULTI-SPECIFIC ANTIBODIES AND USES THEREOF FIELD OF THE INVENTION
The present disclosure generally relates to novel multi-specific antibodies targeting CD276 and one or more additional targets.
BACKGROUND
B7-H3 (CD276, UniProt IDs for human amino acid sequence: Q5ZPR3 and mouse amino acid sequence: Q8VE98) is an important newly found immune checkpoint member of the B7 and CD28 families, which is a type I transmembrane co-stimulatory molecule, existing 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 has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. However, B7-H3 protein is expressed at high frequency on many different cancer types (60%of all cancers) ("B7-H3: an attractive target for antibody-based immunotherapy" . Clinical Cancer Research: clincanres. 2584.2020) .
The function of B7-H3 has been controversial. It was classified as either a co-stimulatory molecule for T cell activation that inhibits tumor antigen-specific immune responses, or the non-immunological role such as promoting migration, tumor growth, invasion, metastasis, malignant stage, recurrence rate, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism. The receptor for B7-H3 has been reported to be triggering receptor expressed on myeloid cell (TREM) -like transcript 2 (TLT-2, or TREML2) , which binds B7-H3 and costimulates activation of CD8 T cells in particular. B7-H3 is also reported an inhibitor for NK cells and osteoblastic cells by ligating unknown  receptor (s) . (The contrasting role of B7-H3, PNAS July 29, 2008, 105 (30) 10277-10278) .
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 observed tumor growth suppression in vitro and in vivo. CD276 is also reported to be expressed in hematological 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 can also be a potential target for treating hematological cancers.
Despite of the development of therapeutics targeting the CD276, there is a significant need for antibodies targeting CD276 as well as one or more additional targets, such as PD-L1.
BRIEF SUMMARY OF THE INVENTION
Throughout the present disclosure, the articles “a, ” “an, ” and “the” are used herein 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 more than one antibody.
The present disclosure provides novel anti-CD276 antibody molecules, amino acid and nucleotide sequences thereof, and uses thereof.
In one aspect, the present disclosure provides an multi-specific antibody or an 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.
In some embodiments, the first binding domain comprises 1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, 41-43, 49-51, 57-59, 65-67, 73-75, 81-83, 89-91, 97-99, 105-107, 113-115, 121-123, 129-131, 137-139, 145-147, 153-155, 161-163, 169-171, 177-179, 185-187, 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, and/or 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 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, 124-126, 132-134, 140-142, 148-150, 156-158, 164-166, 172-174, 180-181, 188-190, 196-198, 204-206, 212-214, 220-222, 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.
In some embodiments, the first binding domain comprises a heavy chain variable region selected from the group consisting of:
a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 1-3;
b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9-11;
c) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 17-19;
d) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 25-27;
e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 33-35;
f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 41-43;
g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 49-51;
h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 57-59;
i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 65-67;
j) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 73-75;
k) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 81-83;
l) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 89-91;
m) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 97-99;
n) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 105-107;
o) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 113-115;
p) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 121-123;
q) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 129-131;
r) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 137-139;
s) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 145-147;
t) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 153-155;
u) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 161-163;
v) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 169-171;
w) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 177-179;
x) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 185-187;
y) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 193-195;
z) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 201-203;
aa) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 209-211;
bb) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 217-219;
cc) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 225-227;
dd) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 233-235;
ee) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 241-243;
ff) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 249-251;
gg) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 257-259;
hh) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 265-267;
ii) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 273-275;
jj) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 281-283;
kk) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 289-291;
ll) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 297-299;
mm) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 305-307;
nn) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 313-315;
oo) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 321-323;
pp) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 329-331;
qq) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 337-339; and
rr) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9, 374 and 375.
In some embodiments, the first binding domain provided herein comprises a light chain variable region selected from the group consisting of:
a) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 4-6;
b) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 12-14;
c) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20-22;
d) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 28-30;
e) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 36-38;
f) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 44-46;
g) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 52-54;
h) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 60-62;
i) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 68-70;
j) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 76-78;
k) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 84-86;
l) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 92-94;
m) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 100-102, 108-110;
n) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 108-110;
o) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 116-118;
p) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 124-126;
q) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 132-134;
r) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 140-142;
s) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 148-150;
t) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 156-158;
u) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 164-166;
v) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 172-174;
w) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 180-181;
x) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 188-190;
y) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 196-198;
z) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 204-206;
aa) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 212-214;
bb) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 220-222;
cc) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 228-230;
dd) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 236-238;
ee) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 244-246;
ff) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 252-254;
gg) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 260-262;
hh) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 268-270, ;
ii) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 276-278;
jj) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 284-286;
kk) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 292-294;
ll) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 300-302;
mm) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 308-310;
nn) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 316-318;
oo) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 324-326;
pp) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 332-334;
qq) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 340-342;
rr) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 376, 13 and 14; and
ss) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 377, 45 and 46.
In some embodiments, the first binding domain comprises:
a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;
b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;
c) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22;
d) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30;
e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38;
f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46;
g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54;
h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62;
i) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70;
j) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78;
k) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86;
l) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94;
m) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102;
n) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110;
o) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118;
p) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126;
q) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 134;
r) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142;
s) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150;
t) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158;
u) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166;
v) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174;
w) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 179; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 180, SEQ ID NO: 181, and SEQ ID NO: 182;
x) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 187; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 189, and SEQ ID NO: 190;
y) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198;
z) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203; and a light  chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 204, SEQ ID NO: 205, and SEQ ID NO: 206;
aa) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 209, SEQ ID NO: 210, and SEQ ID NO: 211; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 214;
bb) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222;
cc) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 225, SEQ ID NO: 226, and SEQ ID NO: 227; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 228, SEQ ID NO: 229, and SEQ ID NO: 230;
dd) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 236, SEQ ID NO: 237, and SEQ ID NO: 238;
ee) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 244, SEQ ID NO: 245, and SEQ ID NO: 246;
ff) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 252, SEQ ID NO: 253, and SEQ ID NO: 254;
gg) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 260, SEQ ID NO: 261, and SEQ ID NO: 262;
hh) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 265, SEQ ID NO: 266, and SEQ ID NO: 267; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 268, SEQ ID NO: 269, and SEQ ID NO: 270;
ii) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 273, SEQ ID NO: 274, and SEQ ID NO: 275; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278;
jj) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 281, SEQ ID NO: 282, and SEQ ID NO: 283; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 284, SEQ ID NO: 285 and SEQ ID NO: 286;
kk) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 289, SEQ ID NO: 290, and SEQ ID NO: 291; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 292, SEQ ID NO: 293, and SEQ ID NO: 294;
ll) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 297, SEQ ID NO: 298, and SEQ ID NO: 299; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 300, SEQ ID NO: 301, and SEQ ID NO: 302;
mm) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 305, SEQ ID NO: 306, and SEQ ID NO: 307; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310;
nn) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 313, SEQ ID NO: 314, and SEQ ID NO: 315; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 316, SEQ ID NO: 317, and SEQ ID NO: 318;
oo) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 321, SEQ ID NO: 322, and SEQ ID NO: 323; and a light  chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 324, SEQ ID NO: 325, and SEQ ID NO: 326;
pp) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 332, SEQ ID NO: 333, and SEQ ID NO: 334;
qq) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 377, SEQ ID NO: 45, and SEQ ID NO: 46.
In some embodiments, the first binding domain comprises a heavy chain variable region 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 the homologue sequences of at least 80% (e.g., at  least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereof.
In some embodiments, the first binding domain comprises a light chain variable region 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: 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 the homologue sequences of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereof.
In some embodiments, the first binding domain comprises SEQ ID NO: 378 (EVQLVESGGGLXQPGXSLRLSCXTSGFTLSDYYMSWVRQXPGKGLEWVXF MRNKANXYTTEYSASVRGRFTISRDTSKSXIYLQMNSLXXEDTAVYYCVRDR XGRPFAYWGQGTLVTVSS) , wherein the X at position i (i=12, 16, 23, 40, 49, 57, 80, 89, 90 and 103) of SEQ ID NO: 378 is referred as XHi, wherein XH12 is V or I, XH16 is G or R, XH23 is A or T, XH40 is A or P, XH49 is G or S, XH57 is A or G, XH80 is I or T, XH89 is R or K, XH90 is A or T, XH103 is D or E; and a light chain variable region comprising SEQ ID NO: 379 (DIXMTQSPXSLXXXXGXXXXIXCKSSQSLLNXINQKNFLTWYXQKPGXXPX LLIYWASTRESGVPXRFSGSGSGTDFTLXISXXXXEDLXXYYCQNDYTYPLTF GQGTKLEIK) , 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 referred as XLi, wherein XL3 is V or Q, XL9 is D, L or S, XL12 is A, S or P, XL13 is A or V, XL14 is S or T, XL15 is L, V or P, XL17 is D or E, XL18 is R or P, XL19 is A or V, XL20 is S or T,  XL22 is N, T or S, XL32 is A or S, XL43 is Q or L, XL48 is Q or K, XL49 is A, P or S, XL51 is K or Q, XL66 is S or D, XL80 is K or T, XL83 is R or S, XL84 is L or V, XL85 is Q or E, XL86 is A or P, XL90 is A or G, XL91 is T or V.
In some embodiments, the first binding domain comprises a heavy chain variable region comprising SEQ ID NO: 380 (QVQLQESGPGLVKPSXTLSLTCXVXGYSITSDYAWNWIRQXPGKGLEWIGYI SHSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSLGRRWY FDVWGQGTTVTVSS) , wherein the X at position i (i=16, 23, 25 and 41) of SEQ ID NO:380 is referred as XHi, wherein XH16 is E or Q, XH23 is A or T, XH25 is S or Y, XH41 is H or P; and a light chain variable region comprising SEQ ID NO: 381 (DIXMTQSPXSLXXXXGXXXXIXCKSSQSLLXSSTQKNYLAWYXQKPGXXPX LLIYFASTRDSGVPXRFSGSGSGTDFTLXISXXXXEDLXXYFCQQHYIIPFTFG QGTKLEIK) , 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 referred as XLi, wherein XL3 is V or Q, XL9 is D, L or S, XL12 is A, S or P, XL13 is A or V, XL14 is S or T, XL15 is L, V or P, XL17 is D or E, XL18 is R or P, XL19 is A or V, XL20 is S or T, XL22 is N, T or S, XL31 is N or Q, XL43 is Q or L, XL48 is Q or K, XL49 is A, P or S, XL51 is K or Q, XL66 is S or D, XL80 is K or T, XL83 is R or S, XL84 is L or V, XL85 is Q or E, XL86 is A or P, XL90 is A or G, XL91 is T or V.
In some embodiments, the first binding domain comprises: (i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences shown 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 1, 2, or 3 CDR sequences shown 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 1, 2, or 3 CDR sequences shown as SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences shown as SEQ ID NO: 44 or 377 (QSLLQSSTQKNY) , SEQ ID NO: 45, and SEQ ID NO: 46, respectively.
In some embodiments, the first binding domain comprises a heavy chain variable region comprising a sequence selected from SEQ ID NO: 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 NO: 352, 354, 356, 359, 361, 363, 366, 368, 369, 371, 372 and 373.
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.
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.
In some embodiments, the first binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to human CD276. In some embodiments, the substitution is 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.
In some embodiments, the multi-specific antibody or antigen-binding fragment thereof further comprises an activating receptor binding domain, optionally a constant region of human Ig, or optionally a constant region of human IgG. In some embodiments, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4.
In some embodiments, the second target is PD-L1.
In some embodiments, the second binding domain comprises 1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 384-386, and/or 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 387-389. In some embodiments, the second binding domain comprises a heavy chain variable region comprising three CDR sequences set forth as SEQ ID NOs: 384, 385 and 386, respectively; and a light chain variable region comprising three CDR sequences set forth as SEQ ID NOs: 387, 388 and 389, respectively.
In some embodiments, the second binding domain comprises a heavy chain variable set forth as SEQ ID NO: 382, and/or a light chain variable region set forth as SEQ ID NO: 383.
In some embodiments, the second binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to human PD-L1. In some embodiments, the substitution is 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.
In some embodiments, the second binding domain comprising an scFv structure of VH-linker-VL. In some embodiments, the linker comprises a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
In some embodiments, the N-termination of the second binding domain is operably linked to the C-termination of the activating receptor binding domain.
In some embodiments, the multi-specific antibody or antigen-binding fragment thereof provided herein is a bi-specific or tri-specific antibody.
In some embodiments, the multi-specific antibody or antigen-binding fragment is linked to one or more conjugates. In some embodiments, the conjugate is covalently attached either directly or via a linker. In some embodiments, the conjugate comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
In one aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof provided herein.
In one aspect, the present disclosure provides a vector comprising the isolated polynucleotide provided herein.
In one aspect, the present disclosure provides a host cell comprising the vector provided herein.
In one aspect, the present disclosure provides a pharmaceutical composition comprising the multi-specific antibody or antigen-binding fragment thereof provided herein or the polynucleotide encoding the multi-specific antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
In one aspect, the present disclosure provides a method of expressing the multi-specific antibody or antigen-binding fragment thereof provided herein, comprising culturing the host cell provided herein under the condition at which the vector provided herein is expressed.
In one aspect, the present disclosure provides a method of treating a disease or condition in a subject that would benefit from modulation of CD276 activity, comprising administering to the subject a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof provided herein or the pharmaceutical composition provided herein.
In some embodiments, the disease or condition is a CD276 related disease or condition.
In some embodiments, the disease or condition is cancer, adaptive immune disease, autoimmune disease, inflammatory disease, or infectious disease.
In some embodiments, the cancer is adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a renal metastatic cancer, a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, a soft-tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a thyroid metastatic cancer, and a uterine cancer, optionally, wherein the cancer is chemoresistant.
In some embodiments, the disease or condition is hematological cancer selected from B-cell lymphomas, such as 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.
In some embodiments, the subject is human.
In some embodiments, the method provided herein comprises administering to the subject a therapeutically effective amount of one or more therapeutic agent. In some embodiments, said therapeutic agent is a chemotherapeutic agent, a radiation therapeutic agent, a hormonal therapeutic agent, a toxin or an immunotherapeutic agent.
In some embodiments, the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
In some embodiments, said method further comprises administration of one or more additional cancer therapies selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, hormonal therapy, and surgery.
In one aspect, the present disclosure provides a method of modulating CD276 activity in a CD276-expressing cell, comprising exposing the CD276-expressing cell to the antibody or antigen-binding fragment thereof provided herein.
In one aspect, the present disclosure provides a method of modulating PD-1/PD-L1 pathway activity in a PD-L1-expressing cell, comprising exposing the PD-L1-expressing cell to the multi-specific antibody or antigen-binding fragment thereof provided herein.
In one aspect, the present disclosure provides use of the multi-specific antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
In some embodiments, the medicament further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is a chemotherapeutic agent, a radiation therapeutic agent, a hormonal therapeutic agent, a toxin or an immunotherapeutic agent.
BRIEF DESCFRIPTION OF THE DRAWINGS
The drawings are for illustration purposes only not for limitation.
Figure 1 shows the binding affinity of the anti-CD276 antibodies provided herein on SKOV3 cells as measured by FACs analysis.
Figure 2A-2E show binding of 6-D8-E7-A11 to several cancer cell lines that express B7H3 as measured by FACS analysis.
Figure 3A-3C show ADCC effect of the anti-CD276 antibodies provided herein on SKOV3 cells.
Figure 4A and 4B show CDC effect of the anti-CD276 antibodies provided herein on CHO-S-hCD276 cells.
Figure 5A-5E show indirect ADC cytotoxicity effect of the anti-CD276 antibodies provided herein on SKOV3 cells.
Figure 6 shows in vivo efficacy of the anti-CD276 antibodies provided herein in inhibiting the tumor growth in the mouse model inoculated with MC-38-hCD276 (B7H3) tumor cells.
Figure 7 shows effects on tumor growth in subcutaneous calu-6 model in balb/c nude mice (mean± sem) .
Figure 8 shows IL2 release by T cell activation in MLR assay.
Figure 9 shows IFNγ release by T cell activation in MLR assay.
Figure 10 shows the binding affinity on SKOV3 of humanized antibodies derived from 30-C7-C11-D4.
Figure 11 shows binding affinity on SKOV3 of humanized antibodies derived from 10-G6-C4-B2.
Figure 12 shows effects on tumor growth in subcutaneous Calu-6 model in Balb/c nude mice (Mean± SEM) .
Figure 13 shows effects of test articles on tumor growth in subcutaneous MC-38-hCD276 (B7H3) model in C57BL/6 Mice (Mean ± SEM) .
Figure 14 shows effects of test articles on tumor growth in subcutaneous MC-38-hCD276 (B7H3) Model in C57BL/6 Mice (Mean± SEM) .
Figure 15 shows effects of test articles on tumor growth in subcutaneous MC-38-hCD276 (B7H3) Model in C57BL/6 Mice (Mean± SEM) .
DETAILED DESCRIPTION OF THE INVENTION
The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.
Definitions
The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, or monovalent antibody that binds to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (VH) and a first, second, and third constant region (CH1, CH2, CH3, respectively) ; mammalian light chains are classified as λ or κ, while each light chain consists of a variable region (VL) and a constant region. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions 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 the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, and HCDR3) . CDR boundaries for the antibodies and antigen-binding domains disclosed herein may be defined or identified by the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., 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, A.M., J. Mol. Biol., 196, 901 (1987) ; N. R. Whitelegg et al, Protein Engineering, v13(12) , 819-824 (2000) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83
; Kabat E. A. 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 flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support 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 assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (gamma1 heavy chain) , IgG2 (gamma2 heavy chain) , IgG3 (gamma3 heavy chain) , IgG4 (gamma4 heavy chain) , IgA1 (alpha1 heavy chain) , or IgA2 (alpha2 heavy chain) .
The term “antibody molecule” as used herein refers to an antigen-binding protein or polypeptide comprising at least one antibody fragment (such as CDR, and/or variable region sequence) . An antibody molecule includes, for example, a monoclonal antibody, an antibody fragment or domain, a fusion protein comprising an  antibody fragment or domain, a polypeptide complex comprising an antibody fragment or domain, and so on.
The term “antigen-binding domain” (e.g. CD276-binding domain or PD-L1 binding domain) as used herein 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 domain include, without limitation, a Fab, a Fab', a F (ab') 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a single-chain antibody molecule (scFv) , a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. 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 to a framework region from one or more different human antibodies. For more and detailed formats of antigen-binding domain are described in Spiess et al, 2015, and Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) , which are incorporated herein by entirety reference.
“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
“Fab'” refers to a Fab fragment that includes a portion of the hinge region.
“F (ab') 2” refers to a dimer of Fab’ .
A “fragment difficult (Fd) ” with regard to an antibody refers to the amino-terminal half of the heavy chain fragment that can be combined with the light chain to form Fab. For example, Fd fragment may consists of the VH and CH1 domains
“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. A number of Fv designs have been provided, including dsFvs, in which the  association between the two domains is enhanced by an introduced disulphide bond; and scFvs can be formed using a peptide linker to bind the two domains together as a single polypeptide. Fvs constructs containing a variable domain of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced.
“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 one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .
A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage 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') ” comprises three peptide chains: two VH moieties linked by a peptide linker (e.g., a long flexible linker) and bound to two VL moieties, respectively, via disulfide bridges. In some embodiments, dsFv-dsFv'is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
“Fc” with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding. The Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
“Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two VH domains and no 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 Camelidae (camels, dromedaries, and llamas) . Although devoid of  light chains, camelized antibodies have an authentic 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 a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .
A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
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. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.
The term “chimeric” as used herein, means an antibody or antigen-binding domain, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
The term “humanized” as used herein means that the antibody or antigen-binding domain comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
The term “operably link” or “operably linked” refers to a juxtaposition, with or without a spacer or a linker or an intervening sequence, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to  function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity. For another example, an antigen-binding domain can be operably linked to another antigen-binding domain with an intervening sequence there between, and such intervening sequence can be a spacer or can comprise a much longer sequence such as a constant region of an antibody. The term may also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.
The term “fusion” or “fused” when used with respect to amino acid sequences (e.g. peptide, polypeptide or protein) refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence which does not exist naturally. A fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
An “antigen” as used herein refers to a compound, composition, peptide, polypeptide, protein or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma) , or injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody) , including those induced by heterologous antigens.
The term “CD276 protein” or “B7-H3 protein” as used herein is intended to encompass any form of CD276, for example, 1) native unprocessed CD276 molecule,  “full-length” CD276 chain or naturally occurring variants of CD276, including, for example, splice variants or allelic variants; 2) any form of CD276 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CD276 subunit generated through recombinant method.
The term “anti-CD276 antibody” , “anti-CD276 binding domain” or “CD276-binding domain” refers to an antibody or antigen-binding domain that is capable of specifically binding to CD276 (e.g. human, monkey or mouse CD276) .
“PD-L1” as used herein refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192: 1027) . Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3. PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells. The binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation. Accordingly, PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.
The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
In certain embodiments, the antibody molecules or antigen-binding domains provided herein specifically bind to CD276 and/or PD-L1 with a binding affinity (KD) of ≤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, ≤4x10-9M) . KD used herein refers to the ratio of the dissociation rate to the association rate (koff/kon) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. In certain embodiments, the KD value can be appropriately determined by using flow cytometry.
Binding affinity to CD276 and/or PD-L1 can also be represented by “half maximal effective concentration” (EC50) value, which refers to the concentration of an antibody where 50%of its maximal effect (e.g., binding or inhibition etc. ) is observed. The EC50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays. In certain embodiments, the antibodies and the fragments thereof provided herein specifically bind to CD276 and/or PD-L1 at an EC50 (i.e. 50%binding concentration) of no more than 0.05 nM, no more than 0.06 nM, no more than 0.07 nM, no more than 0.08 nM, no more than 0.09 nM, no more than 0.1 nM, no more than 0.2 nM, no more than 0.3 nM, no more than 0.4 nM, no more than 0.5 nM, no more than 0.6 nM, no more than 0.7 nM, no more than 0.8 nM, no more than 0.9 nM, no more than 1 nM, no more than 1.5 nM, no more than 2 nM, no more than 2.5 nM, no more than 3.5 nM, no more than 3 nM, no more than 4 nM, no more than 4.5 nM, no more than 5 nM, no more than 6 nM, no more than 7 nM, no more than 8 nM, no more than 9 nM, or no more than 10 nM, by flow cytometry assay.
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 may be greater than 85%, or greater than 90%.
The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitope) . Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on the protein and the small segments of the contiguous amino acids can be digested from an antigen binding with major histocompatibility complex (MHC) molecules or retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding domain blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding domain may be considered to bind the same/closely related epitope as the reference antibody.
A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) . As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
The term “homolog” and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.
“Percent (%) sequence identity” with respect to 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 to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al, J. Mol. Biol., 215: 403–410 (1990) ; Stephen F. et al, Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D. G. et al, Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A. et al, Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.
“Effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex, Fc receptor and effector cell (e.g., macrophage) . Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and antibody-dependent cellular phagocytosis (ADCP) induced by binding of Fc region of  an antibody to phagocytosis. It has become well established that the specific glycan structures associated with the conserved bi-antennary glycan in the Fc-CH2 domain can strongly influence the interaction with the FcyRs that mediate ADCC and ADCP and with Clq binding, the initial binding event leading to CDC (see Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015; 25: 1325-34) .
“Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
The term “subject” or “individual” or “animal” or “patient” as used herein refers to human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease or disorder. Mammalian subjects include humans, monkeys, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for  controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector.
The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
A “CD276-related” disease or condition as used herein refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of CD276. In some embodiments, the CD276 related condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease.
A “PD-L1-related” disease or condition as used herein refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased expression or activities of PD-L1. In some embodiments, the PD-L1 related disease or condition is accompanied with a suppressed immune system. In some embodiments, the PD-L1 related disease or condition is immune-related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease.
“Cancer” as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration 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 cancer or tumors include adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell  tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a renal metastatic cancer, a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, a soft-tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a thyroid metastatic cancer, and a uterine cancer.
In certain embodiments, the hematological malignancies includes B-cell lymphomas, 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.
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 a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.
In certain embodiments, the cancer is chemoresistant. The term “chemoresistant cancer” as used herein refers to a type of cancer that are not responsive to the effects of chemotherapy. For example, a cancer that has been  responding to a chemotherapy or a combination of different chemotherapies suddenly begins to grow can be referred to as a chemoresistant cancer.
The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
Multi-specific antibody or antigen-binding fragments thereof
In one aspect, the present disclosure provides a multi-specific antibody or antigen-binding fragment thereof comprises multiple antigen binding domains and an activating receptor binding domain.
i. Multiple antigen binding domains
In some embodiments, the multiple antigen binding domains comprise at least a first binding domain targeting a first antigen and a second binding domain targeting a second antigen, wherein one of the first antigen and the second antigen is an immune checkpoint molecule or a tumor antigen, while the other one is one or more additional targets.
In some embodiments, the one or more additional targets comprise a target which is another immune checkpoint molecule (e.g., PD-L1) . In some other embodiments, the one or more additional targets comprise targets relating to immune cell (e.g., T cells or B cells) function. In some embodiments, the one or more additional targets comprises a target selected from the group consisting of CD40, CD3, CD28, etc.
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.
CD276 binding domain
In certain embodiments, the CD276 binding domain comprises one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CD276 antibody 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-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, 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 are derived from human, monkey or mouse. In certain embodiments, the CD276 is a recombinant CD276 or a CD276 expressed on a cell surface.
All of these 43 anti-CD276 antibodies thereof provided herein are mouse monoclonal antibodies. Table 1 shows the CDR sequences of these 43 anti-CD276 antibodies according to IMGT numbering system. The heavy chain and light chain variable region sequences are also provided below.
Table 1. CDR sequences of the anti-CD276 antibodies




Heavy or light chain variable region sequences of 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-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, 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 antibodies are provided in Table 2.
Table 2. Variable region sequences of the anti-CD276 antibodies







CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs in the CD276 binding domain comprising the CDRs from anti-CD276 antibody 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-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, 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, or 21-B3-B1-H5, yet substantially retain the specific binding affinity to CD276.
In certain embodiments, the CD276 binding domain comprises a heavy chain CDR3 sequence of one of 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-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, 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 certain 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. Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81) . The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) as well as desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263: 551-67) .
In certain embodiments, the CD276 binding domain comprise suitable framework region (FR) sequences, as long as the antibodies and/or antigen-binding fragments thereof can specifically bind to CD276. The CDR sequences provided in Table 1 are obtained from mouse antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
In certain embodiments, sequences of the CD276 binding domain are PTM optimized. As used herein, PTM optimization refers to post-translation modification, aiming at avoiding potential aggregation, activity loss or other risk. Exemplary PTM optimized CD276 binding domain includes the antigen-binding domain of 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, wherein all the CDR regions are underlined.
Table 3. CDR sequences and variable region sequences of the anti-CD276 antibodies with PTM optimization
In certain embodiments, the CD276 binding domain is humanized. A humanized antibody or antigen-binding fragment is desirable in its reduced immunogenicity in human. A humanized antibody is chimeric in its variable regions, as non-human CDR sequences are grafted to human or substantially human FR sequences. Humanization of an antibody or antigen-binding fragment can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536) .
Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art. In an illustrative  example, “best-fit” approach can be used, where 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 the human scaffold for grafting the non-human CDR sequences (see, for example, 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 may be used for the grafting of the non-human CDRs (see, for example, 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 the CDR sequences which are non-human. In some embodiments, the variable region FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
Table 4 below shows the heavy chain and light chain variable region amino acid sequences of humanized antibodies for 10-G6-C4-B2 and 30-C7-C11-D4, which are designated as hVH2-hVL1-10, hVH3-hVL3-10, hVH4-hVL1-10, hVH4-hVL2-10, hVH5-hVL1-10, hVH5-hVL3-10, 10-G6-C4-B2_hVH2-VL1_PTM, 10-G6-C4-B2_hVH3-VL3_PTM, 10-G6-C4-B2_hVH4-VL1_PTM, 10-G6-C4-B2_hVH4-VL2_PTM, 10-G6-C4-B2_hVH5-VL1_PTM, 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-C11-D4_hVH2-hVL1_PTM, 30-C7-C11-D4_hVH3-hVL2_PTM, 30-C7-C11-D4_hVH3-hVL5_PTM, 30-C7-C11-D4_hVH4-hVL2_PTM and 30-C7-C11-D4_hVH4-hVL5_PTM, wherein all the CDR regions are underlined.
Table 4. Variable region amino acid sequences of the humanized antibodies




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 FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived from different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody. In some embodiments, the humanized CD276 domain comprises human heavy chain HFR1-4, and/or light chain LFR1-4.
In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or its fragment closely approximate the non-human parent antibody structure, so as to optimize binding characteristics (for example, increase binding affinity) . In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises 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 the FR sequences of a heavy or a light chain variable domain. In some embodiments, such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains. In certain embodiments, one or more amino acids of the human FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequences are back mutated to the corresponding amino acid (s) of the parent non-human antibody so as to increase binding affinity.
PD-L1 binding domain
In some embodiments, the variable regions of the PD-L1 binding domain comprised in the multi-specific antibody or antigen-binding fragments thereof disclosed herein can be any of those PD-L1 antibody heavy chain variable regions and light chain variable regions known in the art. In certain embodiments, the PD-L1 binding domain comprises the heavy chain variable region and the light chain variable region from YN035 recited in WO2019196309A1.
Table 5. CDR sequences of the PD-L1 binding domain
Table 6. Variable region sequences of the PD-L1 binding domain
ii. Activating receptor binding domain
The multi-specific antibody or antigen-binding fragments thereof further comprises an activating receptor-binding domain. As used herein, the term “activating receptor” refers to receptors (e.g., FcγR) expressed on an immune effector cell (e.g., a phagocytic cell, such as a macrophage) , which upon activation by binding to, for example, Fc domain, mediates at least one effector functions of the immune effector cell (e.g., a phagocytic cell, such as a macrophage) or pro-inflammatory response. In  certain embodiments, the immune effector cell provided herein co-expresses CD276 and one or more additional targets (e.g., PD-L1) .
In certain embodiments, the activating receptor is an FcγR, and the activating receptor-binding domain is an Fc domain. Fc domain can activate Fc receptors (FcRs) on macrophages to drive a phosphorylation cascade propagated by the receptors’ immunoreceptor tyrosine-based activation motifs (ITAMs) . 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 featured with a conserved amino acid sequence motif, which consists of paired YXXL/I motifs (wherein Y, L and I refer to Tyrosine, Lysine and Isoleucine respectively) and separated by a defined interval of six to eight amino acids. Phosphorylation of residues within the ITAM recruits several signaling molecules for phagocytosis activation. Accordingly, the activating receptor can be any receptor expressed on an immune effector cell that can be bound and activated to induce phagocytosis via an ITAM-comprising intracellular phagocytosis signaling domain.
In other embodiments, the activating receptor is a receptor involved in a different phagocytic signaling or mechanism, such as, Akt mediated signaling cascade (via CD19, CD28, CSFR or PDGFR receptor) , clustering of a group of receptors on an immune effector cell (e.g., macrophage) that potentiates phagocytosis (via, for example, integrins or selectins) , or antigen mediated cytotoxicity (via FcDR1 (CD89) receptor or CD206) .
For example, activating receptors that are relevant to effector functions such as phagocytosis can be fragment crystallizable γ receptors (FcγRs) , TREM2, lectin, 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, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, dectin 1, RAGE (SR-E1) , LRPl, LRP2, ASGP, SR-PSOX, 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. In certain embodiments, the activating receptor is FcγR.
In certain embodiments, the activating receptor that can generate pro-inflammatory signals upon activation, include without limitation, PI3K, FcγRl, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, BAH. Tyro3, Axl, Traf6, Syk, MyD88, Zap70, FcεRl, Fc αRl, BAFF-R, DAP 12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.
The term “activating receptor-binding domain” , as used herein, refers to domain (e.g., a portion of an antibody) that is capable of specifically binding to an activating receptor on an immune effector cell and such binding leads to activation of the receptor as well as downstream signaling thereof (e.g., effector function or pro-inflammatory response of the immune cell) . For example, the activating receptor-binding domain comprises an Fc domain of an antibody or a variant thereof. In certain embodiments, the Fc domain can be derived from IgG1 or IgG4.
In certain embodiments, the activating receptor-binding domain of the multi-specific molecules provided herein binds and activates activating receptor selected from the group consisting of fragment crystallizable γ receptors (FcγRs) , TREM2, lectin, 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, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, dectin 1, RAGE (SR-E1) , LRPl, LRP2, ASGP, SR-PSOX, 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, DAP 12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.
In certain embodiments, the activating receptor-binding domain of the multi-specific molecule provided herein comprises a Fc domain or a variant thereof, which activates Fc receptors (FcRs) on macrophages, such as FcγRII.
In some embodiments, the activating receptor-binding domain comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.
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 human IgG1, IgG2, IgG3, or IgG4.
Human IgG isotypes (the subclasses of mature gamma globulin class G antibodies; IgG1, IgG2, IgG3 and IgG4) exhibit differential capacity 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 engagement of such effector functions is based on selectivity for Fc receptors on distinct immune cells and the ability to bind C1q thereby activating the assembly of a membrane attack complex (MAC) . Among the various isotypes, relative affinity for Fcγ receptors, which include FcγRI, FcγRIIa/b/c, and FcγRIIIa/b is high for IgG1 and IgG3. However, Fcγ affinity for IgG2 is considerably lower with the exception of FcγRIIa H131 polymorphism and IgG4 only has measurable affinity for FcγRI.
In certain embodiments, the activating receptor-binding domain is derived from human IgG1 isotype, which could induce ADCC, CDC or ADCP, or a constant region of IgG4 or IgG2 isotype, which has reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing tumor antigen (e.g., CD276) . Effector functions can be evaluated using various assays such as Fc receptor binding assay, C1q binding assay, and cell lysis assay.
In certain embodiments, the activating receptor-binding domain is derived from a constant region of mouse IgG2 isotype, which could induce ADCC, CDC or ADCP.
iii. Configuration of the multi-specific antibody or antigen-binding  fragments thereof
In certain embodiments, the multi-specific antibody or antigen-binding fragments thereof provided herein is a recombinant protein comprising multiple antigen binding domains described throughout the specification, wherein each of the multiple antigen binding domains has specific binding affinity to each targeted antigen, and is connected with each other by one or more linkers. The one or more linkers may have cognate peptides that exhibit complementary binding with each other. For example, the first binding domain of the multi-specific antibody or  antigen-binding fragments thereof provided herein is fused with the first of a pair of cognate peptides, while the second binding domain thereof is fused with the second of the pairs of cognate peptides; as such, the first binding domain and the second binding domain can be connected by the pair of cognate peptides through the complementary binding between each of the pair of cognate peptides.
In certain embodiments, the pair of cognate peptides comprises two heavy chains of an antibody or any complementary portion thereof, a pair of light chain and heavy chain complementary to each other of an antibody or any complementary portion thereof, leucine zipper domains that exhibit complementary binding with each other (e.g., the zipper sequences within the binding regions of c-Fos and c-June proteins) , or synthetic peptides designed to specifically bind to each other via synthetic connectors.
The multiple antigen binding domains (e.g., the first binding domain and the second binding domain) and the activating receptor binding domain of the multi-specific antibody or antigen-binding fragments thereof provided herein can also be connected via chemical binding, such as crosslinking (e.g., BS2G crosslinker  (Bis [Sulfosuccmimidyl] glutarate) , BS3 crosslinker (Bis [sulfosuccinimidyl] suberate) , Sulfo-DSS, DST crosslinker (Disuccinimidyl tartrate) , BMPS (N- (B-Maleimidopropyloxy) succinimide ester; MBS crosslinker (mMaleimidobenzoyl-N-hydroxysuccinimide ester) ; or PDPH crosslinker (3- [2-Pyridyldithio] propionylhydrazide) ) .
In certain embodiments, the first binding domain (e.g., CD276 binding domain or PD-L1 binding domain) is linked to N-termination of the activating receptor-binding domain (e.g., Fc domain) . In certain embodiments, the first binding antibody domain (e.g., CD276 binding domain or PD-L1 binding domain) comprises a Fab domain, optionally, the Fab domain comprises a heavy chain linked to one of the N-termination of the activating receptor-binding domain (e.g., Fc domain) .
In certain embodiments, both of the first binding domain and the second binding domain comprise a Fab domain, optionally, each of the Fab domains comprises a heavy chain linked to each N-termination of the activating receptor-binding domain (e.g., Fc domain) , respectively.
In certain embodiments, the second binding domain (e.g., CD276 binding domain or PD-L1 binding domain) is linked to the activating receptor-binding domain (e.g., Fc domain) or to the first binding domain. In certain embodiments, the second binding domain comprises an scFv structure.
In certain embodiments, the second binding domain is linked to the C-termination of light chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) . In certain embodiments, the second binding domain is linked to the N-termination of light chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) . In certain embodiments, the second binding domain is linked to the C-termination of heavy chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) . In certain embodiments, the second binding domain is linked to the N-termination of heavy  chain of the first binding domain (e.g., the first binding domain comprising a Fab domain) .
As used herein, the term “linked to” refers to covalent or non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds) between two components.
In certain embodiments, the second binding domain is linked to C-termination of the activating receptor-binding domain (e.g., Fc domain) . In certain embodiments, the first binding domain (e.g., the first binding domain comprising a Fab domain) is linked to the N-termination of activating receptor-binding domain, and the second binding domain is linked to the C-termination of activating receptor-binding domain. In such embodiments, the first binding domain (e.g., CD276 binding domain or PD-L1 binding domain) and the activating receptor binding domain (e.g., Fc domain) of the multi-specific antibody or antigen-binding fragments thereof disclosed herein together form an intact antibody structure. In some embodiments, the second binding domain comprises an scFv structure from N-termination to C-termination comprising a second heavy chain variable region, a first linker and a second light chain variable region. In certain embodiments, the first linker has a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
In some embodiments, the N-termination of the second binding domain is linked to the C-termination of the activating receptor binding domain via a second linker. In certain embodiments, the second linker has a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
In certain embodiments, the multi-specific antibody or antigen-binding fragments thereof disclosed herein comprises two second binding domains, each of which is operably linked to each C-termination of the activation receptor binding domain, respectively.
In some embodiments, the multi-specific antibody or antigen-binding fragments thereof comprises four chains, which from N-termination to C-termination have the structures as following:
Chains 1 and 4: VL1-CL;
Chains 2 and 3: VH1-CH1-hing region-CH2-CH3-linker 2-VH2-linker 1-VL2;
wherein VH1 stands for the heavy chain variable region of the first binding domain, VL1 stands for the light chain variable region of the first binding domain; VH2 stands for the heavy chain variable region of the second binding domain, VL2 stands for the light chain variable region of the second binding domain; linker 1 stands for the first linker; and linker 2 stands for the second linker.
In some certain embodiments, the first binding domain is a CD276 binding domain and the second binding domain is a PD-L1 binding domain. In some other certain embodiments, the first binding domain is a PD-L1 binding domain and the second binding domain is a CD276 binding domain.
Antibody Variants
The present disclosure also encompasses various variants of the multi-specific antibodies and/or antigen-binding fragments thereof provided herein. In certain embodiments, the present disclosure encompasses various types of variants of an exemplary antibody provided herein, i.e., the antibody with 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.
In certain embodiments, the antibody variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, 3 or 5, one or more FR sequences, the heavy or light chain variable region sequences  provided in Table 2, 4 or 6, and/or the activating receptor binding domain (e.g. Fc region) . Such variants retain specific binding affinity to CD276 and/or PD-L1 of their parent antibodies, but have one or more desirable properties conferred by the modification (s) or substitution (s) . For example, the antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function (s) , improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues) .
The parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) . Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) , and the modified antibodies are produced and screened for the interested property. If substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution. The potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc. ) .
Affinity variant
Affinity variant may contain modifications or substitutions in one or more CDR sequences as provided in Table 1, 3 or 5, one or more FR sequences, or the heavy or light chain variable region sequences in provided in Table 2, 3, 4 or 6. The affinity variants retain specific binding affinity to CD276 and/or PD-L1 of the parent antibody, or even have improved CD276 and/or PD-L1 specific binding affinity over the parent antibody. In certain embodiments, at least one (or all) of the substitution (s) in the CDR sequences, FR sequences, or variable region sequences comprises a conservative substitution.
A skilled artisan will understand that in the CDR sequences provided in Table 1 or 5, and FR sequences, one or more amino acid residues may be substituted yet the resulting antibody or antigen-binding fragment still retain the binding affinity to CD276 and/or PD-L1, or even have an improved binding affinity. Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to CD276 and/or PD-L1. For another example, computer software can be used to virtually simulate the binding of the antibodies to CD276 and PD-L1, and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity, or targeted for substitution to provide for a stronger binding.
In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences. In certain embodiments, an affinity variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences in total.
In certain embodiments, the multi-specific antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1 or 5, and in the meantime retain the binding affinity to CD276 and/or PD-L1 at a level similar to or even higher than its parent antibody.
In certain embodiments, the multi-specific 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 that (or those) listed in Table 2, 4 or 6, and in the meantime retain the binding affinity to CD276 and/or PD-L1 at a level similar to or even higher than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have  been substituted, inserted, or deleted in a sequence selected from that (or those) listed in Table 1. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs) .
Glycosylation variant
The multi-specific antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment thereof.
The multi-specific antibody or antigen binding fragment thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, 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-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
Cysteine-engineered variant
The multi-specific antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
A free cysteine residue is one which is not part of a disulfide bridge. A cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
Fc Variant
The multi-specific antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.
In certain embodiments, the multi-specific antibodies or antigen-binding fragments comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) . Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with 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) .
In certain embodiments, the multi-specific antibodies or antigen-binding fragments comprise one or more amino acid substitution (s) that alters the antibody-dependent cellular cytotoxicity (ADCC) . Certain amino acid residues at CH2 domain of the Fc region can be substituted to provide for enhanced ADCC activity. Alternatively or additionally, carbohydrate structures on the antibody can be changed to enhance ADCC activity. Methods of altering ADCC activity by antibody engineering have been described in the art, see for example, Shields RL. et al., J Biol  Chem. 2001.276 (9) : 6591-604; Idusogie EE. et al., J Immunol. 2000.164 (8) : 4178-84; Steurer W. et al., J Immunol. 1995, 155 (3) : 1165-74; Idusogie EE. et al., J Immunol. 2001, 166 (4) : 2571-5; Lazar GA. et al., PNAS, 2006, 103 (11) : 4005-4010; Ryan MC. et al., Mol. Cancer Ther., 2007, 6: 3009-3018; Richards JO, . et al., Mol Cancer Ther. 2008, 7 (8) : 2517-27; Shields R. L. et al, J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278: 3466-3473.
In certain embodiments, the multi-specific antibodies or antigen-binding fragments comprise one or more amino acid substitution (s) that alters Complement Dependent Cytotoxicity (CDC) , for example, by improving or diminishing C1q binding and/or CDC (see, for example, 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 concerning other examples of Fc region variants.
In certain embodiments, the multi-specific antibodies or antigen-binding fragments thereof comprise one or more amino acid substitution (s) in the interface of the Fc region to facilitate and/or promote heterodimerization. These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731,168.
Conjugates
In some embodiments, the multi-specific antibodies and antigen-binding fragments thereof is linked to one or more conjugates, optionally, wherein the conjugate is covalently attached either directly or via a linker. A conjugate is a non-proteinaceous moiety that can be attached to the 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, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse  and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugates may be linked to the multi-specific antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods. In some embodiments, the conjugate comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
In certain embodiments, the multi-specific antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate.
In certain embodiments, the multi-specific antibodies may be linked to a conjugate indirectly, or through another conjugate. For example, the multi-specific antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin. The conjugate can be a toxin (e.g., a chemotherapeutic agent) , a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) .
A “toxin” can be any agent that is detrimental to cells or that can damage or kill cells. Examples of toxin include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) ,  cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g., dactinomycin (formerly actinomycin) , bleomycin, mithramycin, and anthramycin (AMC) ) , and anti-mitotic agents (e.g., vincristine and vinblastine) .
Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotopes (e.g. 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32P, other lanthanides, luminescent labels) , chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection.
In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety which helps increase half-life of the antibody. Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. 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 if more than one polymer are attached, they can be the same or different molecules.
In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead.
In certain embodiments, the multi-specific antibodies and/or antigen-binding fragments thereof provided herein is used for a base for a conjugate.
Polynucleotides and Recombinant Methods
The present disclosure provides isolated polynucleotides that encode the multi-specific antibodies and antigen-binding fragments thereof. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes  encoding the heavy and light chains of the antibody) . The encoding DNA may also be obtained by synthetic methods.
The isolated polynucleotide that encodes the multi-specific antibodies and antigen-binding fragments thereof can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: 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.
In some embodiments, the vector system includes mammalian, bacterial, yeast systems, etc, and comprises plasmids 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, and other laboratorial and commercially available vectors. Suitable vectors may include, plasmid, or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) .
Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for multi-specific antibody-encoding  vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424) , K. bulgaricus (ATCC 16,045) , K. wickeramii (ATCC 24,178) , K. waltii (ATCC 56,500) , K. drosophilarum (ATCC 36,906) , K. thermotolerans, and K. marxianus; yarrowia (EP 402,226) ; Pichia pastoris (EP 183,070) ; Candida; Trichoderma reesia (EP 244,234) ; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (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 liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .
Host cells are transformed with the above-described expression or cloning vectors for multi-specific antibody production 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, the antibody may be produced by homologous recombination known in the art.
The host cells used to produce the multi-specific antibodies or antigen-binding fragments provided herein may 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 the 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. No. 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. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary 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 GENTAMYCINTM drug) , trace elements (defined as inorganic compounds usually 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 that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously  used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, the multi-specific antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The multi-specific antibodies and antigen-binding fragments thereof prepared from the 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 the preferred purification technique.
In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J.  5: 1567 1575 (1986) ) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX. TM. resin (J.T. Baker, Phillipsburg, N.J. ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step (s) , the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
Pharmaceutical Composition
The present disclosure further provides pharmaceutical compositions comprising the multi-specific antibodies or antigen-binding fragments thereof and one or more pharmaceutically acceptable carriers.
Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
Suitable components 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 hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a composition comprising an antibody or antigen-binding fragment and conjugates as provided herein decreases oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain embodiments compositions are provided that comprise one or more multi-specific antibodies or antigen-binding fragments thereof as 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 an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
To further illustrate, pharmaceutical 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, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at 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 carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering 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. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose  containers that include phenols or cresols, 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 buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.
In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other  pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the anti-CD276 antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.
Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
Methods of Use
The present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof as provided herein to a subject in need thereof, thereby treating or preventing a CD276 and/or PD-L1 related disease or condition. In some embodiment, the CD276-related disease or condition is cancer, autoimmune disease, inflammatory disease, adaptive immune disease or infectious disease.
Examples of cancer include but are not limited to, non-small cell lung cancer (squamous/nonsquamous) , small cell lung cancer, renal cell cancer, colorectal  cancer, colon cancer, ovarian cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast 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, myelomas, mycoses fungoids, merkel cell cancer, hepatocellular carcinoma (HCC) , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid malignancy, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, chronic myelocytic (granulocytic) leukemia, chronic myelogenous 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 effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary CNS lymphoma, spinal axis tumor, brain stem glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.
In certain embodiments, the cancer is adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer,  a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a renal metastatic cancer, a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, a soft-tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a thyroid metastatic cancer, and a uterine cancer. In certain embodiments, the cancer is chemoresistant.
In certain embodiments, the disease or condition is hematological cancer chosen from B-cell lymphomas. Examples of B-cell lymphomas includes 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.
Autoimmune diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is 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) , Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos'disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (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, pemacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS) , also known as systemic sclerosis (SS) ) , Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.
Inflammatory disorders, include, for example, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury. In some embodiments, the CD276 associated conditions are inflammatory diseases such as systemic lupus erythematosus (SLE) , intestinal mucosal inflammation, wasting disease associated with colitis, multiple  sclerosis, viral infections, rheumatoid arthritis, osteoarthritis, Cohn's disease, and inflammatory bowel disease, psoriasis, systemic scleroderma, autoimmune diabetes and the like.
Infectious disease include, but are not limited to, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, Trichuriasis, Trypanosomiasis, helminth infection, infection of hepatitis B (HBV) , hepatitis C (HCV) , herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papilloma virus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, Kaposi West sarcoma associated herpes virus epidemics, thin ring virus (Torquetenovirus) , human T lymphotrophic viruse I, human T lymphotrophic viruse II, varicella zoster, JC virus or BK virus.
In certain embodiments, the subject is human.
In another aspect, methods are provided to treat a disease or condition in a subject that would benefit from modulation of CD276 activity, comprising administering a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof as provided herein to a subject in need thereof. In yet another aspect, methods are provided to treat a disease or condition in a subject that would benefit from modulation of PD-1/PD-L1 pathway activity, comprising administering a therapeutically effective amount of the multi-specific antibody or  antigen-binding fragment thereof as provided herein to a subject in need thereof. The term “disease or condition” as used herein can be used interchangeably with the term “CD276 and/or PD-L1 related disease or condition” .
The 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 for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
In certain embodiments, an multi-specific antibody or antigen-binding fragment thereof as provided herein may be administered at a therapeutically effective dosage 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 dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage 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 administration dosage may change over the course of treatment. For example, in certain embodiments, the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
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.
The multi-specific antibodies and antigen-binding fragments thereof disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
In some embodiments, the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents. In some embodiments, the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered alone or in combination with a second therapeutic agent. For example, the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with a second therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug. In certain embodiments, the multi-specific antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with an antagonist of one or more immunoinhibitory molecule, e.g., CD24, CD47, SIRPα, PD-L1, or the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M) . The term “antagonist” as used herein comprises can refer to any small molecule, small or micro RNAs, or antibodies or antigen-binding fragments thereof that blocks or inhibits binding of CD24, CD47, SIRPα, PD-L1 or B2M to their respective binding partners so as to prevent elicit of immunoinhibitory signals.
In certain of these embodiments, a multi-specific antibody or antigen-binding fragment thereof as 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, and in certain of these embodiments the multi-specific antibody or antigen-binding fragment thereof and the additional  therapeutic agent (s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment thereof and a second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians’ Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002) ) or protocols well known in the art.
In some embodiments, the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
In another aspect, the present disclosure also provides a method of modulating CD276 activity in a CD276-expressing cell, comprising exposing the CD276-expressing cell to the multi-specific antibody or antigen-binding fragment thereof provided herein. In yet another aspect the present disclosure also provides a method of modulating PD-1/PD-L1 pathway activity in a PD-L1-expressing cell, comprising exposing the PD-L1-expressing cell to the multi-specific antibody or antigen-binding fragment thereof provided herein.
In another aspect, the present disclosure also provides use of the multi-specific antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.
EXAMPLES
EXAMPLE 1: Hybridoma development
1. Methods
1.1 Immunization and serum titer determination
1.1.1 Immunogen and immunization strategies
Cell immunization
CHO-Scells 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 immunogen.
Protein immunization
Recombinant human CD276 protein (SEQ ID NO: 346) : the recombinant human CD276 protein was prepared by digesting the human CD276 protein with enterokinase, and the extracellular domain of human CD276 was fused with 6xHis tag and DDDDK (SEQ ID NO: 345) .
Recombinant human CD276 protein (SEQ ID NO: 346) :
Balb/c and SJL mice were immunized as shown below. The primary immunization was followed by several boosts until animals developed satisfactory antiserum titers suitable for hybridoma development.
1.1.2 Immunization schedules
Immunization schedule (Group 1)
Immunization schedule (Group 2)
Immunization schedule (Group 3)
Immunization schedule (Group 4)
Immunization schedule (Group 5)
1.1.3 Test bleed antiserum analysis
Screening-Test bleeds were performed and evaluated by testing using FACS on CHO-Scell line stably over-expressing human and/or mouse CD276 (CHO-S-hCD276 and/or CHO-S-mCD276) .
Screening-Test bleeds were performed and evaluated by testing using Elisa with extra-cellular domain of recombinant human CD276 protein.
1.2 Hybridoma Generation and screening
1.2.1 Cell fusion and screening
Fusion-Splenocyte fusions were performed on the mice which responded the best to the immunizations as determined by test bleed FACS. The lymphocytes from spleens and lymph nodes were fused to a Sp2/0 cell line using an optimized electrofusion protocol. Multiple fusions were performed to ensure success of the cell fusion.
Screening and expansion-The fusion was plated (2 x 104 to 105 per well) into a stack of 96-well plates. Plates were monitored for growth and fed weekly. Wells with cell growth were screened by primary screening assays in 10-14 days with FACS and/or other feasible assays such as Elisa. Multiple fusions for each targeting antigen were performed and screened. The positive parental clones which showed positive binding with CHO-S-CD276 and positive Elisa signal from primary screening were expanded into 24-well plates for secondary screening.
Additional antibody screening-Following primary screening, positive parental clones expanded into 24-well plates were screened again by the assay described in the hybridoma screening funnel below.
Hybridomas of interest were chosen to proceed to subcloning.
1.2.2 Hybridoma subcloning, screening and cryopreservation
Subcloning-The parental hybridomas with desired reactivity and isotypes from the screening funnel above were then subcloned by multiple rounds of limiting dilution or single cell sorting until monoclones were obtained.
Screening &Expansion-The subcloning plates were screened by protein or cell-based Elisa, and the subclones with good binding ability were expanded to 24-wells for confirmation tests. The specificity and cross-reactivity of these subclones were confirmed with FACS analysis. Briefly, parental CHO-Scells, CHO-S-hCD276, CHO-Scell line stably over-expressing Macaca_fascicularis CD276, CHO- K1 S, cell line stably over-expressing mouse CD276 were incubated with antibodies produced by each subclone respectively. Fluorescent dye-conjugated secondary antibody was used to detect the binding of the primary antibody with the cells. Median fluorescence intensity was measured by FACS analysis.
Cryopreservation-The desired subclonal cell lines were sequenced and further expanded into culture flasks for cryopreservation. 4-6 vials per cell line at 0.5-13.0 x106 cells/vial were initially cryopreserved. Master cell bank and working cell bank were established for the selected most valuable cell lines if desired.
2. Results
We discovered 43 antibodies with unique sequences showing positive binding with CHO-Scell stably over-expressing human CD276 protein (CHO-S-hCD276) but not binding to parental CHO-Scells, suggesting these antibodies are human CD276 recognizing antibody. Among which 42 antibodies could bind with cynomolgus monkey CD276, and 11 antibodies could bind with mouse CD276 protein. The MFI of the mouse antibodies staining CHO-S, CHO-S-hCD276, CHO-S-mCD276, CHO-S-Macaca_fascicularis CD276 (CHO-S-cynoCD276) , detected by FACS were summarized in the table below (Table 7) .
Table 7. MFI of antibodies binding with different cell lines

EXAMPLE 2: Antibody characterization: Affinity
1. Methods
1.1 Cell based binding affinity on SKOV3 cancer cell line 
Sequences of 31 mouse antibodies from Table 5 were selected to generate and produce human IgG1 chimeric antibodies. The binding affinity of these antibodies and bench mark antibody, Enoblituzumab (see US8802091, MGA271,  specifically with a construct designated as hBRCA84D-2) and MGC018 (an antibody-drug-conjugate, in which the 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 in both dividing and non-dividing cells, thereby causing cell death) with human patient derived ovarian cancer cell line, SKOV3, was determined by FACS analysis.
The protocol for FACs analysis is described as follows:
1. Digested cells using Trypsin (1X) ; Centrifuged the harvested cells at 300g for 3 min and discarding the supernatant.
2. Washed the cells 2 times with FACS buffer by centrifuging at 300 g for 3 min and discarding the supernatant.
3. Resuspended the cells, and 2*105 cell/well was seeded into the assay plate in 50 μl FACS buffer, then added 50 μl primary antibody (primary antibody final concentration: (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) . Incubated at 4℃ for 1 hour.
4. Washed the cells twice by using the condition in step 2. Resuspended the cells with 100 μl/well diluted 2nd antibody, incubated at 4℃ for 1 hour in the dark.
5. Washed the cells twice by using the condition in step 2. Resuspended the cells with 100 μl/well FACS buffer. Kept the cells in dark for FACS analysis.
The binding affinity of the selected antibodies on SKOV3 are higher, lower or comparable with bench mark antibody Enoblituzumab (see Table 8 and Figure 1) .
Table 8. Binding affinity of the chimeric antibodies on SKOV3

2. Protein based Affinity test by Biacore (ChemPartner)
Test condition:
Analyte: B7H3
Running buffer: HBS-EP+
Flow Rate: 30 μL/min
Capture: Abs, 10 μL/min for 60 s
Injection of serial diluted B7H3
Contact time: 180 s
Dissociation time: 400 s
Regeneration: pH=1.5 Gly, 30 μL/min for 30 s
Method: Multiple cycle kinetics/affinity using capture
Machine Model: Biacore 8K (GE)
Analysis Temperature: 25℃
Table 9. shows the binding affinity of the chimeric antibodies on hB7H3 by Biacore (ChemPartner)

3. B7H3’s expression pattern on several cancer cell lines
B7H3 expression were detected by FACS with 6-D8-E7-A11. High expression level of B7H3 on several cancer cell lines, such as BxPC3 (Pancreatic) , MCF7 (Breast) , Dentroit562 (Head and neck) , RKO (Colon) and SUN620 (Gastric) , were found (see Figure 2) .
EXAMPLE 3. Antibody characterization: ADCC
1. Methods
In order to determine the ADCC of the anti-CD276 antibodies, 1x105/well SKOV3 were seeded to 96 wells Flat-bottom sterile plate, then 2x104 Jurkat-NFAT-Luciferase-CD16 were added as effector cell. After that, serial diluted antibodies were added to each well and the plate were incubated at 37℃, 5%CO2 for 18 hours.  Finally, the luciferase activity was detected to evaluate the ADCC activity of antibodies.
2. Result
All our antibodies showed potent ADCC effect on SKOV3 cells (ahuman ovarian cancer cell line) . Some of the antibodies showed lower or comparable EC50 compared with bench mark antibody, Enoblituzumab (Table 10 and figure 3) , indicating that they are more potent in mediating ADCC effect on SKOV3 cells than Enoblituzumab (MGA271) .
Table 10. EC50 of antibody induced ADCC effect on SKOV3

EXAMPLE 4. Antibody characterization: CDC
1. Methods
In order to determine the CDC of the anti-CD276 antibodies, CHO-S-hCD276 cells were resuspended in cell culture medium at 4E5 cells/mL and were then added into a 96-well opaque wall plate at 50 μL/well. Anti-CD276 antibodies were diluted with complete F-12K medium and added to the 96-well opaque wall plate at 50 μL/well. Human serum complement was diluted with cell culture medium and was added to the same plate at 50 μL/well. The mixture was incubated for 2 hours in a CO2 incubator at 37℃. CellTiter-Glo reagent for determining the cell cytotoxicity was added at 50 μL/well and the mixture was incubated for 10 mins at R.T. Luminescence signal of viable cells on a microplate reader was recorded.
2. Result
All the anti-CD276 antibodies showed potent CDC effect on CHO-S-hCD276 cells and lower EC50 compared with benchmark antibody, Enoblituzumab (MGA271) (see Table 10 and Figure 4) .
Table 11. Max CHO-S-hCD276 killing percentage and EC50 of antibody induced CDC effect

EXAMPLE 5. Antibody characterization: Indirect ADC cytotoxicity
1. Methods
Fab-ZAP is a chemical conjugate of goat anti-human monovalent antibody (asecondary antibody) and the ribosome-inactivating protein, saporin. Fab-ZAP is used to determine the internalization ability of antibodies. In this assay, 80 μL SKOV-3 cells were plated at 2000 cells/well in a 96-well plate and incubated overnight at 37℃. Anti-CD276 antibodies were then added at 40 μL/well. Fab-ZAP human dilution were added at 40 μL/well and incubated for 96 hours in a CO2 incubator at 37℃. CellTiter-Glo reagent for determining the cell cytotoxicity were added at 100 μL/well and incubated for 10 mins at R.T. Luminescence signal of viable cells on a microplate reader were recorded.
2. Result
All the anti-CD276 antibodies showed potent indirect ADC effect on SKOV3 cells, with lower or comparable IC50 compared with bench mark antibody, MGC018 (Table 12, Figure 5) , indicating that they are potential candidates for making ADCs.
Table 12. IC50 of indirect ADC cytotoxicity on SKOV3

EXAMPLE 6. Antibody in vivo efficacy in the treatment of 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 the in vivo efficacy. A bispecific antibody using B7H3 mAb and PD-L1 mAb (SHANGHAI ORIGINCELL MEDICAL TECHNOLOGY CO., LTD; see WO2019196309A1, YN035) with mouse IgG2a was also constructed in the format of IgG (H) scFV. The N terminal of the anti-PD-L1 scFv VH followed by (GGGGS) 3-VL was linked to the C terminal of the Fc region of the B7H3 antibody. As for Bis-25-C8-D7-C5 reverse, the N terminal of the anti-B7H3 scFv VH followed by (GGGGS) 3-VL was linked to the C terminal of the Fc region of the PD-L1 antibody.
That is, the naming of the bi-specific antibodies follows the following pattern.
Taking the bispecific antibody based on the CD276 antibody 25-C8-D7-C5 and PD-L1 (i.e., YN035) for 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.
The heavy chain variable region amino acid sequence of YN035 (with CDR regions underlined) :
The light chain variable region amino acid sequence of YN035 (with CDR regions underlined) :
1. The MC-38-hCD276 (B7H3) tumor cells were maintained in vitro with DMEM medium supplemented with 10%fetal bovine serum at 37℃ in an atmosphere of 5%CO2 in air. The cells in exponential growth phase were harvested and quantitated by cell counter before tumor inoculation.
2. Each mouse was inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 10^6) in 0.1 ml of PBS for tumor development. The date of randomization was denoted as day 0, and dosing starts from day 0.
3. The randomization will start when the mean tumor size reaches approximately 50-60 mm3.60 mice were enrolled in this study. All animals were randomly allocated to 7 study groups. Randomization was performed based on randomized block design.
4. Tumor volumes were measured twice per week in two dimensions using a caliper, and the volume were expressed in mm3 using the formula: V = (L x W x W) /2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L) . Dosing as well as tumor size and body weight measurements were conducted in a Laminar Flow Cabinet (see Table 13, Figure 6) .
5. Atezolizumab (an anti-PD-L1 antibody, also named Tecentriq or MPDL3280A, see WO2010077634A1) and Antengene-084M (MGA271’s Fab sequence (see US8802091B) constructed with mouse IgG2a) were used as control.
Table 13. Study design for in vivo efficacy test.
2. Result
As shown in Figure 6, some of the anti-CD276 antibodies showed potent in vivo efficacy. In particular, 25-C8-D7-C5 mouse IgG2a and Bis-6-D8-E7-A11-reverse mouse IgG2a showed higher tumor inhibition than Antengene-084M mouse IgG2a.
EXAMPLE 7: ADC in vivo efficacy test of the antibodies
1. Study design and Methods
To evaluate the ADC potential of the antibodies, antibody conjugations with VC-MMAE in ChemPartner were performed. A serial of VC-MMAE conjugated antibodies were obtained. The details are shown in Table 14.
Table 14. Summary of the antibody conjugations
Each Balb/c nude mouse for Calu-6 study was inoculated subcutaneously in the right front flank region with Calu-6 tumor cells (5 x 106) in 0.1 ml of PBS for tumor development. The randomization starts when the mean tumor size reaches approximately 122 mm3. 60 mice were enrolled in the study. The date of randomization was denoted as day 0, dosing starts from day 0.
2. Results
The therapeutic efficacy of test articles in the treatment of subcutaneous human lung cancer Calu-6 in Balb/c nude mice was investigated in this study. No obvious body weight loss, mortality or toxic response was observed in the designed dosing regimens during the efficacy study. At 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 a single agent show a  significant anti-tumor efficacy against Calu-6 model in Balb/c nude mice, respectively. The results are shown in Figure 7 and Table 15.
Table 15. Antitumor Activity of Test Compounds in Subcutaneous Calu-6 Model in Balb/c Nude Mice
Note: a: Mean ± SEM; b: TGI, T/C and p values were compared with group 1 tumor volume on day 24 by using Dunnett’s tests.
EXAMPLE 8: T cell activation (MLR assay) test of the antibodies
1. Study design and Methods
DC induction
Monocyte cells were re-suspended at 5 x 106 in 3 ml complete medium supplemented with 2 U/ml of dendritic cell culture factor and then culture cells in 6-well plate. At the second day, 2 ml/well of fresh complete medium supplemented with 2 U/ml of dendritic cell culture factor were added for another three days culture. Then monocytes differentiated into immature dendritic cells (iDC) . After stimulated 48 hours with 2 U/ml of dendritic cell mature factor, iDCs would differentiate into mature dendritic cells.
Antibody Digestion
Combined 25 μg of hIgG1 and 3 μl of 10X Glyco Buffer, and then added PBS to make a 30 μl total reaction volume. 1 μl of IdeZ Protease was added, then  incubated at 37℃ for 30 minutes. 10 μl protein A/G beads were added in 1 mL PBS, washed 2 times. The beads were resuspended with reaction solution from the above step.
The tube was put on a magnet for 1 min RT, the F (ab) 2 is in supernatant and the Fc was captured by beads.
MLR assay
0.2 million cells/well T cells and 0.02 million cells/well mDCs were seeded.
Cells were treated PBS, IgG1, 25-C8-D7-C5, 30-C7-C11-D4, BMK (MGA271) or their Fab (final concentration 5 μg/ml) . After 24 hours, supernatants were collected and human IL-2 and IFNγ was tested with Elisa.
2. Results
As shown in Figures 8 and 9, 30-C7-C11-D4 can activate T cell activation while 25-C8-D7-C5 and BMK (MGA271) cannot.
EXAMPLE 9: Antibody humanization and PTM optimization
9.1 Cell-based affinity test of the humanized antibodies by FACS
10-G6-C4-B2 and 30-C7-C11-D4 were chosen to perform humanization and PTM optimization. The affinity of humanized candidates and optimized PTM sequences were evaluated by FACS.
The results are shown in Figures 10 and 11. All sequence showed comparable affinity with parental antibody.
9.2 Protein based affinity test by Biacore (ChemPartner)
Test condition:
Analyte: B7H3. Running buffer: HBS-EP+. Flow Rate: 30 μL/min. Capture: Abs, 10 μL/min for 60 s. Injection of serial diluted B7H3. Contact time 180s, dissociation time 400 s. Regeneration: pH=1.5 Gly, 30 μL/min for 30 s.  Method: Multiple cycle kinetics/affinity using capture. Machine Model: Biacore 8K (GE) . Analysis Temperature: 25℃.
The results are shown in Table 16.
Table 16. Binding affinity of the humanized antibodies on hB7H3 by Biacore (ChemPartner)
9.3 ADC therapeutic capability test of the humanized 10-G6-C4-B2 antibodies in vivo
The in vivo ADC therapeutic capability of the humanized 10-G6-C4-B2 antibodies were evaluated. Antibody conjugations with VC-MMAE in ChemPartner were constructed. A serial of VC-MMAE conjugated antibodies were obtained, the details of which were shown in Table 17.
Table 17. Summary of the antibody conjugations

Each Balb/c nude mouse for Calu-6 study was inoculated subcutaneously in the right front flank region with Calu-6 tumor cells (5 x 106) in 0.1 ml of PBS for tumor development. The randomization starts when the mean tumor size reaches approximately 122 mm3.60 mice were enrolled in the study. The date of randomization was denoted as day 0, dosing starts from day 0.
The therapeutic efficacy of test articles in the treatment of subcutaneous human lung cancer Calu-6 in Balb/c nude mice was investigated in this study. No obvious body weight loss, mortality or toxic response was observed in the designed dosing regimens during the efficacy study.
The results are shown in Figure 12 and Table 18. At day 26, almost all the test articles at 3 mg/kg as a single agent show a significant anti-tumor efficacy against Calu-6 model in Balb/c nude mice, respectively.
Table 18. Antitumor activity of test compounds in subcutaneous Calu-6 model in Balb/c nude mice

Note: Mean ± SEM; TGI, T/C and p values were compared with group 1 tumor volume on day 26 using Kruskal-Wallis test.
EXAMPLE 10: In vivo efficacy of the bispecific antibodies
Each mouse (C57BL/6) was inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 106) in 0.1 ml of PBS for tumor development. The randomization starts when the mean tumor size reaches approximately 139 mm3.66 mice were enrolled in the study. The date of randomization was denoted as day 0, dosing starts from the day of randomization (day 0) .
At day 19, Bis-30-C7-C11-D4 at 3 mg/kg, 25-C8-D7-C5-igG2a at 2.25 mg/kg combined with YN035-IgG2a at 2.25 mg/kg show a significant anti-tumor efficacy against MC-38-hCD276 (B7H3) murine colon carcinoma in C57BL/6 mice, respectively, as shown in Table 19 and Figure 13.
Table 19. Antitumor activity of test compounds in subcutaneous MC-38-hCD276 (B7H3) model in C57BL/6 mice
Note: Mean ± SEM; TGI, T/C and p values were compared with group 1 tumor volume on day 19 using Kruskal-Wallis test.
Each mouse (C57BL/6) was inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 106) in 0.1 ml of PBS for tumor development. The randomization starts when the mean tumor size reaches approximately 100 mm3.30 mice were enrolled in the study. The date of randomization was denoted as day 0, dosing starts from the day of randomization (day 0) .
At day 19, Bis-25-C8-D7-C5 at 3 mg/kg and Bis-30-C7-C11-D4 at 3 mg/kg as a single agent showed a significant anti-tumor efficacy against MC-38-hCD276 (B7H3) murine colon carcinoma in C57BL/6 mice, respectively, as shown in Table 20 and Figure 14.
Table 20. Antitumor activity of test compounds in subcutaneous MC-38-hCD276 (B7H3) model in C57BL/6 mice
Note: Mean ± SEM; TGI, T/C and p values were compared with group 1 tumor volume on day 19 by using Dunnett’s tests.
EXAMPLE 11: In vivo efficacy of the bispecific antibodies incorporated with PTM optimization
The humanized antibodies incorporated PTM optimization were constructed and expressed for the in vivo efficacy. A bispecific antibody using B7H3 mAb 30-C7-C11-D4 and PD-L1 mAb (SHANGHAI ORIGINCELL MEDICAL TECHNOLOGY CO., LTD; see WO2019196309A1, YN035) with human IgG1 was also constructed in the format of IgG (H) scFV. The N terminal of the anti-PD-L1 scFv VH followed by (GGGGS) 3-VL was linked to the C terminal of the Fc region of the B7H3-antibody.
Each mouse (C57BL/6) was inoculated subcutaneously in the right rear flank region with MC-38-hCD276 (B7H3) tumor cells (1 x 106) in 0.1 ml of PBS for tumor development. The randomization starts when the mean tumor size reaches approximately 100 mm3.54 mice were enrolled in the study. The date of randomization was denoted as day 0, dosing starts from the day of randomization (day 0) .
Bis-30-C7-C11-D4_hVH3-hVL2-PTM and Bis-30-C7-C11-D4_hVH4-hVL5-PTM showed comparable tumor growth inhibition with parental Bis-30-C7-C11-D4, as shown in Table 21 and Figure 15.
Table 21. Antitumor activity of test compounds in subcutaneous MC-38-hCD276 (B7H3) model in C57BL/6 mice
Note: Mean ± SEM; TGI, T/C and p values were compared with group 1 tumor volume on day 19 by using Dunnett’s tests.

Claims (45)

  1. A multi-specific antibody or an 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 multi-specific antibody or an antigen-binding fragment thereof of claim 1, wherein the first binding domain comprises 1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 1-3, 9-11, 17-19, 25-27, 33-35, 41-43, 49-51, 57-59, 65-67, 73-75, 81-83, 89-91, 97-99, 105-107, 113-115, 121-123, 129-131, 137-139, 145-147, 153-155, 161-163, 169-171, 177-179, 185-187, 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, and/or 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 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, 124-126, 132-134, 140-142, 148-150, 156-158, 164-166, 172-174, 180-181, 188-190, 196-198, 204-206, 212-214, 220-222, 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 multi-specific antibody or antigen-binding fragment thereof of claim 1, wherein the first binding domain comprises a heavy chain variable region selected from the group consisting of:
    a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 1-3;
    b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9-11;
    c) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 17-19;
    d) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 25-27;
    e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 33-35;
    f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 41-43;
    g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 49-51;
    h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 57-59;
    i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 65-67;
    j) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 73-75;
    k) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 81-83;
    l) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 89-91;
    m) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 97-99;
    n) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 105-107;
    o) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 113-115;
    p) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 121-123;
    q) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 129-131;
    r) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 137-139;
    s) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 145-147;
    t) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 153-155;
    u) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 161-163;
    v) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 169-171;
    w) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 177-179;
    x) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 185-187;
    y) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 193-195;
    z) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 201-203;
    aa) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 209-211;
    bb) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 217-219;
    cc) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 225-227;
    dd) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 233-235;
    ee) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 241-243;
    ff) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 249-251;
    gg) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 257-259;
    hh) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 265-267;
    ii) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 273-275;
    jj) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 281-283;
    kk) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 289-291;
    ll) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 297-299;
    mm) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 305-307;
    nn) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 313-315;
    oo) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 321-323;
    pp) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 329-331;
    qq) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 337-339; and
    rr) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 9, 374 and 375.
  4. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the first binding domain comprises a light chain variable region selected from the group consisting of:
    a) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 4-6;
    b) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 12-14;
    c) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 20-22;
    d) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 28-30;
    e) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 36-38;
    f) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 44-46;
    g) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 52-54;
    h) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 60-62;
    i) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 68-70;
    j) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 76-78;
    k) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 84-86;
    l) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 92-94;
    m) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 100-102
    n) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 108-110;
    o) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 116-118;
    p) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 124-126;
    q) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 132-134;
    r) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 140-142;
    s) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 148-150;
    t) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 156-158;
    u) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 164-166;
    v) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 172-174;
    w) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 180-181;
    x) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 188-190;
    y) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 196-198;
    z) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 204-206;
    aa) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 212-214;
    bb) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 220-222;
    cc) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 228-230;
    dd) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 236-238;
    ee) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 244-246;
    ff) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 252-254;
    gg) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 260-262;
    hh) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 268-270, ;
    ii) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 276-278;
    jj) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 284-286;
    kk) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 292-294;
    ll) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 300-302;
    mm) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 308-310;
    nn) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 316-318;
    oo) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 324-326;
    pp) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 332-334;
    qq) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 340-342;
    rr) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 376, 13 and 14; and
    ss) a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NOs: 377, 45 and 46.
  5. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the first binding domain comprises:
    a) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a light chain  variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6;
    b) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14;
    c) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22;
    d) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30;
    e) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38;
    f) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46;
    g) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54;
    h) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62;
    i) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70;
    j) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78;
    k) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86;
    l) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 92, SEQ ID NO: 93, and SEQ ID NO: 94;
    m) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102;
    n) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 108, SEQ ID NO: 109 and SEQ ID NO: 110;
    o) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118;
    p) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123; and a light  chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126;
    q) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 134;
    r) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142;
    s) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150;
    t) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 156, SEQ ID NO: 157, and SEQ ID NO: 158;
    u) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166;
    v) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174;
    w) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 179; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 180, SEQ ID NO: 181, and SEQ ID NO: 182;
    x) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 186, and SEQ ID NO: 187; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 189, and SEQ ID NO: 190;
    y) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 196, SEQ ID NO: 197 and SEQ ID NO: 198;
    z) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 201, SEQ ID NO: 202, and SEQ ID NO: 203; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 204, SEQ ID NO: 205, and SEQ ID NO: 206;
    aa) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 209, SEQ ID NO: 210, and SEQ ID NO: 211; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 212, SEQ ID NO: 213, and SEQ ID NO: 214;
    bb) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 217, SEQ ID NO: 218, and SEQ ID NO: 219; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 220, SEQ ID NO: 221, and SEQ ID NO: 222;
    cc) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 225, SEQ ID NO: 226, and SEQ ID NO: 227; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 228, SEQ ID NO: 229, and SEQ ID NO: 230;
    dd) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 233, SEQ ID NO: 234, and SEQ ID NO: 235; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 236, SEQ ID NO: 237, and SEQ ID NO: 238;
    ee) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 244, SEQ ID NO: 245, and SEQ ID NO: 246;
    ff) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 252, SEQ ID NO: 253, and SEQ ID NO: 254;
    gg) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 260, SEQ ID NO: 261, and SEQ ID NO: 262;
    hh) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 265, SEQ ID NO: 266, and SEQ ID NO: 267; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 268, SEQ ID NO: 269, and SEQ ID NO: 270;
    ii) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 273, SEQ ID NO: 274, and SEQ ID NO: 275; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 276, SEQ ID NO: 277, and SEQ ID NO: 278;
    jj) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 281, SEQ ID NO: 282, and SEQ ID NO: 283; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 284, SEQ ID NO: 285 and SEQ ID NO: 286;
    kk) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 289, SEQ ID NO: 290, and SEQ ID NO: 291; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 292, SEQ ID NO: 293, and SEQ ID NO: 294;
    ll) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 297, SEQ ID NO: 298, and SEQ ID NO: 299; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 300, SEQ ID NO: 301, and SEQ ID NO: 302;
    mm) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 305, SEQ ID NO: 306, and SEQ ID NO: 307; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 308, SEQ ID NO: 309, and SEQ ID NO: 310;
    nn) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 313, SEQ ID NO: 314, and SEQ ID NO: 315; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 316, SEQ ID NO: 317, and SEQ ID NO: 318;
    oo) a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID NO: 321, SEQ ID NO: 322, and SEQ ID NO: 323; and a light chain variable region comprising 1, 2, or 3 CDR sequences selected from SEQ ID SEQ ID NO: 324, SEQ ID NO: 325, and SEQ ID NO: 326;
    pp) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 332, SEQ ID NO: 333, and SEQ ID NO: 334;
    qq) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 340, SEQ ID NO: 341, and SEQ ID NO: 342;
    rr) a heavy chain variable region comprising 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 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 1, 2, or 3 CDR sequences selected from SEQ ID NO: 377, SEQ ID NO: 45, and SEQ ID NO: 46.
  6. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the first binding domain comprises a heavy chain  variable region 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 the homologue sequences of at least 80%sequence identity thereof.
  7. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the first binding domain comprises a light chain variable region 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: 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 the homologue sequences of at least 80%sequence identity thereof.
  8. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, wherein the first binding domain comprises SEQ ID NO: 378 (EVQLVESGGGLXQPGXSLRLSCXTSGFTLSDYYMSWVRQXPGKGLEWV XFMRNKANXYTTEYSASVRGRFTISRDTSKSXIYLQMNSLXXEDTAVYY  CVRDRXGRPFAYWGQGTLVTVSS) , wherein the X at position i (i=12, 16, 23, 40, 49, 57, 80, 89, 90 and 103) of SEQ ID NO: 378 is referred as XHi, wherein XH12 is V or I, XH16 is G or R, XH23 is A or T, XH40 is A or P, XH49 is G or S, XH57 is A or G, XH80 is I or T, XH89 is R or K, XH90 is A or T, XH103 is D or E; and a light chain variable region comprising SEQ ID NO: 379 (DIXMTQSPXSLXXXXGXXXXIXCKSSQSLLNXINQKNFLTWYXQKPGX XPXLLIYWASTRESGVPXRFSGSGSGTDFTLXISXXXXEDLXXYYCQNDY TYPLTFGQGTKLEIK) , 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 referred as XLi, wherein XL3 is V or Q, XL9 is D, L or S, XL12 is A, S or P, XL13 is A or V, XL14 is S or T, XL15 is L, V or P, XL17 is D or E, XL18 is R or P, XL19 is A or V, XL20 is S or T, XL22 is N, T or S, XL32 is A or S, XL43 is Q or L, XL48 is Q or K, XL49 is A, P or S, XL51 is K or Q, XL66 is S or D, XL80 is K or T, XL83 is R or S, XL84 is L or V, XL85 is Q or E, XL86 is A or P, XL90 is A or G, XL91 is T or V.
  9. The multi-specific antibody or antigen-binding fragment thereof of claim 9, wherein the first binding domain comprises a heavy chain variable region comprising SEQ ID NO: 380 (QVQLQESGPGLVKPSXTLSLTCXVXGYSITSDYAWNWIRQXPGKGLEWI GYISHSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSL GRRWYFDVWGQGTTVTVSS) , wherein the X at position i (i=16, 23, 25 and 41) of SEQ ID NO: 380 is referred as XHi, wherein XH16 is E or Q, XH23 is A or T, XH25 is S or Y, XH41 is H or P; and
    a light chain variable region comprising SEQ ID NO: 381 (DIXMTQSPXSLXXXXGXXXXIXCKSSQSLLXSSTQKNYLAWYXQKPGX XPXLLIYFASTRDSGVPXRFSGSGSGTDFTLXISXXXXEDLXXYFCQQHYI IPFTFGQGTKLEIK) , 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 referred as XLi, wherein XL3 is V or Q, XL9 is D, L or S, XL12 is A, S or P, XL13 is A or V, XL14 is S or T, XL15 is L, V or P, XL17 is D or E, XL18 is R or P, XL19 is A or V, XL20 is S or T, XL22 is N, T or S, XL31 is N or Q, XL43 is Q or L, XL48 is  Q or K, XL49 is A, P or S, XL51 is K or Q, XL66 is S or D, XL80 is K or T, XL83 is R or S, XL84 is L or V, XL85 is Q or E, XL86 is A or P, XL90 is A or G, XL91 is T or V.
  10. The multi-specific antibody or antigen-binding fragment thereof of claim 9, wherein the first binding domain comprises:
    (i) a heavy chain variable region comprising 1, 2, or 3 CDR sequences shown 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 1, 2, or 3 CDR sequences shown 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 1, 2, or 3 CDR sequences shown as SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively; and a kappa light chain variable region comprising 1, 2, or 3 CDR sequences shown as SEQ ID NO: 44 or 377 (QSLLQSSTQKNY) , SEQ ID NO: 45, and SEQ ID NO: 46, respectively.
  11. The multi-specific 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 NO: 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 NO: 352, 354, 356, 359, 361, 363, 366, 368, 369, 371, 372 and 373.
  12. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, wherein 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.
  13. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the first binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to human CD276.
  14. The multi-specific antibody or antigen-binding fragment thereof of claim 13, wherein the substitution is 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. The multi-specific antibody or antigen-binding fragment thereof of any of the preceding claims, which further comprises an activating receptor binding domain, and, optionally, the activating receptor binding domain is a constant region of human Ig, or a constant region of human IgG, and, optionally, the first binding domain is an intact antibody.
  16. The multi-specific antibody or antigen-binding fragment thereof of claim 15, wherein the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4.
  17. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the second target is PD-L1.
  18. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the second binding domain comprises 1, 2, or 3 heavy chain complementarity determining region (CDR) sequences selected from the group consisting of: SEQ ID NOs: 384-386, and/or 1, 2, or 3 light chain CDR sequences selected from the group consisting of: SEQ ID NOs: 387-389.
  19. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable region comprising three CDR sequences set forth as SEQ ID NOs: 384, 385 and 386, respectively; and a light chain variable region comprising three CDR sequences set forth as SEQ ID NOs: 387, 388 and 389, respectively.
  20. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable set forth as SEQ ID NO: 382, and/or a light chain variable region set forth as SEQ ID NO: 383.
  21. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the second binding domain further comprises one or  more amino acid residue substitutions or modifications yet retains specific binding affinity to human PD-L1.
  22. The multi-specific antibody or antigen-binding fragment thereof of claim 16, wherein the substitution is 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 multi-specific antibody or antigen-binding fragment thereof of claim 16, wherein the second binding domain comprising an scFv structure of VH-linker-VL.
  24. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the linker comprises a sequence of (GGGGS) 3 (SEQ ID NO: 390) .
  25. The multi-specific antibody or antigen-binding fragment thereof of claim 16, wherein the N-termination of the second binding domain is operably linked to the C-termination of the activating receptor binding domain.
  26. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, which is a bi-specific or tri-specific antibody.
  27. The multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, which is linked to one or more conjugates, optionally, wherein the conjugate is covalently attached either directly or via a linker.
  28. The multi-specific antibody or antigen-binding fragment thereof of claim 25, wherein the conjugate comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
  29. A pharmaceutical composition comprising the multi-specific antibody or antigen-binding fragment thereof of any one of the preceding claims, and a pharmaceutically acceptable carrier.
  30. An isolated polynucleotide encoding the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-27.
  31. A vector comprising the isolated polynucleotide of claim 31.
  32. A host cell comprising the vector of claim 32.
  33. A method of expressing the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-27, comprising culturing the host cell of claim 33 under the condition at which the vector of claim 32 is expressed
  34. A method of treating a disease or condition in a subject that would benefit from modulation of CD276 activity and/or PD-L1, comprising administering to the subject a therapeutically effective amount of the multi-specific antibody or antigen-binding fragment thereof of any of claims 1-27 or the pharmaceutical composition of any one of claims 28-30.
  35. The method of claim 35, wherein the disease or condition is a CD276 and/or PD-L1 related disease or condition.
  36. The method of claim 35, wherein the disease or condition is cancer, adaptive immune disease, autoimmune disease, inflammatory disease, or infectious disease.
  37. The method of claim 37, wherein the cancer is adrenal gland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, a brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a desmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasia, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid  tumor, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a renal metastatic cancer, a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, a soft-tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a thyroid metastatic cancer, and a uterine cancer, optionally, wherein the cancer is chemoresistant.
  38. The method of claim 37, wherein the disease or condition is hematological cancer chosen from B-cell lymphomas, 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. The method of claim 35, wherein the subject is human.
  40. The method of claim 35, comprising administering to the subject a therapeutically effective amount of one or more therapeutic agent.
  41. The method of claim 35, wherein said therapeutic agent is a chemotherapeutic agent, a radiation therapeutic agent, a hormonal therapeutic agent, a toxin or an immunotherapeutic agent.
  42. The method of any of claims 35-42, wherein the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
  43. The method of claim 35-43, wherein said method further comprises administration of one or more additional cancer therapies selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, hormonal therapy, and surgery.
  44. Use of the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-27 in the manufacture of a medicament for treating a CD276 and/or PD-L1 related disease or condition in a subject.
  45. Use of the multi-specific antibody or antigen-binding fragment thereof of any one of claims 1-27 in a method for treating a CD276 and/or PD-L1 related disease or condition in a subject.
PCT/CN2023/130106 2022-11-11 2023-11-07 Novel multi-specific antibodies and uses thereof WO2024099295A1 (en)

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CN2022131463 2022-11-11

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