Novel Multispecific Molecules

Abstract

The present disclosure provides multispecific molecules specific for SIPR-alpha and one or more target antigens, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and uses thereof. The present disclosure provides multispecific molecules comprising a SIRP-alpha binding domain, an activating receptor binding domain comprising an Fe domain, and a target antigen binding domain.

Description

[Technical field]

[0001]

[0001] This disclosure relates generally to novel multispecific molecules specific for SIRP-α and tumor-associated antigens. [Background technology]

[0002]

[0002] Signal regulatory protein alpha (SIRPα) is an inhibitory receptor that is expressed primarily on myeloid and dendritic cells. SIRPα contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) cytoplasmic domain. The immunoregulatory activity of SIRPα on myeloid cells is activated by binding to its ligand CD47, which induces tyrosine phosphorylation of the ITIM cytoplasmic domain of SIRPα with subsequent recruitment of SH2-containing tyrosine phosphatases (SHP-1 / 2). SHP-1 / 2 then participate in inhibitory signaling events via protein dephosphorylation, ultimately resulting in the inhibition of phagocytosis in macrophages (Barclay AN, Van den Berg TK. The interaction between signal regulatory protein alpha (SIRPα) and CD47: structure, function, and therapeutic target. Annu Rev Immunol. 2014;32:25-50; Oldenborg PA,et al. Role of CD47 as a Marker of self on red blood cells. Science. 2000;288(5473):2051-2054). Thus, CD47 binding to SIRPα results in the delivery of a "don't eat me" signal, inhibiting phagocytosis.

[0003]

[0003] CD47 is ubiquitously expressed on normal cells and is upregulated on many cancer cells. High CD47 expression is a mechanism used by cancer cells to evade the immune system and correlates with poor clinical outcome (Willingham SB, et al. The CD47-signal regulatory protein-α (SIRPα) interaction is a therapeutic target for human solid tumors. Proc Natl Acad Sci USA. 2012; 109(17): 6662-6667; Zhao XW, et al. CD47-signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction. Proc Natl Acad Sci USA. 2011; 108(45): 18342-18347; Majeti R, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute Myeloid leukemia stem cells. Cell. 2009; 138(2): 286-299). Bispecific macrophage-enhancing (BiME) antibodies specific for SIRPα and a second antigen (e.g., a target antigen) have been designed to achieve enhanced phagocytosis properties on cancer cells, see, e.g., WO2015138600A2, which is incorporated by reference herein. Furthermore, more BiME molecules constructed with different properties and formats of SIRPα antibodies need to be studied and compared in more depth. Summary of the Invention [Problem to be solved by the invention]

[0004]

[0004] Therefore, there is a need to develop improved BiME antibodies or multispecific molecules that are specific for SIRPα, have reduced side effects, and are safer. [Means for solving the problem]

[0005]

[0005] Throughout this 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.

[0006]

[0006] The present disclosure presents novel multispecific molecules specific for SIRP-α and tumor-associated antigens, their amino acid and nucleotide sequences, and uses thereof.

[0007] In one aspect, the present disclosure provides a method for producing a medicament for use in a method for treating a vascular endotheli (a) a SIRP alpha binding domain; and (b) an activating receptor binding domain; (c) a target antigen-binding domain that binds to a target antigen expressed on a target cell that co-expresses the target antigen and CD47; wherein the multispecific molecule selectively induces an effector function of an immune effector cell in the presence of a target antigen, and the immune effector cell co-expresses SIRP-alpha and an activating receptor.

[0007]

[0008] In certain embodiments, the multispecific molecules presented herein comprise a SIRP-alpha binding domain presented herein, an activating receptor binding domain comprising an Fc domain presented herein, and a target antigen binding domain presented herein.

[0008]

[0009] In any of these embodiments, the target binding domain is a claudin 18.2 binding domain or a PD-L1 binding domain.

[0010] In certain embodiments, the target cell that co-expresses the target antigen and CD47 is a cancer cell, an infected cell, or a diseased cell of interest that is to be eliminated by the effector function of an immune effector cell.

[0009]

[0011] In certain embodiments, the multispecific molecules induce minimal immune effector cell effector function in the absence of target antigen.

[0012] In certain embodiments, the effector function induced by the multispecific molecule in the absence of target antigen is no more than 10% of the effector function induced in the presence of target antigen.

[0010]

[0013] In certain embodiments, the immune effector cell is a bone marrow cell, optionally, the immune effector cell is a macrophage cell, a monocyte, a neutrophil, an eosinophil, a phagocyte, or a basophil, optionally, the immune effector cell is a macrophage cell.

[0011]

[0014] In certain embodiments, the effector function comprises phagocytosis by immune effector cells of cells that co-express an antigen and CD47.

[0015] In certain embodiments, the activating receptor is a crystallizable gamma receptor fragment (FcγR), TREM2, lectin, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, CD68, CD205, CD206, FcDR1, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CD64, CD32a, CD16a, CD89, CD19, CD28, CSFR, PDGFR, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, Dectin 1, RAGE (SR-E1), LRP1, LRP2, ASGP, SR-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, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, BAH.Tyro3, ​​Ax1, Traf6, Syk, MyD88, Zap70, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b, and optionally the activating receptor is FcγR.

[0012]

[0016] In certain embodiments, the activating receptor binding domain comprises an Fc domain, optionally wherein the Fc domain is derived from IgG1 or IgG4.

[0017] In certain embodiments, the SIRP-alpha binding domain is capable of substantially blocking the interaction between SIRP-alpha and CD47.

[0013]

[0018] In certain embodiments, the SIRP-alpha binding domain has the ability to completely block the interaction between SIRP-alpha and CD47.

[0019] In certain embodiments, the SIRP-alpha binding domain is capable of substantially blocking SHP-1 induction mediated by the interaction between SIRP-alpha and CD47.

[0014]

[0020] In certain embodiments, the SIRP-alpha binding domain is capable of completely blocking SHP-1 induction mediated by the interaction between SIRP-alpha and CD47.

[0015]

[0021] In certain embodiments, the SIRP-alpha binding domain has minimal intrinsic activity to induce effector function of an immune effector cell.

[0022] In certain embodiments, the SIRP-alpha binding domain and the activating receptor binding domain are in close proximity to enable the multispecific molecule to bind to both SIRP-alpha and activating receptors co-expressed on the same immune effector cell.

[0016]

[0023] In certain embodiments, the SIRP-alpha binding domain and / or the target antigen binding domain comprises an antibody domain or an antibody mimetic domain, optionally comprising an antibody mimetic domain comprising a fibronectin domain, a Z domain of protein A (Affibody), a gamma B crystallographic domain, a ubiquitin domain, a cystatin domain, a Sac7d domain, a triple helix coiled-coil domain, a lipocalin domain, an A domain of a membrane receptor, an ankyrin repeat motif, an SH3 domain of Fyn, a Kunitz domain of a protease inhibitor, a type III domain of fibronectin (minibody), or carbohydrate binding module 32-2.

[0017]

[0024] In certain embodiments, the antibody domain comprises a Fab, a VHH, a single chain Fv (scFv), a diabody, a Fab', a F(ab')2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a F(ab)2, a scFv dimer (bivalent diabody), a camelized single domain antibody, a nanobody, a tetrabody, a domain antibody, or a bivalent domain antibody.

[0018]

[0025] In certain embodiments, the multispecific molecules presented herein comprise a multispecific antibody comprising a target antigen-binding antibody domain, a SIRP-a binding antibody domain, and an Fc domain.

[0019]

[0026] In certain embodiments, the target antigen-binding antibody domain is linked to the N-terminus of the Fc domain.

[0027] In certain embodiments, the target antigen-binding antibody domain comprises a Fab domain, optionally comprising a heavy chain linked to one of the N-terminal ends of an Fc domain.

[0020]

[0028] In certain embodiments, the multispecific molecule comprises two target antigen-binding antibody domains, each of which comprises a Fab domain, and optionally each of the Fab domains comprises a heavy chain linked respectively to the N-terminus of an Fc domain.

[0021]

[0029] In certain embodiments, the SIRP-alpha binding domain is linked to an Fc domain or a target antigen-binding antibody domain.

[0030] In certain embodiments, the SIRP-alpha binding domain is linked to the C-terminus of the Fc domain.

[0022]

[0031] In certain embodiments, the SIRP-alpha binding domain is linked to the N-terminus of an Fc domain, provided that the SIRP-alpha binding domain and the target antigen-binding antibody domain are not linked to the same N-terminus of the Fc domain.

[0023]

[0032] In certain embodiments, the SIRP-alpha binding domain is linked to the C-terminus of the light chain of the target antigen-binding Fab domain.

[0033] In certain embodiments, the SIRP-alpha binding antibody domain is linked to the N-terminus of the Fc domain.

[0024]

[0034] In certain embodiments, the SIRP-alpha binding antibody domain comprises a Fab domain, optionally comprising a heavy chain linked to one of the N-terminal ends of an Fc domain.

[0025]

[0035] In certain embodiments, the antibody comprises two SIRP-alpha binding antibody domains, each of which comprises a Fab domain, and optionally each of the Fab domains comprises a heavy chain respectively linked to the respective N-terminus of an Fc domain.

[0026]

[0036] In certain embodiments, the target antigen binding domain is linked to an Fc domain or a SIRP-alpha binding antibody domain.

[0037] In certain embodiments, the target antigen binding domain is linked to the N-terminus of an Fc domain, provided that the target antigen binding domain and the SIRP-alpha binding domain are not linked to the same N-terminus of the Fc domain.

[0027]

[0038] In certain embodiments, the target antigen binding domain is linked to the C-terminus of the light chain of the SIRP-alpha binding Fab domain.

[0039] In certain embodiments, the target antigen comprises a tumor surface antigen.

[0028]

[0040] In certain embodiments, the tumor surface antigen is PD-L1, claudin 18.2, BCMA, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, EGFR, HER2, HER3, CD117, C-Met, EGFR, EGFRvIII, ERBB3, ERBB4, VEGFR1, VEGFR2, ROR1, PTHR2, B7-H1(PD-L1), B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, Trop-2, GPC-3, EPCAM, DLL-3, Nectin-4, Claudin 6, Muc-1, PSMA, GD3, FAP, CEA, or EphA2.

[0029]

[0041] The multispecific molecules presented herein may be in any suitable format. An illustrative example is presented as follows: In certain embodiments, the multispecific molecules presented herein comprise a target antigen-binding antibody comprising two heavy chains and two light chains, and the C-terminus of each of the light chains is fused to an anti-SIRPα scFv (i.e., a SIRPα-binding domain). The target antigen-binding antibody comprises a target antigen-binding domain and an Fc domain. An illustrative example is shown in Figure 2A.

[0030]

[0042] In certain embodiments, the multispecific molecules provided herein comprise a target antigen-binding antibody comprising two heavy chains and two light chains, wherein the C-terminus of each of the heavy chains is fused to an anti-SIRPα scFv (i.e., a SIRPα-binding domain). The target antigen-binding antibody comprises a target antigen-binding domain and an Fc domain. An illustrative example is shown in FIG. 2B.

[0031]

[0043] In certain embodiments, the multispecific molecule provided herein comprises an anti-SIRPα antibody comprising two heavy chains and two light chains, wherein the C-terminus of each of the light chains is fused to an scFv (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in FIG. 2C.

[0032]

[0044] In certain embodiments, the multispecific molecule provided herein comprises an anti-SIRPα antibody comprising two heavy chains and two light chains, wherein the C-terminus of each of the heavy chains is fused to an scFv (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 2D.

[0033]

[0045] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, wherein the C-terminus of each of the light chains is fused to a single domain antibody (sdAb) (i.e., a target antigen-binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα-binding domain and an Fc domain. An illustrative example is shown in FIG. 6A.

[0034]

[0046] In certain embodiments, the multispecific molecule presented herein comprises an anti-SIRPα antibody comprising two heavy chains and two light chains, wherein the C-terminus of each of the heavy chains is fused to a single domain antibody (sdAb) (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 6B.

[0035]

[0047] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, wherein the N-terminus of each of the heavy chains is fused to a single domain antibody (sdAb) (i.e., a target antigen-binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα-binding domain and an Fc domain. An illustrative example is shown in FIG. 6C.

[0036]

[0048] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, wherein the N-terminus of each of the light chains is fused to a single domain antibody (sdAb) (i.e., a target antigen-binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα-binding domain and an Fc domain. An illustrative example is shown in Figure 6D.

[0037]

[0049] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα binding domain (e.g., a Fab) and a single domain antibody (sdAb), each capable of binding to a target antigen, each fused to the N-terminus of a polypeptide chain of an Fc domain. An illustrative example is shown in Figure 6E.

[0038]

[0050] In certain embodiments, the multispecific molecules presented herein comprise two heavy chains, each comprising a single domain antibody (sdAb) capable of binding to a target antigen fused to the N-terminus of a polypeptide chain of an Fc domain, and further comprising at least one anti-SIRPα binding domain fused to the C-terminus of one of the polypeptide chains of the Fc domain. In certain embodiments, the multispecific molecules presented herein comprise one anti-SIRPα binding domain fused to the C-terminus of one of the polypeptide chains of the Fc domain. An illustrative example is shown in Figure 6F.

[0039]

[0051] In certain embodiments, the multispecific molecules presented herein comprise two anti-SIRPα binding domains, each fused to the C-terminus of one of the polypeptide chains of the Fc domain. An illustrative example is shown in Figure 6G.

[0040]

[0052] In certain embodiments, the SIRP-alpha binding domain comprises: a) HCDR1 comprising the sequence X1YYMH (SEQ ID NO: 161), HCDR2 comprising the sequence RIDPEDX2EX3KYAPKFQG (SEQ ID NO: 162), and 15 an HCDR3 comprising the sequence X4X5Y (SEQ ID NO: 163), and / or an LCDR1 comprising the sequence SASSSVSSSYLY (SEQ ID NO: 26), an LCDR2 comprising the sequence STSNLAS (SEQ ID NO: 27), and an LCDR3 comprising the sequence X6QWSSYPYT (SEQ ID NO: 164); or b) HCDR1 containing the sequence TYGMS (SEQ ID NO: 35), HCDR2 containing the sequence WINTYSGVX7TX8ADDFKG (SEQ ID NO: 165), and DPHX9YGX 10 SPAWFX 11 HCDR3 comprising the sequence of Y (SEQ ID NO: 166), and / or X 12 ASQX 13 VGIX 14 LCDR1 containing the sequence of VA (SEQ ID NO: 188), LCDR2 containing the sequence of SASNRYT (SEQ ID NO: 39), and QQYSX 16 YPX17 an LCDR3 comprising the sequence of T (SEQ ID NO: 189); or c) HCDR1 comprising the sequence EYVLS (SEQ ID NO: 41), HCDR2 comprising the sequence EIYPGTITTYYNEKFKG (SEQ ID NO: 42), and HCDR3 comprising the sequence FYDYDGGWFAY (SEQ ID NO: 43), and / or LCDR1 comprising the sequence SASSSVSSSDLH (SEQ ID NO: 44), LCDR2 comprising the sequence GTSNLAS (SEQ ID NO: 45), and LCDR3 comprising the sequence QQWSGYPWT (SEQ ID NO: 46). wherein X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, X6 is Y or H, X7 is S or P, X8 is Y or C, X9 is Y or S, 10 is N or S, and X 11 is P or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is S or absent, and X 16 is S or A, and X 17 is F or L.

[0041]

[0053] In certain embodiments, the SIRP-alpha binding domain comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 23, an HCDR2 comprising the sequence of SEQ ID NO: 24 or SEQ ID NO: 198, and an HCDR3 comprising the sequence of SEQ ID NO: 25; and / or an LCDR1 comprising the sequence of SEQ ID NO: 26, an LCDR2 comprising the sequence of SEQ ID NO: 27, and an LCDR3 comprising the sequence of SEQ ID NO: 28; or b) an HCDR1 comprising the sequence of SEQ ID NO: 29, an HCDR2 comprising the sequence of SEQ ID NO: 30, and an HCDR3 comprising the sequence of SEQ ID NO: 31; and / or an LCDR1 comprising the sequence of SEQ ID NO: 32, an LCDR2 comprising the sequence of SEQ ID NO: 33, and an LCDR3 comprising the sequence of SEQ ID NO: 34; or c) an HCDR1 comprising the sequence of SEQ ID NO: 35, an HCDR2 comprising the sequence of SEQ ID NO: 36, and an HCDR3 comprising the sequence of SEQ ID NO: 37; and / or an LCDR1 comprising the sequence of SEQ ID NO: 38, an LCDR2 comprising the sequence of SEQ ID NO: 39, and an LCDR3 comprising the sequence of SEQ ID NO: 40; or d) HCDR1 comprising the sequence of SEQ ID NO: 47, HCDR2 comprising the sequence of SEQ ID NO: 48, and HCDR3 comprising the sequence of SEQ ID NO: 49; and / or LCDR1 comprising the sequence of SEQ ID NO: 50, LCDR2 comprising the sequence of SEQ ID NO: 51, and LCDR3 comprising the sequence of SEQ ID NO: 52.

[0042]

[0054] In certain embodiments, the SIRP-alpha binding domain comprises HCDRs and LCDRs identical to those of an anti-SIRP-alpha antibody selected from the group consisting of C25, C15, C42, C59, and C73; a) C25 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:1 and / or a light chain variable region comprising the sequence of SEQ ID NO:2; b) C15 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 11 and / or a light chain variable region comprising the sequence of SEQ ID NO: 12; c) C42 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the sequence of SEQ ID NO: 14; d) C59 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 15 and / or a light chain variable region comprising the sequence of SEQ ID NO: 16; and e) C73 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:17 and / or a light chain variable region comprising the sequence of SEQ ID NO:18.

[0043]

[0055] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 is EVQLVQSGAEVKKPGATVKISCKX 20 SGFNIK (SEQ ID NO: 190) or a homologous sequence having at least 80% sequence identity thereto; and / or b) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191), or a homologous sequence having at least 80% sequence identity thereto; and / or c) The HFR3 sequence is RVTITADTSTX 21 TAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 192), or a homologous sequence having at least 80% sequence identity thereto; and / or d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193), or a homologous sequence having at least 80% sequence identity thereto; and / or e) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194), or a homologous sequence having at least 80% sequence identity thereto; and / or f) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto; and / or g) LFR3 is GIPARFSGSGSGTDX 22 TLTISSLEPEDFAVYYC (SEQ ID NO: 196), or a homologous sequence having at least 80% sequence identity thereto; and / or h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197) or a homologous sequence having at least 80% sequence identity thereto; In the formula, X 20 is A or V, and X 21 is N or D, and X 22 is Y or F.

[0044]

[0056] In certain embodiments, the SIRP-alpha binding domain comprises: f) a heavy chain variable region comprising the sequence of SEQ ID NO: 1 and / or a light chain variable region comprising the sequence of SEQ ID NO: 2, or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 3 and / or a light chain variable region comprising the sequence of SEQ ID NO: 4, or h) a heavy chain variable region comprising the sequence of SEQ ID NO:5 and / or a light chain variable region comprising the sequence of SEQ ID NO:6, or i) a heavy chain variable region comprising the sequence of SEQ ID NO: 7, and / or a light chain variable region comprising the sequence of SEQ ID NO: 8, or j) a heavy chain variable region comprising the sequence of SEQ ID NO: 9 and / or a light chain variable region comprising the sequence of SEQ ID NO: 10, or k) a heavy chain variable region comprising the sequence of SEQ ID NO: 11, and / or a light chain variable region comprising the sequence of SEQ ID NO: 12, or l) a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the sequence of SEQ ID NO: 14, or m) a heavy chain variable region comprising the sequence of SEQ ID NO: 15, and / or a light chain variable region comprising the sequence of SEQ ID NO: 16, or n) a heavy chain variable region comprising the sequence of SEQ ID NO: 17, and / or a light chain variable region comprising the sequence of SEQ ID NO: 18, or o) a heavy chain variable region comprising the sequence of SEQ ID NO: 159, and / or a light chain variable region comprising the sequence of SEQ ID NO: 160 Includes.

[0045]

[0057] In certain embodiments, the target antigen binding domain comprises a claudin 18.2 binding domain.

[0058] In certain embodiments, the claudin 18.2 binding domain comprises: p) an HCDR1 comprising the sequence of SEQ ID NO: 77, an HCDR2 comprising the sequence of SEQ ID NO: 78, and an HCDR3 comprising the sequence of SEQ ID NO: 79; and / or an LCDR1 comprising the sequence of SEQ ID NO: 80, an LCDR2 comprising the sequence of SEQ ID NO: 81, and an LCDR3 comprising the sequence of SEQ ID NO: 82 or SEQ ID NO: 225, or q) an HCDR1 comprising the sequence of SEQ ID NO: 83, an HCDR2 comprising the sequence of SEQ ID NO: 84, and an HCDR3 comprising the sequence of SEQ ID NO: 85; and / or an LCDR1 comprising the sequence of SEQ ID NO: 86, an LCDR2 comprising the sequence of SEQ ID NO: 87, and an LCDR3 comprising the sequence of SEQ ID NO: 88, or r) an HCDR1 comprising the sequence of SEQ ID NO: 89, an HCDR2 comprising the sequence of SEQ ID NO: 90, and an HCDR3 comprising the sequence of SEQ ID NO: 91; and / or an LCDR1 comprising the sequence of SEQ ID NO: 92, an LCDR2 comprising the sequence of SEQ ID NO: 93, and an LCDR3 comprising the sequence of SEQ ID NO: 94, or s) HCDR1 comprising the sequence of SEQ ID NO: 95, HCDR2 comprising the sequence of SEQ ID NO: 96, and HCDR3 comprising the sequence of SEQ ID NO: 97; and / or LCDR1 comprising the sequence of SEQ ID NO: 98, LCDR2 comprising the sequence of SEQ ID NO: 99, and LCDR3 comprising the sequence of SEQ ID NO: 100, or t) HCDR1 comprising the sequence of SEQ ID NO: 101, HCDR2 comprising the sequence of SEQ ID NO: 102, and HCDR3 comprising the sequence of SEQ ID NO: 103; and / or LCDR1 comprising the sequence of SEQ ID NO: 104, LCDR2 comprising the sequence of SEQ ID NO: 105, and LCDR3 comprising the sequence of SEQ ID NO: 106. Includes.

[0046]

[0059] In certain embodiments, the claudin 18.2 binding domain comprises HCDRs and LCDRs identical to an anti-claudin 18.2 antibody selected from the group consisting of hu26.H1L1, hu26.H1L2(S92A), hu28.H1L2, C10, C29, and C30; u) hu26.H1L1 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 65, and / or a light chain variable region comprising the sequence of SEQ ID NO: 66; v) hu26.H1L2(S92A) comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 65 and / or a light chain variable region comprising the sequence of SEQ ID NO: 224; w) hu28.H1L2 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 69 and / or a light chain variable region comprising the sequence of SEQ ID NO: 70; x) C10 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 71 and / or a light chain variable region comprising the sequence of SEQ ID NO: 72; y) C29 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 73 and / or a light chain variable region comprising the sequence of SEQ ID NO: 74; and z) C30 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the sequence of SEQ ID NO: 76.

[0047]

[0060] In certain embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises an amino acid sequence selected from the group consisting of EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167) and QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 168), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 is WVRQAPGKGLEWVX 18 (SEQ ID NO: 169) and WVRQAPGQGLEWMG (SEQ ID NO: 170), or a homologous sequence having at least 80% sequence identity thereto; c) The HFR3 sequence is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAX 23 (SEQ ID NO: 171) and RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 172), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises an amino acid sequence selected from the group consisting of DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174) and DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 175), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 is WYQQKPGX 26 X 27 PKX 19 LIY (SEQ ID NO: 176), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 is GVPSRFSGSGSGTEX 24TLTISSLQPEDFATYYC (SEQ ID NO: 178) and GVPDRFSGSGSGTDFTLTISSLQAEDVAVYHC (SEQ ID NO: 179), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 is FGX 25 GTKLEIK (SEQ ID NO: 180), or a homologous sequence having at least 80% sequence identity thereto; In the formula, X 18 is S or A, and X 19 is L or A, and X 23 is T or K, and X 24 is Y or F, and X 25 is Q or G, and X 26 is Q or K, and X 27 is P or A.

[0048]

[0061] In certain embodiments, the claudin 18.2 binding domain comprises: aa) a heavy chain variable region comprising the sequence of SEQ ID NO: 65 or 68, and / or a light chain variable region comprising the sequence of SEQ ID NO: 66 or 67 or 224, or bb) a heavy chain variable region comprising the sequence of SEQ ID NO: 69 and / or a light chain variable region comprising the sequence of SEQ ID NO: 70, or cc) a heavy chain variable region comprising the sequence of SEQ ID NO: 71 and / or a light chain variable region comprising the sequence of SEQ ID NO: 72, or dd) a heavy chain variable region comprising the sequence of SEQ ID NO: 73, and / or a light chain variable region comprising the sequence of SEQ ID NO: 74, or ee) a heavy chain variable region comprising the sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the sequence of SEQ ID NO: 76 Includes.

[0049]

[0062] In certain embodiments, the target antigen binding domain comprises a PD-L1 binding domain.

[0063] In certain embodiments, the PD-L1 binding domain comprises: ff) HCDR1 comprising the sequence of SEQ ID NO: 119, HCDR2 comprising the sequence of SEQ ID NO: 120 and HCDR3 comprising the sequence of SEQ ID NO: 121, or gg) HCDR1 comprising the sequence of SEQ ID NO: 122, HCDR2 comprising the sequence of SEQ ID NO: 123, and HCDR3 comprising the sequence of SEQ ID NO: 124, or hh) an HCDR1 comprising the sequence of SEQ ID NO: 125, an HCDR2 comprising the sequence of SEQ ID NO: 126, and an HCDR3 comprising the sequence of SEQ ID NO: 127, or ii) an HCDR1 comprising the sequence of SEQ ID NO: 128, an HCDR2 comprising the sequence of SEQ ID NO: 129, and an HCDR3 comprising the sequence of SEQ ID NO: 130, or jj) an HCDR1 comprising the sequence of SEQ ID NO: 131, an HCDR2 comprising the sequence of SEQ ID NO: 132, and an HCDR3 comprising the sequence of SEQ ID NO: 133, or kk) HCDR1 comprising the sequence of SEQ ID NO: 134, HCDR2 comprising the sequence of SEQ ID NO: 135, and HCDR3 comprising the sequence of SEQ ID NO: 136, or ll) HCDR1 comprising the sequence of SEQ ID NO: 137, HCDR2 comprising the sequence of SEQ ID NO: 138, and HCDR3 comprising the sequence of SEQ ID NO: 139, or mm) an HCDR1 comprising the sequence of SEQ ID NO: 140, an HCDR2 comprising the sequence of SEQ ID NO: 141, and an HCDR3 comprising the sequence of SEQ ID NO: 142, or nn) HCDR1 comprising the sequence of SEQ ID NO: 143, HCDR2 comprising the sequence of SEQ ID NO: 144, and HCDR3 comprising the sequence of SEQ ID NO: 145, or oo) HCDR1 comprising the sequence of SEQ ID NO: 146, HCDR2 comprising the sequence of SEQ ID NO: 147, and HCDR3 comprising the sequence of SEQ ID NO: 148, or pp) HCDR1 comprising the sequence of SEQ ID NO: 149, HCDR2 comprising the sequence of SEQ ID NO: 150, and HCDR3 comprising the sequence of SEQ ID NO: 151, or HCDR1 comprising the sequence of SEQ ID NO: 152, HCDR2 comprising the sequence of SEQ ID NO: 153, and HCDR3 comprising the sequence of SEQ ID NO: 154. Includes.

[0050]

[0064] In certain embodiments, the PD-L1 binding domain comprises HCDRs identical to those of an anti-PD-L1 antibody selected from the group consisting of: C71, C71v38, C239, C492, C570, 570h3, C446, C2811, C1778, C1793, C2855, C2713, and C2719; qq) C71 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 107; rr) C71v38 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 108; ss) C239 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 109; tt) C492 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 110; uu) C570 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 111; vv) 570h3 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 223; ww) C446 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 112; xx) C2811 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 113; yy) C1778 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 114; zz) C1793 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 115; aaa) C2855 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 116; bbb) C2713 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 117, and ccc) C2719 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:118.

[0051]

[0065] In certain embodiments, the PD-L1 binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 107-118, and 223.

[0066] In certain embodiments, ddd) the SIRP-alpha binding domain further comprises a substitution or modification of one or more amino acid residues whilst still retaining specific binding to human SIRPα; and / or eee) the claudin 18.2 binding domain further comprises a substitution or modification of one or more amino acid residues while still retaining specific binding to claudin 18.2; and / or fff) The PD-L1 binding domain may further comprise a substitution or modification of one or more amino acid residues whilst still retaining specific binding to PD-L1.

[0052]

[0067] In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of the heavy chain variable region or the light chain variable region and / or in one or more of the non-CDR sequences.

[0053]

[0068] In certain embodiments, the multispecific molecules presented herein are humanized.

[0069] In certain embodiments, the multispecific molecules presented herein are linked to one or more conjugate moieties.

[0054]

[0070] In certain embodiments, the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification moiety, or other anti-cancer agent.

[0055]

[0071] In another aspect, the present disclosure provides pharmaceutical compositions comprising the multispecific molecules presented herein and one or more pharma- ceutically acceptable carriers.

[0072] In another aspect, the present disclosure provides isolated polynucleotides encoding the multispecific molecules provided herein.

[0056]

[0073] In another aspect, the disclosure provides a vector comprising an isolated polynucleotide provided herein.

[0074] In another aspect, the disclosure provides a host cell comprising the vectors provided herein.

[0057]

[0075] In another aspect, the present disclosure provides kits comprising a multispecific molecule as presented herein and / or a pharmaceutical composition as presented herein and a second therapeutic agent.

[0076] In another aspect, the present disclosure provides a method of expressing a multispecific molecule as presented herein, comprising culturing a host cell as presented herein under conditions in which a vector as presented herein is expressed.

[0058]

[0077] In another aspect, the present disclosure provides a method of treating a disease, disorder, or condition that may benefit from inducing phagocytosis of target cells in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

[0059]

[0078] In another aspect, the present disclosure provides a method of treating a disease, disorder, or condition associated with a target antigen in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

[0060]

[0079] In another aspect, the disclosure provides a method of treating a SIRPα-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

[0061]

[0080] In another aspect, the present disclosure provides a method of treating a CD47-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

[0062]

[0081] In certain embodiments, the subject is a human.

[0082] In certain embodiments, the subject has been diagnosed with or is at risk for a disease, disorder, or condition selected from the group consisting of immune-related diseases or disorders, tumors and cancers, autoimmune diseases, and infectious diseases.

[0063]

[0083] In certain embodiments, the immune-related disease or disorder is selected from the group consisting of systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), vasculitis, myasthenia gravis, idiopathic pulmonary fibrosis, Crohn's disease, asthma, rheumatoid arthritis, graft-versus-host disease, spondyloarthritic disorders (e.g., ankylosing spondylitis, psoriatic arthritis, sporadic acute enteropathic arthritis associated with inflammatory bowel disease, reactive arthritis, Behcet's syndrome, undifferentiated spondyloarthropathy, anterior uveitis, and juvenile idiopathic arthritis), multiple sclerosis, endometriosis, glomerulonephritis, sepsis, diabetes, acute coronary syndrome, ischemia-reperfusion, psoriasis, progressive systemic sclerosis, atherosclerosis, Sjogren's syndrome, scleroderma, or inflammatory autoimmune myositis.

[0064]

[0084] In certain embodiments, the tumors and cancers are optionally selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mycosis, mycoses ... Mediastinal large B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, etc., nasopharyngeal carcinoma, and HHV8-related primary effusion lymphoma, Hodgkin's lymphoma, neoplasms of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis tumors, brain stem glioma, etc., anal cancer, appendix cancer, star tumors, etc. cytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary gland cancer, vaginal cancer, thyroid cancer, throat cancer The solid tumor or hematological malignancy is selected from the group consisting of head and neck cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma, or metastasis thereof.

[0065]

[0085] In certain embodiments, administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0086] In certain embodiments, the methods presented herein further comprise the step of administering a therapeutically effective amount of a second therapeutic agent.

[0066]

[0087] In certain embodiments, the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer agents, radiation therapy agents, immunotherapy agents, anti-angiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormonal therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0067]

[0088] In another aspect, the present disclosure provides for the use of a multispecific molecule as presented herein, and / or a pharmaceutical composition as presented herein, in the manufacture of a medicament for treating, preventing, or alleviating a disease, disorder, or condition in a subject that may benefit from inducing phagocytosis of target cells.

[0068]

[0089] In another aspect, the present disclosure provides a method of inducing phagocytosis of a target cell in a subject, comprising administering to the subject a multispecific molecule as presented herein and / or a pharmaceutical composition as presented herein in a dose effective to induce phagocytosis of the target cell.

[0069]

[0090] In certain embodiments, the subject is a human.

[0091] In certain embodiments, the subject has been diagnosed with or is at risk for a disease, disorder, or condition selected from the group consisting of immune-related diseases or disorders, tumors and cancers, autoimmune diseases, and infectious diseases.

[0070]

[0092] In another aspect, the present disclosure provides a method for inducing phagocytosis of target cells in vitro, comprising contacting target cells with a sample of SIRPα-positive phagocytic cells in the presence of a multispecific molecule as provided herein and / or a pharmaceutical composition as provided herein, thereby inducing phagocytosis of the target cells by the SIRPα-positive phagocytic cells.

[0071]

[0093] In certain embodiments, the target cell is a cell that expresses a target antigen.

[0094] In another aspect, the present disclosure provides a method for inducing removal of a target cell that co-expresses a target antigen and CD47 by phagocytosis, comprising contacting the target cell with a multispecific molecule presented herein in the presence of phagocytic immune cells.

[0072]

[0095] In another aspect, the present disclosure provides a method for selectively inducing phagocytosis in a subject against target cells that co-express a target antigen and CD47 over cells that do not express the target antigen, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.

[0073]

[0096] In another aspect, the present disclosure provides a method of increasing levels of M1 macrophages in the tumor microenvironment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein. [Brief description of the drawings]

[0074] [Figure 1]

[0097] FIG. 1 shows a schematic diagram of bispecific macrophage engager (BiME) antibodies that enhance macrophage phagocytic activity against cancer cells expressing tumor-associated antigens (TAA). [Figure 2A]

[0098] FIG. 1 shows a schematic diagram of an anti-claudin 18.2 / SIRPα bispecific molecule (ES028-001) comprising an antibody targeting claudin 18.2 fused to two anti-SIRPα scFvs at the C-terminus of the light chain (LC). [Figure 2B]

[0099] FIG. 1 shows a schematic diagram of an anti-claudin 18.2 / SIRPα bispecific molecule (ES028-005) comprising an antibody targeting claudin 18.2 fused to two anti-SIRPα scFvs at the C-terminus of the heavy chain (HC). [Figure 2C][000100] FIG. 1 shows a schematic diagram of an anti-claudin18.2 / SIRPα bispecific molecule (ES028-009) containing an antibody targeting SIRPα fused to two anti-claudin18.2 scFvs at the C-terminus of the light chain (LC). [Figure 2D] [000101] Figure 1 shows a schematic diagram of an anti-claudin18.2 / SIRPα bispecific molecule (ES028-013) containing an antibody targeting SIRPα fused to two anti-claudin18.2 scFvs at the C-terminus of the heavy chain (LC). [Figure 3A] [000102] Representative bispecific antibodies were able to bind to Raji / h-claudin 18.2 cells by FACS detection. Anti-claudin 18.2 antibody was used as a control. [Figure 3B] [000103] Figure 1 shows that representative bispecific antibodies were able to bind to CHO-K1 / SIRPα cells by FACS detection. Anti-SIRPα antibody was used as a control. [Figure 4A] [000104] Figure 1 shows that anti-claudin18.2 / SIRPα bispecific antibody stimulates mouse BMDM phagocytosis better than single or combined treatments against MC38 / hCD47 / hclaudin18.2 cells. Anti-CD47 antibody is used as a control. [Figure 4B] [000105] Figure 1 shows that anti-claudin18.2 / SIRPα bispecific antibody does not stimulate mouse BMDM phagocytosis against MC38 / hCD47 cells that do not express Claudin18.2. Anti-CD47 antibody is used as a control. [Diagram 5] [000106] Figure 1 shows a comparison of mouse BMDM phagocytosis towards MC38 / hCD47 / hClaudin18.2 cells for several anti-claudin18.2 / SIRPα bispecific antibody isotypes. Anti-CD47 antibody is used as a control. [Figure 6A][000107] Figure 1 shows a schematic diagram of an anti-PDL1 / SIRPα bispecific molecule (ES019-020) containing one antibody targeting SIRPα fused to two anti-PDL1 sdAbs at the C-terminus of the light chain (LC). [Figure 6B] [000108] Figure 1 shows a schematic diagram of an anti-PDL1 / SIRPα bispecific molecule (ES019-024) containing one antibody targeting SIRPα fused to two anti-PDL1 sdAbs at the C-terminus of the heavy chain (HC). [Figure 6C] [000109] Figure 1 shows a schematic diagram of an anti-PDL1 / SIRPα bispecific molecule (ES019-025) containing one antibody targeting SIRPα fused to two anti-PDL1 sdAbs at the N-terminus of the heavy chain (HC). [Figure 6D] [000110] Figure 1 shows a schematic diagram of an anti-PDL1 / SIRPα bispecific molecule (ES019-026) containing one antibody targeting SIRPα fused to two anti-PDL1 sdAbs at the N-terminus of the light chain (LC). [Figure 6E] [000111] Schematic diagram of an anti-PDL1 / SIRPα bispecific molecule (ES019-029) containing one asymmetric antibody with one Fab arm targeting SIRPα and the other arm containing two anti-PDL1 sdAbs; the heterodimer is linked by a knob-in-hole mutation in the Fc region. [Figure 6F] [000112] Schematic diagram of anti-PDL1 / SIRPα bispecific molecule (ES019-072) containing one asymmetric antibody with two anti-PDL1 sdAbs fused to the N-terminus of Fc and one anti-SIRPα Fab fused to the C-terminus of Fc, the heterodimer is linked by knob-in-hole mutations in the Fc region. [Figure 6G] [000113] Figure 1 shows a schematic diagram of an anti-PDL1 / SIRPα bispecific molecule (ES019-073 or ES019-079) containing one asymmetric antibody with two anti-PDL1 sdAbs fused to the N-terminus of Fc and two anti-SIRPα Fabs fused to the C-terminus of Fc. [Figure 7A] [000114] Representative bispecific antibodies ES019-020, ES019-024, ES019-025, and ES019-026 were able to bind to Raji / hPDL1 cells by FACS detection. Anti-PDL1 antibody was used as a control. [Figure 7B] [000115] Representative bispecific antibodies ES019-020, ES019-024, ES019-025, and ES019-026 were able to bind to CHO-K1 / SIRPα cells by FACS detection. Anti-SIRPα antibody was used as a control. [Figure 8] [000116] Representative bispecific antibodies ES019-020, ES019-024, ES019-025, and ES019-026 were able to activate Jurkat T cells by Jurate / PD1 reporter cell assay. Anti-PDL1 antibody was used as a control. [Figure 9A] [000117] Figure 1 shows that anti-PDL1 / SIRPα bispecific antibodies ES019-020, ES019-024, ES019-025, ES019-026, and ES019-029 stimulate human monocyte-derived macrophage phagocytosis of K562 / hPDL1 cells better than single treatments or similar to combination treatments. Anti-CD47 antibody is used as a control. [Figure 9B] [000118] Figure 1 shows that anti-PDL1 / SIRPα bispecific antibodies do not stimulate phagocytosis of human monocyte-derived macrophages towards K562 (PDL1 negative) cells similar to single or combined treatments. Anti-CD47 antibody is used as a control. [Figure 9C] [000119] Figure 1 shows that anti-PDL1 / SIRPα bispecific antibody does not stimulate phagocytosis of human monocyte-derived macrophages towards Jurkat cells (SIRPγ positive) similarly to single or combination treatments. Anti-CD47 antibody is used as a control. [Figure 10][000120] Figure 1 shows the in vivo anti-tumor efficacy of ES028 BiME (e.g., ES028-001, ES028-005, and ES028-009) in the MC38 / hClaudin18.2 / hSIRPα syngenic model. [Figure 11A] [000121] Figure 1 shows that combinations based on different SIRPα antibodies or SIRPα bispecific antibodies induced phagocytosis of Raji / hPDL1 cells. [Figure 11B] [000122] Figure 1 shows that different SIRPα antibody-based combinations or PDL1 / SIRPα bispecific antibodies induced phagocytosis of Raji (PDL1 negative) cells. [Figure 12] [000123] Figure 13 shows the binding affinity of chimeric antibodies C15, C25, C42, C59, C73, and hu1H9G4 to CHOK1-hSIRPα v1 and CHOK1-hSIRPα v2. [Figure 13] [000124] Figure 1 shows the IC50 values ​​and blocking rates (%) of human CD47 / SIRPα v1 interaction and human CD47 / SIRPα v2 interaction for each of the chimeric antibodies C15, C25, C42, C59, C73, and hu1H9G4. [Figure 14] [000125] This figure shows the IC50 values ​​and blocking rates (%) of SHP-1 transduction for each of the chimeric antibodies C15, C25, C42, C59, C73, and hu1H9G4. [Figure 15] [000126] Figure showing the binding affinity of humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060, and C25 to CHOK1-hSIRPα v1 and CHOK1-hSIRPα v2. [Figure 16] [000127] Figure 1 shows the binding kinetics of humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060, and C25 to human SIRPα v1 and human SIRPα v2. [Figure 17][000128] Figure 1 shows the IC50 values ​​and blocking rates (%) of human CD47 / SIRPα v1 interaction and human CD47 / SIRPα v2 interaction for humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060, and C25, as measured by competitive ELISA assay. [Figure 18] [000129] Figure 1 shows the IC50 values ​​and blocking rates (%) of human CD47 / SIRPα v1 and human CD47 / SIRPα v2 interactions for humanized antibodies hu025.023, hu025.060, and C25, as measured by competitive FACS assay. [Figure 19] [000130] This figure shows the IC50 values ​​and blocking rates (%) of SHP-1-introduced humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060, as well as C25. [Figure 20A] [000131] Figure 14 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v1 / v2 in the presence of anti-SIRPα antibodies 025c, 015c, 042c, 059c, or 073c alone or in combination with rituximab. [Figure 20B] [000132] Figure 2 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v1 / v1 in the presence of anti-SIRPα antibodies 025c or 042c alone or in combination with different concentrations of anti-PD-L1 antibodies. [Figure 20C] [000133] Figure 2 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v2 / v2 in the presence of anti-SIRPα antibodies 025c, 042c, or 073c alone or in combination with different concentrations of anti-PD-L1 antibodies. [Figure 21A][000134] Figure 2 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v1 / v1 in the presence of anti-SIRPα antibodies 025c, hu025.023, or hu025.060 alone or in combination with different concentrations of anti-PD-L1 antibodies. [Figure 21B] [000135] Figure 14 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v1 / v1 in the presence of anti-SIRPα antibodies 025c, hu025.023, or hu025.060 alone or in combination with different concentrations of rituximab. [Figure 21C] [000136] Figure 2 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v2 / v2 in the presence of anti-SIRPα antibodies 025c, hu025.023, or hu025.060 alone or in combination with different concentrations of anti-PD-L1 antibodies. [Figure 21D] [000137] Figure 14 shows the phagocytic index of Raji cells by human macrophages expressing hSIRPα v2 / v2 in the presence of anti-SIRPα antibodies 025c, hu025.023, or hu025.060 alone or in combination with different concentrations of rituximab. [Figure 22] [000138] Figure 1 shows the blocking rate (%) of different concentrations of anti-SIRPα antibodies 035, 050, and 025 (% blocking) for blocking the interaction between SIRPα and CD47. [Figure 23A] [000139] Figure 23 shows the epitope binding potential of anti-SIRPα antibodies 025c (Figure 23A), 042c (Figure 23B), 073c (Figure 23C), hu1H9G4 (Figure 23D), HEFLB (Figure 23E) as measured by HDX-MS. All antibodies are human IgG4 chimeric antibodies with the S228P mutation. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 23E] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] [000140] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. Therefore, the particular modifications discussed should not be construed as limitations on the scope of the present disclosure. It is obvious to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of the present disclosure, and such equivalent embodiments are understood to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein in their entirety by reference.

[0076] [000141]Definition [000142] The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, single domain antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. Naturally occurring, unaltered IgG antibodies contain two heavy chains (H) and two light chains (L). Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable region (V H ) and the first, second, and third constant regions (C H1 , C H2 , C H3 ), mammalian light chains are classified as lambda or kappa, while each light chain is composed of a variable region (V L) and constant regions. Antibodies have 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 bonds. Each arm of the Y contains 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 complementarity determining regions (CDRs) (light chain CDRs containing LCDR1, LCDR2, and LCDR3, and heavy chain CDRs containing HCDR1, HCDR2, and HCDR3). The boundaries of the CDRs for the antibodies and antigen-binding domains disclosed herein may be defined or specified by the rules 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(1989);Kabat EAet 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 inserted between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to several classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0077] [000143] The term "antibody" as used herein may also encompass single domain antibodies, such as heavy chain antibodies. "Heavy chain antibody" or "HCAb" refers to an antibody that comprises two VH domains and has 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); WO 94 / 04678; WO 94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies are originally derived from camelids (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies have a bona fide antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0078] [000144] The term "antigen binding domain" as used herein refers to an antibody fragment formed from a portion of an antibody that contains one or more CDRs or any other antibody fragment that binds to an antigen but does not contain the intact native antibody structure. Examples of antigen binding domains include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen binding domain has the ability to bind to the same antigen as that bound by the parent antibody. In certain embodiments, an antigen-binding domain may comprise one or more CDRs from a particular human antibody grafted onto framework regions derived from one or more different human antibodies. More detailed formats of antigen-binding domains are described in Spiess et al., 2015 (supra), and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.

[0079] [000145] As used herein, the term "phagocytosis" refers to the process of cellular uptake of particulate matter (>0.5 Dm) within a plasma membrane envelope. Phagocytosis includes different variants, such as efferocytosis, which is associated with the uptake of apoptotic cells, necroptosis and pyroptosis, which are associated with the uptake of necrotic cells resulting from infection and inflammation, and phagocytosis, which is associated with the uptake of exogenous particles.

[0080] [000146] "Antigen," as used herein, refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate the production of antibodies or an immune cell (e.g., T cell or myeloid cell) response in cell culture or in animals, and includes compositions that are added to cell cultures (e.g., hybridomas, etc.) or injected or absorbed into animals, or expressed on the cell surface (e.g., those that contain cancer-specific proteins, etc.). Antigens react with the products of specific humoral or cellular immunity (e.g., antibodies, etc.).

[0081] [000147] "Fab," when referring to an antibody, refers to the portion of an antibody that is composed of a single light chain (both variable and constant regions) linked by disulfide bonds to a single heavy chain variable region and a first constant region. "F(ab)2" refers to a dimer of Fab.

[0082] [000148] "Fab'" refers to a Fab fragment that contains part of the hinge region. [000149] "F(ab')2" refers to a Fab' dimer. [000150] "Fragment difficult (Fd)" with respect to antibodies refers to the amino terminal half of a heavy chain fragment that can combine with a light chain to form Fab. For example, an Fd fragment can be composed of the VH and CH1 domains.

[0083] [000151] "Fv" with respect to antibodies refers to the smallest fragment of an antibody that carries a complete antigen-binding site. Fv fragments consist of the variable region of a single light chain bound to the variable region of a single heavy chain. Several Fv designs have been provided, including dsFvs, which are enhanced by disulfide bonds that introduce association between the two domains, but scFvs can also be formed using a peptide linker that links the two domains together as a single polypeptide. Fv constructs have also been generated that contain variable domains of immunoglobulin heavy or light chains associated with the variable and constant domains of the corresponding immunoglobulin heavy or light chains. Fvs have also been multimerized to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0084] [000152] "Single chain Fv antibody" or "scFv" refers to an engineered antibody that is composed of a light chain variable region and a heavy chain variable region linked together directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). ScFvs can serve as basic modules for developing multimeric structures (dimers: "diabodies", trimers: "triabodies", tetramers: "tetrabodies").

[0085] [000153] A "diabody" or "dAb" refers to a small antibody fragment with two antigen-binding sites, the fragment having a V L V associated with the domain H Domain Included (V H -V L Or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14):6444-8 (1993); EP 404097; WO 93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0086] [000154] "dsFv" refers to a disulfide-stabilized Fv fragment in which 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 two VFs linked by a peptide linker (e.g., a long flexible linker). H The V moiety is connected to two V L In some embodiments, the dsFv-dsFv' comprises three peptide chains each linked to a different moiety. In some embodiments, the dsFv-dsFv' is bispecific, with each disulfide-paired heavy and light chain having a different antigen specificity.

[0087] [000155] The term "valent" as used herein refers to the presence of a specified number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment having only one single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple (i.e., two or more) antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0088] [000156] "Nanobody" refers to an antibody fragment that is composed of a VHH domain derived from a heavy chain antibody, and two constant domains, CH2 and CH3. [000157] "Domain antibody" or "single domain antibody" or "sdAb" refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain cases, two or more VH domains are covalently linked 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.

[0089] [000158] "Fc," with respect to an antibody, refers to the portion of an antibody that is composed of the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is involved in various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and phagocytosis.

[0090] [000159] The term "chimeric" as used herein refers to an antibody or antigen-binding domain having a portion of the heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In another illustrative example, a chimeric antibody may contain a FR region derived from a human and a CDR region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0091] [000160] The term "humanized," as used herein, means that an antibody or antigen-binding domain contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

[0092] [000161] The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest in a relationship that allows them to function in the intended manner, with or without spacers or linkers or intervening sequences. When used in reference to a polypeptide, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function. For example, an antibody variable region may be operably linked to a constant region to provide a stable product with antigen-binding activity. In another example, an antigen-binding domain may be operably linked to another antigen-binding domain by a sequence located between them, but such a sequence located between them may be a spacer or may include a much longer sequence, such as the constant region of an antibody. The term may also be used in reference to a polynucleotide. In one case, when a polynucleotide encoding a polypeptide is operably linked to a control sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequence is linked to allow for controlled expression of the polypeptide from the polynucleotide.

[0093] [000162] The terms "fusion" or "fused," when used in reference to amino acid sequences (e.g., peptides, polypeptides, or proteins), refer to the combination of two or more amino acid sequences into a single amino acid sequence, for example, by chemical conjugation or recombinant means. A fused amino acid sequence can be produced by the genetic recombination of two coding polynucleotide sequences, but can also be expressed by methods in which a construct containing a recombinant polynucleotide is introduced into a host cell.

[0094] [000163] "SIRPα" is used interchangeably with "SIRP alpha" or "SIRP-alpha" and as used herein refers to a regulatory membrane glycoprotein from the signal-regulatory protein (SIRP) family that is primarily expressed by myeloid cells (e.g., macrophages, granulocytes, myeloid dendritic cells, mast cells, and their precursors, such as hematopoietic stem cells (HSCs)), dendritic cells, and also by stem cells or neurons. The structure of SIRPα includes an extracellular domain and a cytoplasmic domain. The extracellular domain of SIRPα is composed of a membrane-distal Ig variable-like (IgV) fold and two membrane-proximal Ig constant-like (IgC) folds. The IgV domain of SIRPα is involved in binding the extracellular Ig domain of CD47. In certain embodiments, SIRPα is human SIRPα. The gene encoding human SIRPα is a polymorphic gene, and several variants have been described in the human population. The most common protein variants are SIRPα v1 and SIRPα v2 (Accession Nos. NP_542970 (P78324) and CAA71403). SIRPα, as used herein, may be from other animal species, such as mouse and cynomolgus monkey, among others. Exemplary sequences of Mus Musculus (mouse) SIRPα proteins are disclosed in NCBI Ref Seq No. NP_031573, or BAA20376.1, or BAA13521.1. Exemplary sequences of Cynomolgus (monkey) SIRPα proteins are disclosed in NCBI Ref Seq No. NP_001271679.

[0095] [000164] "PD-L1" as used herein refers to programmed cell death ligand 1 (PD-L1, see e.g., Freeman et al. (2000) J. Exp. Med. 192:1027). A representative amino acid sequence of human PD-L1 is disclosed under NCBI Accession No. NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is set forth under NCBI Accession No. NM_014143.3. PD-L1 is expressed in the 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. Binding of PD-L1 to its receptor induces signal transduction and suppresses TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 is thought to play an important role in suppressing the immune system during certain events, such as pregnancy, autoimmune diseases, tissue allogeneic transplantation, etc., and also allows tumor or cancer cells to bypass immune checkpoints and evade the immune response.

[0096] [000165] "CLDN18", as used herein, refers to claudin 18, including any variants thereof (CLDN18.1 and CLDN18.2, including conformations, isoforms, and interspecies homologs of CLDN18 expressed naturally by cells or expressed by cells transfected with the CLDN18 gene). In certain embodiments, CLDN18 is human CLDN18. CLDN18, as used herein, may be from other animal species, such as human, mouse, and cynomolgus monkey, among others. The terms "CLDN18", "CLDN-18", "CLDN 18", "claudin 18", "claudin-18", or "claudin 18" may be used interchangeably in the present disclosure. Unless otherwise specified, CLDN18, as used herein, refers to the CLDN18 protein.

[0097] [000166] "CLDN18.1" is a splice variant of CLDN18 and includes post-translationally modified variants, isoforms, and interspecies homologs of CLDN18.1 that are naturally expressed by cells or expressed on cells transfected with the CLDN18.1 gene. The terms "CLDN18.1", "CLDN-18.1", "CLDN 18.1", "claudin 18.1", "claudin-18.1", or "claudin 18.1" may be used interchangeably in this disclosure. Unless otherwise specified, CLDN18.1 as used herein refers to the CLDN18.1 protein. An exemplary sequence of the human CLDN18.1 protein is disclosed in NCBI Ref Seq No. NP_057453.1.

[0098] [000167] "CLDN18.2" is a splice variant of CLDN18 and includes post-translationally modified variants, isoforms, and interspecies homologs of CLDN18.2 that are naturally expressed by cells or expressed on cells transfected with the CLDN18.2 gene. The terms "CLDN18.2", "CLDN-18.2", "CLDN 18.2", "claudin 18.2", "claudin-18.2", or "claudin 18.2" may be used interchangeably in this disclosure. Unless otherwise specified, CLDN18.2 as used herein refers to the CLDN18.2 protein. An exemplary sequence of the human CLDN18.2 protein is disclosed in NCBI Ref Seq No. NP_001002026.1.

[0099] [000168] The terms "specific binding" or "specifically binds to" as used herein refer to a non-random binding reaction between two molecules, such as between an antibody or antigen-binding domain thereof and an antigen. In certain embodiments, the antibody molecules or antigen-binding domains provided herein have a binding affinity of ≦10 -6 M (e.g., ≦5×10 -7 M, ≤2×10 -7 M, ≦10 -7 M, ≤5×10 -8 M, ≤2×10 -8M, ≦10 -8 M, ≤5×10 -9 M, ≤4×10 -9 Binding affinity (K D ) and specifically binds to human SIRPα, human claudin 18.2, and / or human PD-L1. D is the ratio of the dissociation rate to the association rate (k off / k on ) and may be determined by using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS, etc.). In certain embodiments, K D The value may be suitably determined by using flow cytometry.

[0100] [000169] The term "epitope," as used herein, refers to a particular atom or group of amino acids on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids (also called linear or contiguous epitopes) or from non-contiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitopes). Epitopes formed from contiguous amino acids are generally arranged linearly along the primary amino acid residues on the protein, and small segments of contiguous amino acids can be digested from the antigen binding site by major histocompatibility complex (MHC) molecules or are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Epitopes generally include at least 3, and more usually at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within that antigen, or to closely related epitopes. For example, an antibody or antigen-binding domain may be considered to bind to the same / closely related epitope as a reference antibody if it blocks at least 85%, or at least 90%, or at least 95% of the binding of the reference antibody to the antigen.

[0101] [000170] The term "amino acid," as used herein, refers to an organic compound that comprises an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain unique to each amino acid. In this disclosure, the names of amino acids are also represented as standard one-letter or three-letter codes and are summarized as follows:

[0102] [Table 1]

[0103] [000171] "Conservative substitution", in the context of an amino acid sequence, refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can occur between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in the conformational structure of a protein and thus may retain the biological activity of the protein.

[0104] [000172] The terms "homolog" and "homologous" as used herein are interchangeable and refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0105] [000173] "Percent (%) sequence identity," when it comes to amino acid (or nucleic acid) sequences, is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for the purpose of determining the percentage of amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Bioinformatics Institute website, see also Higgins D Get al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or can customize the parameters for alignment as appropriate, for example by selecting a suitable algorithm.

[0106] [000174] As used herein, the term "myeloid cells" refers to normal or neoplastic cells found in blood, bone marrow, other hematopoietic or other non-hematopoietic compartments of the body. In particular, the term "myeloid cells" is used herein to mean cell lineages derived from bone marrow, including monocytes (which give rise to macrophages and dendritic cells), polymorphonuclear neutrophils, eosinophils, basophils, and mast cells, as well as the monocyte / macrophage lineage and the distinct dendritic cell lineage. The term refers to cells of the myeloid lineage found in all stages of their differentiation, and thus includes hematopoietic blasts, i.e., hematopoietic cells that are committed to the myeloid lineage but are still in an early stage of differentiation. Examples include myeloblasts, among others. The term "myeloid cells" also includes cell lineages, e.g., in the bone marrow, that have the capacity to differentiate into myeloid progenitor cells, i.e., myelomonocytic progenitors, proerythroblasts, or immature nucleoblasts, etc. "Treating" a condition or "treatment" thereof, as used herein, includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, producing a complete or partial regression of the condition, curing the condition, or some combination thereof.

[0107] [000175] The terms "subject" or "individual" or "animal" or "patient," as used herein, refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, farm animals, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0108] [000176] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted to cause expression of the protein. A vector can be used to transform, transduce, or transfect a host cell, causing expression of the genetic elements it carries in the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). A vector may contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector may contain an origin of replication. A vector may also contain substances that aid its entry into cells, including, but not limited to, viral particles, liposomes, or protein coatings. A vector may be an expression vector or a cloning vector.

[0109] [000177] The phrase "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector has been introduced. [000178] "Cancer" is used interchangeably with "tumor" herein and refers to any medical condition characterized by malignant cell proliferation or neoplasia, abnormal growth, invasion, or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies), such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. Examples of cancers or tumors include hematopoietic malignancies, cancers of the oral cavity (e.g., of the lip, tongue, or pharynx), digestive tract (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver, and biliary tract, pancreas, respiratory system, such as the larynx or lung (small cell and non-small cell), bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tubes, uterus, cervix, testes, ovaries, or prostate), urethra (e.g., bladder or kidney), brain, and endocrine glands, such as the thyroid gland. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer, In certain embodiments, the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0110] [000179] The term "pharmaceutical acceptable" refers to the specified carrier, vehicle, diluent, excipient(s), and / or salt generally being chemically and / or physically compatible with the other ingredients comprising the formulation, as well as physiologically compatible with the recipient thereof.

[0111] [000180]A. Multispecific molecules [000181] In one embodiment, the present disclosure provides a multispecific molecule comprising a SIRP-alpha binding domain, an activating receptor binding domain, and a target antigen binding domain. The multispecific molecule provided herein is configured to bind to 1) an activating receptor (e.g., FcγR expressed on a phagocytic cell), 2) a target antigen expressed on a target cell, and 3) an immune effector cell (e.g., a phagocytic cell, such as a monocyte or macrophage). The target antigen can be, for example, a tumor surface antigen, an inflammatory antigen, or an antigen of an infectious microorganism.

[0112] [000182] Without wishing to be bound by any theory, it is believed that the SIRP-alpha binding domain and the activating receptor binding domain may engage and provide multiple activation signals by cross-linking with SIRP-alpha and activating receptors on immune effector cells (e.g., phagocytic cells) and then cross-linking with target cells via interactions between the target antigen binding domain and the target antigen on the target cells. Such a design allows the immune effector cells to become activated and engage and be introduced into the target microenvironment, triggering phagocytosis and killing of target cells (e.g., cancer cells, infected cells, or damaged or diseased cells). Thus, the multispecific molecules of the present disclosure are also referred to as multispecific macrophage engagers. The engagers presented herein are particularly advantageous in selectively removing unwanted cells (e.g., cancer cells, etc.), while normal cells, even those expressing CD47, are not targeted by macrophage phagocytosis. Thus, the multispecific molecules presented herein exhibit a high degree of selectivity in eliminating abnormal cells from normal cells and therefore have fewer side effects, are less toxic and are safer, for example in cancer treatment.

[0113] [000183] Without wishing to be bound by any theory, in cancer treatment, activation of SIRP-alpha and / or activating receptors (e.g., FcγR) redirects tumor associated macrophages (TAMs) from a tumor-promoting state to an anti-tumor state. Thus, the multispecific molecule or engager design presented herein also allows for redirection of pre-existing and / or newly introduced tumor associated macrophages (TAMs) within the tumor microenvironment to enhance cancer killing. In other words, the multispecific molecule / multispecific macrophage engager presented herein can promote or maintain monocytes or macrophages that can effectively and specifically kill cancer cells, rather than becoming immunosuppressive macrophages and TAMs. Thus, the present disclosure also presents methods to redirect TAMs to anti-tumor macrophages to enhance phagocytosis of cancer cells.

[0114] [000184] Within cancer, there exists a tumor microenvironment that contributes to the establishment of an immunosuppressive environment. The tumor microenvironment is a complex natural milieu of cells that develop with and provide support to tumor cells during the transition to malignancy (Noy et al., Tumor-associated macrophages: from mechanisms to therapy, Immunity. 2014 July 17;41(1):49-61). Factors such as IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes can interfere with innate immune cell function.

[0115] [000185] Monocytes that induce mature macrophages can be attracted and migrate into the tumor microenvironment by numerous factors, with the majority of these monocytes being able to differentiate into TAMs (Zhou et al., (2020) Tumor-Associated Macrophages: Recent Insights and Therapies. Front. Oncol. 10:188). Macrophages present within the tumor microenvironment can also be repolarized by various factors within the tumor microenvironment such that these macrophages become differentiated into tumor-promoting TAMs.

[0116] [000186] TAMs primarily comprise alternatively activated macrophages (M2 phenotype) and a small fraction of classically activated macrophages (M1 phenotype) (Zhou et al., (2020) Tumor-Associated Macrophages: Recent Insights and Therapies. Front. Oncol. 10:188). Macrophages with the M1 phenotype are potent and have the ability to kill pathogens or cancer cells. Macrophages with the M2 phenotype lack the ability to phagocytose tumor cells and may help such tumor cells escape killing and spread to other tissues and organs (Zhou et al., (2020) Tumor-Associated Macrophages: Recent Insights and Therapies. Front. Oncol. 10:188).

[0117] [000187] The SIRP-alpha binding domain of the engagers presented herein can inhibit the downregulation of macrophage phagocytosis mediated by CD47-SIRP alpha axis signaling. Cancer cells typically overexpress CD47, which binds to SIRP alpha expressed on immune cells (e.g., monocytes or macrophages) to trigger a "don't eat me" signal that prevents the cancer cells from being eliminated by immune cells (e.g., monocytes or macrophages). Inhibition of the CD47-SIRP alpha axis can counteract cancer cell-mediated anti-phagocytic activity.

[0118] [000188] The SIRP-alpha binding domains provided herein can inhibit CD47-SIRPalpha axis signaling by blocking CD47-SIRPalpha interaction and / or blocking downstream signaling mediated by CD47-SIRPalpha interaction (e.g., SHP-1 signaling). In certain embodiments, the SIRP-alpha binding domain can include a SIRPalpha blocker, such as the extracellular domain (ECD) of CD47 that recognizes and binds to SIRPalpha, or an antibody or antigen binding domain thereof that recognizes and binds to SIRPalpha.

[0119] [000189] In a more preferred embodiment, the antibody or antigen binding domain thereof that recognizes and binds to SIRPalpha has one or more of the following properties: 1) has the ability to substantially or completely block the interaction between SIRP-alpha and CD47; 2) has the ability to substantially or completely block SHP-1 transduction mediated by the interaction between SIRP-alpha and CD47; 3) has minimal intrinsic activity to induce phagocytosis of monocytes or macrophages; and 4) has the ability to bind to an epitope outside the IgV domain of SIRPalpha.

[0120] [000190] The multispecific molecules provided herein further comprise a target antigen binding domain. Thus, the term "target antigen binding domain" as used herein encompasses any binding domain for a target antigen. The term "target antigen" refers to any cell surface marker that can distinguish cells expressing the target antigen from other cells, including, but not limited to, tumor antigens, or antigens presented on infected cells.

[0121] [000191] In certain embodiments, the multispecific molecules presented herein selectively induce effector function of immune effector cells (co-expressing SIRP-alpha and activating receptors) in the presence of a target antigen.

[0122] [000192] The multispecific molecules presented herein have the ability to bind and activate immune effector cells to mount a selective response against target cells over non-target cells.

[0123] [000193] In certain embodiments, the target cell expresses the target antigen. In certain embodiments, the target cell co-expresses the target antigen and CD47. [000194] In certain embodiments, the multispecific molecules presented herein induce only marginal effector function of immune effector cells in the absence of target antigen. As used herein, the term "marginal" refers to a level of effector function comparable to that induced by an isotype control when referring to effector function. In certain embodiments, the effector function induced by the multispecific molecules presented herein in the absence of target antigen is 10%, 20%, 30%, 40%, 50% or less of the effector function induced in the presence of target antigen.

[0124] [000195] The multispecific molecules or engagers of the present disclosure also include additional structures to aid in the modular and simultaneous engagement of the engager with multiple targets, such as linking elements, such as linkers, cognate peptides, etc., or chemical bonds to link and separate two or more binding domains to provide spatial proximity and flexibility.

[0125] [000196] Incorporation of the additional structures described above into the higher order multispecific macrophage engagers presented herein aids in engager generation, folding, stability, function, and tissue availability.

[0126] [000197] i.SIRPα binding domain [000198] The SIRPα binding domain of the multispecific molecules presented herein has the ability to substantially block the interaction between SIRP-alpha and CD47.

[0127] [000199] "Substantially blocks the interaction" between two interacting molecules means that the antibody has the ability to inhibit binding between the two interacting molecules by at least 50%, or has the ability to inhibit at least 40% of signaling induced by the interaction of the two molecules. Signaling induced by the interaction between SIRP-alpha and CD47 can be characterized by the introduction of SHP1 to the intracellular portion of SIRP-alpha (e.g., the C-terminal tail).

[0128] [000200] In certain embodiments, the SIRPα binding domain of the multispecific molecules provided herein has the ability to completely block the interaction between SIRP-alpha and CD47. The phrase "completely blocks," when referring to two interacting molecules, means at least 80% inhibition of binding between the two interacting molecules, or at least 50% inhibition of signal transduction induced by the interaction of the two molecules.

[0129] [000201] In certain embodiments, the SIRPα binding domain of the multispecific molecules presented herein can completely block the "don't eat me" signal delivered by CD47 binding to SIRPα (which inhibits phagocytosis), either by completely blocking the SIRPα-CD47 interaction or by completely blocking SIRPα-CD47 interaction-mediated downstream signaling (e.g., SHP-1 transduction) regardless of whether it blocks or does not block the effect on the SIRPα-CD47 interaction. SIRP-alpha antibodies that have the ability to completely block the interaction between SIRP-alpha and CD47 are also referred to herein as full blockers.

[0130] [000202] Blocking of the binding interaction between SIRP-alpha and CD47 can be determined by any suitable assay, such as competitive ELISA or competitive FACS assay. Briefly, in the case of competitive ELISA, the soluble extracellular domain (ECD) of SIRP-alpha can be immobilized on a substrate, and a test article SIRP-alpha antibody can be tested at different concentrations for its ability to block a concentration of soluble ECD of CD47 from binding to the immobilized ECD of SIRP-alpha. The binding of the ECD of CD47 to the immobilized ECD of SIRP-alpha can be determined in the absence and presence of the test article SIRP-alpha binding domain, respectively. The binding reduction of CD47 and SIPR-alpha in the presence of the test article SIRP-alpha binding domain can be determined, and thus the percentage of blocking can be determined.

[0131] [000203] In certain embodiments, certain anti-SIRPα binding domains provided herein have a maximum blocking percentage (%) of greater than 90% as measured by competitive ELISA assay. The assay conditions may be similar to those provided in Example 12 of the present disclosure (the concentration of soluble ECD of CD47 is 25 nM and the concentration of soluble ECD of SIRPα is 20 nM). Exemplary full blockers as provided herein are anti-SIRPα binding domains derived from C15, C25, C42, C59, C73, and their humanized antibodies.

[0132] [000204] The term "maximum blocking percentage" is used interchangeably with "highest blocking percentage" and refers to a plateau in the percentage blocking of the interaction between two proteins (e.g., SIRP-alpha and CD47) at increasing concentrations in the presence of a blocker (e.g., a SIRPα binding domain). Generally, the percentage of blocking may increase with increasing concentrations of the SIRP-alpha binding domain, however, a plateau is reached where no further blocking is achievable despite further increases in the concentration of the SIRP-alpha binding domain. The maximum blocking percentage may vary for different assays, such as competitive ELISA assays and competitive FACS assays.

[0133] [000205] In certain embodiments, certain anti-SIRPα binding domains provided herein have a maximum blocking percentage (%) of 10% or less of the maximum blocking percentage (%) as measured by competitive ELISA assay. The assay conditions may be similar to those provided in Example 12 of the present disclosure (the concentration of soluble ECD of CD47 is 25 nM and the concentration of soluble ECD of SIRPα is 20 nM). An exemplary non-blocker as provided herein is C50 and its humanized antibody. Such SIRP-alpha antibodies are also referred to herein as non-blockers.

[0134] [000206] In certain embodiments, certain anti-SIRPα antibodies provided herein have a maximum blocking percentage (%) of 50% or less as measured by competitive ELISA assay. The assay conditions are similar to those provided in Example 12 of the present disclosure. Such SIRP-alpha antibodies are also referred to herein as partial blockers. An exemplary partial blocker as provided herein is C35 and its humanized antibody.

[0135] [000207] In certain embodiments, the SIRPα binding domain of the multispecific molecules provided herein has minimal intrinsic activity of inducing immune effector cell effector function on target cells. In certain embodiments, the target cells are tumor cells, or cancerous cells, infected cells, or specific disease cell types that need to be eliminated by SIRP-alpha mediated effector function, such as phagocytosis. As used herein, the term "intrinsic activity" refers to the ability of the SIRPα binding domain, in the absence of the target antigen binding domain, to induce immune effector cell phagocytosis on target cells that co-express a specific target antigen and CD47. In certain embodiments, the SIRPα binding domain induces 20% or less, 15% or less, or 10% or less phagocytosis on target cells. As used herein, the term "marginal," when referring to the intrinsic activity of inducing phagocytosis, refers to the level of phagocytosis induced by the intrinsic activity of the SIRP-alpha binding domain (comparable to the level induced by an isotype control). In certain embodiments, the phagocytosis induced by the SIRP-alpha binding domain alone, or a molecule containing the SIRP-alpha binding domain alone, is less than 10%, 20%, 30%, 40%, 50% or less of the phagocytosis induced by the engagers provided herein.

[0136] [000208] In certain embodiments, the SIRPα binding domain of the multispecific molecules presented herein comprises an antibody domain or an antibody mimetic domain. [000209] As used herein, the term "antibody domain" refers to an antigen-binding domain derived from an antibody and includes at least one antibody fragment (e.g., CDR and / or variable region sequences, etc.). Antibody domains include, for example, monoclonal antibodies, antibody fragments or domains, fusion proteins including antibody fragments or domains, polypeptide complexes including antibody fragments or domains, etc. In certain embodiments, an antibody domain includes a Fab, a VHH, a single chain Fv (scFv), a diabody, a Fab', a F(ab')2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a F(ab)2, a scFv dimer (bivalent diabody), a camelized single domain antibody, a nanobody, a tetrabody, a domain antibody, or a bivalent domain antibody.

[0137] [000210] As used herein, the term "antibody mimetic domain" refers to an artificial peptide or compound that behaves similarly to an antibody (e.g., can specifically bind to an antigen like an antibody), but is not produced by the immune system and is not structurally related to an antibody. The term "antibody mimetic domain" can also refer to unrelated protein scaffolds containing α-helices, β-sheets, or random coils that can bind to specific targets and can be designed to incorporate novel binding sites through protein engineering strategies.

[0138] [000211] In certain embodiments, the SIRPα binding domain is an intrabody (e.g., a fibronectin domain), a monobody, a linear peptide, the Z domain of protein A (affibody), a gamma B crystallographic domain, a ubiquitin domain, a cystatin domain, a Sac7d domain, a triple helix coiled-coil domain, a lipocalin domain, the A domain of a membrane receptor, an ankyrin repeat motif, the SH3 domain of Fyn, the Kunitz domain of a protease inhibitor, a fibronectin type III domain (minibody), a DARPin domain, or an antibody mimetic domain including carbohydrate-binding module 32-2.

[0139] [000212] In certain embodiments, the SIRPα binding domain comprises one or more (e.g., one, two, three, four, five, or six) CDR sequences of an anti-SIRPα antibody selected from the group consisting of C25, hu025.021, hu025.033, hu025.023, hu025.059, hu025.060, C15, C42, C59, and C73.

[0140] [000213] "C25" or "025c" as used herein refers to a murine antibody having a heavy chain variable region of SEQ ID NO:1 and a light chain variable region of SEQ ID NO:2. [000214] "hu025.021," as used herein, refers to a humanized antibody based on C25, comprising a heavy chain variable region of SEQ ID NO:3 and a light chain variable region of SEQ ID NO:4.

[0141] [000215] "hu025.023," as used herein, refers to a humanized antibody based on C25, comprising a heavy chain variable region of SEQ ID NO:5 and a light chain variable region of SEQ ID NO:6. [000216] "hu025.033," as used herein, refers to a humanized antibody based on C25, comprising a heavy chain variable region of SEQ ID NO:159 and a light chain variable region of SEQ ID NO:160.

[0142] [000217] "hu025.059" as used herein refers to a humanized antibody based on C25, comprising a heavy chain variable region of SEQ ID NO:7 and a light chain variable region of SEQ ID NO:8. [000218] "hu025.060" as used herein refers to a humanized antibody based on C25, comprising a heavy chain variable region of SEQ ID NO:9 and a light chain variable region of SEQ ID NO:10.

[0143] [000219] "C15" or "015c" as used herein refers to a murine antibody having a heavy chain variable region of SEQ ID NO:11 and a light chain variable region of SEQ ID NO:12. [000220] "C42" or "042c" as used herein refers to a murine antibody having a heavy chain variable region of SEQ ID NO:13 and a light chain variable region of SEQ ID NO:14.

[0144] [000221] "C59" or "059c" as used herein refers to a murine antibody having a heavy chain variable region of SEQ ID NO:15 and a light chain variable region of SEQ ID NO:16. [000222] "C73" or "073c" as used herein refers to a murine antibody having a heavy chain variable region of SEQ ID NO:17 and a light chain variable region of SEQ ID NO:18.

[0145] [000223] The SIRP alpha binding domain of the multispecific molecules presented herein can be obtained from anti-SIRP alpha antibodies, the amino acid sequences of the variable regions and CDRs of which are shown in Tables 1-2 below.

[0146] [000224]

[0147] [Table 2-1]

[0148] [Table 2-2]

[0149] [Table 2-3]

[0150] [000225]

[0151] [Table 3-1]

[0152] [Table 3-2]

[0153] [000226] X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, X6 is Y or H, X7 is S or P, X8 is Y or C, X9 is Y or S, and X 10 is N or S, and X 11 is P or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is S or absent, and X 16 is S or A, and X 17 is F or L.

[0154] [000227] Although the CDRs are known to be involved in antigen binding, it has been found that not all six CDRs are essential or unalterable. In other words, it is possible to replace, alter or modify one or more of the CDRs presented herein for the SIRPα binding domain while still substantially retaining the specific binding affinity for SIRPα.

[0155] [000228] The heavy chain CDR3 region is located in the center of the antigen binding site, and therefore is believed to make the strongest contact with the antigen and provide the highest free energy for the affinity of the antibody to the antigen.The heavy chain CDR3 is also believed to be the most highly diverse CDR of the antigen binding site in terms of length, amino acid composition, and conformation due to multiple diversification mechanisms (Tonegawa S.Nature.302:575-81).The diversity in the heavy chain CDR3 is sufficient to generate most of the antibody specificity (Xu JL, Davis MM.Immunity.13:37-45) and the desired antigen binding affinity (Schier R, etc.J Mol Biol.263:551-67).

[0156] [000229] In certain embodiments, the SIRP-alpha binding domain comprises: a) HCDR1 comprising the sequence X1YYMH (SEQ ID NO: 161), HCDR2 comprising the sequence RIDPEDX2EX3KYAPKFQG (SEQ ID NO: 162), and 15 an HCDR3 comprising the sequence X4X5Y (SEQ ID NO: 163), and / or an LCDR1 comprising the sequence SASSSVSSSYLY (SEQ ID NO: 26), an LCDR2 comprising the sequence STSNLAS (SEQ ID NO: 27), and an LCDR3 comprising the sequence X6QWSSYPYT (SEQ ID NO: 164); or b) HCDR1 comprising the sequence TYGMS (SEQ ID NO: 35), HCDR2 comprising the sequence WINTYSGVX7TX8ADDFKG (SEQ ID NO: 165), and DPHX9YGX 10 SPAWFX 11 HCDR3 comprising the sequence of Y (SEQ ID NO: 166), and / or X 12 ASQX 13 VGIX 14 LCDR1 comprising the sequence of VA (SEQ ID NO: 188), LCDR2 comprising the sequence of SASNRYT (SEQ ID NO: 39), and QQYSX 16 YPX 17 an LCDR3 comprising the sequence of T (SEQ ID NO: 189); or c) HCDR1 comprising the sequence EYVLS (SEQ ID NO: 41), HCDR2 comprising the sequence EIYPGTITTYYNEKFKG (SEQ ID NO: 42), and HCDR3 comprising the sequence FYDYDGGWFAY (SEQ ID NO: 43), and / or LCDR1 comprising the sequence SASSSVSSSDLH (SEQ ID NO: 44), LCDR2 comprising the sequence GTSNLAS (SEQ ID NO: 45), and LCDR3 comprising the sequence QQWSGYPWT (SEQ ID NO: 46), wherein X1 is A or D, X2 is G or A, X3 is T or S, X4 is L or Y, X5 is E or A, X6 is Y or H, X7 is S or P, X8 is Y or C, X9 is Y or S, and X 10 is N or S, and X 11 is P or V, and X 12 is E or K, and X 13 is N or I, and X 14 is S or A, and X 15 is S or absent, and X 16 is S or A, and X 17 is F or L.

[0157] [000230] In certain embodiments, the SIRP-alpha binding domain comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 23, an HCDR2 comprising the sequence of SEQ ID NO: 24 or SEQ ID NO: 198, and an HCDR3 comprising the sequence of SEQ ID NO: 25, and / or an LCDR1 comprising the sequence of SEQ ID NO: 26, an LCDR2 comprising the sequence of SEQ ID NO: 27, and an LCDR3 comprising the sequence of SEQ ID NO: 28, or b) an HCDR1 comprising the sequence of SEQ ID NO: 29, an HCDR2 comprising the sequence of SEQ ID NO: 30, and an HCDR3 comprising the sequence of SEQ ID NO: 31, and / or an LCDR1 comprising the sequence of SEQ ID NO: 32, an LCDR2 comprising the sequence of SEQ ID NO: 33, and an LCDR3 comprising the sequence of SEQ ID NO: 34, or c) an HCDR1 comprising the sequence of SEQ ID NO: 35, an HCDR2 comprising the sequence of SEQ ID NO: 36, and an HCDR3 comprising the sequence of SEQ ID NO: 37, and / or an LCDR1 comprising the sequence of SEQ ID NO: 38, an LCDR2 comprising the sequence of SEQ ID NO: 39, and an LCDR3 comprising the sequence of SEQ ID NO: 40, or d) HCDR1 comprising the sequence of SEQ ID NO: 47, HCDR2 comprising the sequence of SEQ ID NO: 48, and HCDR3 comprising the sequence of SEQ ID NO: 49, and / or LCDR1 comprising the sequence of SEQ ID NO: 50, LCDR2 comprising the sequence of SEQ ID NO: 51, and LCDR3 comprising the sequence of SEQ ID NO: 52. Includes.

[0158] [000231] In certain embodiments, the SIRP-alpha binding domain comprises HCDRs and LCDRs identical to an anti-SIRP-alpha antibody selected from the group consisting of C25, C15, C42, C59, and C73; a) C25 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:1 and / or a light chain variable region comprising the sequence of SEQ ID NO:2; b) C15 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 11 and / or a light chain variable region comprising the sequence of SEQ ID NO: 12; c) C42 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the sequence of SEQ ID NO: 14; d) C59 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 15 and / or a light chain variable region comprising the sequence of SEQ ID NO: 16; and e) C73 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:17 and / or a light chain variable region comprising the sequence of SEQ ID NO:18.

[0159] [000232] In certain embodiments, the SIRPα binding domains presented herein comprise any suitable framework region (FR) sequence, so long as the antigen binding domain can specifically bind to SIRPα. In certain embodiments, the CDR sequences of hu025.021, hu025.033, hu025.023, hu025.059, and hu025.060 are obtained from mouse antibody C25, but they can be grafted to any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, etc., among others, using suitable methods known in the art, such as recombinant techniques.

[0160] [000233] In certain embodiments, the SIRPα binding domains presented herein are humanized. Humanized antigen binding domains are desirable due to their reduced immunogenicity in humans. Humanized antigen binding domains are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antigen binding domains can be substantially performed by substituting non-human (e.g., mouse, etc.) CDR genes with the corresponding human CDR genes in human immunoglobulin genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0161] [000234] Suitable human heavy and light chain variable domains can be selected to achieve this goal using methods known in the art. In an illustrative example, a "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 a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al, (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, a framework derived from the consensus sequence of all human antibodies can be used to graft non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0162] [000235] In certain embodiments, the humanized antigen-binding domains presented herein are composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable regions FR, and the constant regions, if present, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be derived from different human immunoglobulin genes, e.g., the FR sequences can be derived from one human antibody and the constant regions can be derived from another human antibody. In some embodiments, the humanized antigen-binding domain comprises human FR1-4.

[0163] [000236] In some embodiments, the FR region derived from a human may contain 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 replaced with the corresponding residues from the non-human parent antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more closely resemble the non-human parent antibody structure. In certain embodiments, the humanized SIRPα binding domains presented herein contain 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 FRs of the heavy or light chain variable domain. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. Table 2.1 shows the FR sequences of humanized antibodies hu025.021, hu025.023, hu025.033, hu025.059, and hu025.060.

[0164] [000237]

[0165] [Table 4-1]

[0166] [Table 4-2]

[0167] In the formula, X 20 is A or V, and X 21 is N or D, and X 22 is Y or F. [000238] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 is EVQLVQSGAEVKKPGATVKISCKX 20 SGFNIK (SEQ ID NO: 190) or a homologous sequence having at least 80% sequence identity thereto; and / or b) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191), or a homologous sequence having at least 80% sequence identity thereto; and / or c) The HFR3 sequence is RVTITADTSTX 21 TAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 192), or a homologous sequence having at least 80% sequence identity thereto; and / or d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193), or a homologous sequence having at least 80% sequence identity thereto; and / or e) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194), or a homologous sequence having at least 80% sequence identity thereto; and / or f) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto; and / or g) LFR3 is GIPARFSGSGSGTDX 22 TLTISSLEPEDFAVYYC (SEQ ID NO: 196), or a homologous sequence having at least 80% sequence identity thereto; and / or h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197) or a homologous sequence having at least 80% sequence identity thereto; In the formula, X 20 is A or V, and X 21 is N or D, and X 22 is Y or F.

[0168] [000239] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; i) HFR1 comprises EVQLVQSGAEVKKPGATVKISCKVSGFNIK (SEQ ID NO: 207), or a homologous sequence having at least 80% sequence identity thereto; and / or j) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191), or a homologous sequence having at least 80% sequence identity thereto; and / or k) the HFR3 sequence comprises RVTITADTSTNTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 208), or a homologous sequence having at least 80% sequence identity thereto; and / or l) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193), or a homologous sequence having at least 80% sequence identity thereto; and / or m) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194) or a homologous sequence having at least 80% sequence identity thereto; and / or n) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto; and / or o) LFR3 comprises GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO: 209), or a homologous sequence having at least 80% sequence identity thereto; and / or p) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197) or a homologous sequence having at least 80% sequence identity thereto.

[0169] [000240] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; q) HFR1 comprises EVQLVQSGAEVKKPGATVKISCKVSGFNIK (SEQ ID NO: 207), or a homologous sequence having at least 80% sequence identity thereto; and / or r) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191), or a homologous sequence having at least 80% sequence identity thereto; and / or s) the HFR3 sequence comprises RVTITADTSTNTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 208), or a homologous sequence having at least 80% sequence identity thereto; and / or t) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193) or a homologous sequence having at least 80% sequence identity thereto; and / or u) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194) or a homologous sequence having at least 80% sequence identity thereto; and / or v) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195), or a homologous sequence having at least 80% sequence identity thereto; and / or w) LFR3 comprises GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 212), or a homologous sequence having at least 80% sequence identity thereto; and / or x) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197), or a homologous sequence having at least 80% sequence identity thereto.

[0170] [000241] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; y) HFR1 comprises EVQLVQSGAEVKKPGATVKISCKVSGFNIK (SEQ ID NO: 207), or a homologous sequence having at least 80% sequence identity thereto; and / or z) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191), or a homologous sequence having at least 80% sequence identity thereto; and / or aa) the HFR3 sequence comprises RVTITADTSTDTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 210), or a homologous sequence having at least 80% sequence identity thereto; and / or bb) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193) or a homologous sequence having at least 80% sequence identity thereto; and / or cc) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194) or a homologous sequence having at least 80% sequence identity thereto; and / or dd) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto; and / or ee) LFR3 comprises GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO: 209), or a homologous sequence having at least 80% sequence identity thereto; and / or ff) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197) or a homologous sequence having at least 80% sequence identity thereto.

[0171] [000242] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; gg) HFR1 comprises EVQLVQSGAEVKKPGATVKISCKASGFNIK (SEQ ID NO: 211), or a homologous sequence having at least 80% sequence identity thereto; and / or hh) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191), or a homologous sequence having at least 80% sequence identity thereto; and / or ii) the HFR3 sequence comprises RVTITADTSTNTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 208), or a homologous sequence having at least 80% sequence identity thereto; and / or jj) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193) or a homologous sequence having at least 80% sequence identity thereto; and / or kk) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194), or a homologous sequence having at least 80% sequence identity thereto; and / or ll) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto; and / or mm) LFR3 comprises GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC (SEQ ID NO: 209), or a homologous sequence having at least 80% sequence identity thereto; and / or nn) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197), or a homologous sequence having at least 80% sequence identity thereto.

[0172] [000243] In certain embodiments, the SIRP-alpha binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; oo) HFR1 comprises EVQLVQSGAEVKKPGATVKISCKASGFNIK (SEQ ID NO: 211), or a homologous sequence having at least 80% sequence identity thereto; and / or pp) HFR2 comprises WVQQAPGKGLEWIG (SEQ ID NO: 191) or a homologous sequence having at least 80% sequence identity thereto; and / or qq) the HFR3 sequence comprises RVTITADTSTNTAYMELSSLRSEDTAVYYCDR (SEQ ID NO: 208), or a homologous sequence having at least 80% sequence identity thereto; and / or rr) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 193) or a homologous sequence having at least 80% sequence identity thereto; and / or ss) LFR1 comprises EIVLTQSPATLSLSPGERATLSC (SEQ ID NO: 194), or a homologous sequence having at least 80% sequence identity thereto; and / or tt) LFR2 comprises WYQQKPGQAPKLWIY (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto; and / or uu) LFR3 comprises GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC (SEQ ID NO: 212), or a homologous sequence having at least 80% sequence identity thereto; and / or vv) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 197), or a homologous sequence having at least 80% sequence identity thereto.

[0173] [000244] In certain embodiments, a SIRPα binding domain presented herein comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 159. In certain embodiments, a SIRPα binding domain presented herein comprises a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, and 160.

[0174] [000245] In certain embodiments, the SIRP-alpha binding domain comprises: f) a heavy chain variable region comprising the sequence of SEQ ID NO: 1 and / or a light chain variable region comprising the sequence of SEQ ID NO: 2, or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 3 and / or a light chain variable region comprising the sequence of SEQ ID NO: 4, or h) a heavy chain variable region comprising the sequence of SEQ ID NO:5 and / or a light chain variable region comprising the sequence of SEQ ID NO:6, or i) a heavy chain variable region comprising the sequence of SEQ ID NO: 7, and / or a light chain variable region comprising the sequence of SEQ ID NO: 8, or j) a heavy chain variable region comprising the sequence of SEQ ID NO: 9 and / or a light chain variable region comprising the sequence of SEQ ID NO: 10, or k) a heavy chain variable region comprising the sequence of SEQ ID NO: 11, and / or a light chain variable region comprising the sequence of SEQ ID NO: 12, or l) a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the sequence of SEQ ID NO: 14, or m) a heavy chain variable region comprising the sequence of SEQ ID NO: 15, and / or a light chain variable region comprising the sequence of SEQ ID NO: 16, or n) a heavy chain variable region comprising the sequence of SEQ ID NO: 17, and / or a light chain variable region comprising the sequence of SEQ ID NO: 18, and / or o) a heavy chain variable region comprising the sequence of SEQ ID NO: 159, and / or a light chain variable region comprising the sequence of SEQ ID NO: 160 Includes.

[0175] [000246] In some embodiments, the SIRPα binding domain provided herein comprises all or a portion of a heavy chain variable domain and / or all or a portion of a light chain variable domain. In one embodiment, the SIRPα binding domain provided herein is a single domain antibody composed of all or a portion of a heavy chain variable domain provided herein. More information on such single domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0176] [000247] In certain embodiments, the SIRPα binding domain further comprises one or more amino acid residue substitutions or modifications while still retaining specific binding to SIRPα. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or in one or more of the non-CDR sequences of the heavy chain variable region or the light chain variable region.

[0177] [000248] ii. Target Antigen Binding Domain [000249] As used herein, the term "target antigen binding domain" refers to an antigen binding domain that targets cells that co-express an antigen and CD47. The target antigen binding domain of the multispecific molecules presented herein can be a tumor antigen binding domain. In certain embodiments, the target antigen comprises a tumor surface antigen. As used herein, the term "tumor surface antigen" refers to an antigen that is primarily presented by tumor cells to distinguish them from non-malignant tissues, and is preferably located on the cell membrane of tumor cells. Tumor surface antigens can take various forms, such as polypeptides (especially glycosylated proteins), or the glycosylation pattern of polypeptides, glycolipids (e.g., gangliosides, such as GM2), or even altered composition of lipids of the cell membrane that can be characteristic of cancer cells. Tumor surface antigens can be antigens that are specifically expressed on cancer cells (eliciting an immune response and / or binding to T cell receptors (e.g., when presented by MHC molecules) or antibodies). In some embodiments, tumor surface antigens induce a humoral response (e.g., involving the production of antigen-specific antibodies). In some embodiments, tumor surface antigens induce a cellular response (e.g., involving T cells, whose receptors specifically interact with the tumor surface antigen). In some embodiments, tumor surface antigens bind to antibodies and may or may not induce a specific physiological response in the organism.

[0178] [000250] In certain embodiments, the tumor surface antigen is, for example, PD-L1, claudin 18.2, BCMA, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, EGFR, HER2, HER3, CD117, C-Met, EGFR, EGFR vIII, ERBB3, ERBB4, VEGFR1, VEGFR2, PTHR2, B7-H1(PD-L1), B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, Trop-2, GPC-3, EPCAM, DLL-3, nectin-4, claudin 6, claudin 18.2, Muc-1, PSMA, GD3, FAP, CEA, or EphA2.

[0179] [000251] Claudin 18.2 binding domain [000252] In certain embodiments, the tumor surface antigen is claudin 18.2. In certain embodiments, the claudin 18.2 binding domain has the ability to specifically bind to claudin 18.2 (e.g., human claudin 18.2, etc.).

[0180] [000253] In certain embodiments, the claudin 18.2 binding domain comprises one or more (e.g., one, two, three, four, five, or six) CDR sequences of an anti-claudin 18.2 antibody selected from the group consisting of hu26.H1L1, hu26.H1L2, hu26.H1L2(S92A), hu26.H3L1, hu26.H3L2, hu28.H1L2, C10, C29, and C30.

[0181] [000254] "hu26.H1L1," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:65 and a light chain variable region of SEQ ID NO:66.

[0182] [000255] "hu26.H1L2," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:65 and a light chain variable region of SEQ ID NO:67.

[0183] [000256] "hu26.H1L2(S92A)" as used herein refers to a humanized monoclonal antibody having a heavy chain variable region of antibody SEQ ID NO:65 and a light chain variable region of SEQ ID NO:224.

[0184] [000257] "hu26.H3L1," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:68 and a light chain variable region of SEQ ID NO:66.

[0185] [000258] "hu26.H3L2," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:68 and a light chain variable region of SEQ ID NO:67.

[0186] [000259] "hu28.H1L2," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:69 and a light chain variable region of SEQ ID NO:70.

[0187] [000260] "C10," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:71 and a light chain variable region of SEQ ID NO:72. [000261] "C29," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:73 and a light chain variable region of SEQ ID NO:74.

[0188] [000262] "C30," as used herein, refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO:75 and a light chain variable region of SEQ ID NO:76. [000263] Table 3 shows the CDR sequences of anti-claudin 18.2 antibodies. The heavy and light chain variable region sequences are also provided in Tables 4 and 5 below.

[0189] [000264]

[0190] [Table 5-1]

[0191] [Table 5-2]

[0192] [000265]

[0193] [Table 6-1]

[0194] [Table 6-2]

[0195] [000266] Although CDRs are known to be involved in antigen binding, it has been found that not all six CDRs are essential or unalterable. In other words, for the claudin 18.2 binding domain, one or more of the CDRs presented herein can be replaced, altered, or modified while still substantially retaining the specific binding affinity to PD-1 (e.g., human claudin 18.2).

[0196] [000267] In certain embodiments, the claudin 18.2 binding domain comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 77, an HCDR2 comprising the sequence of SEQ ID NO: 78, and an HCDR3 comprising the sequence of SEQ ID NO: 79, and / or an LCDR1 comprising the sequence of SEQ ID NO: 80, an LCDR2 comprising the sequence of SEQ ID NO: 81, and an LCDR3 comprising the sequence of SEQ ID NO: 82 or SEQ ID NO: 225, or b) an HCDR1 comprising the sequence of SEQ ID NO: 83, an HCDR2 comprising the sequence of SEQ ID NO: 84, and an HCDR3 comprising the sequence of SEQ ID NO: 85, and / or an LCDR1 comprising the sequence of SEQ ID NO: 86, an LCDR2 comprising the sequence of SEQ ID NO: 87, and an LCDR3 comprising the sequence of SEQ ID NO: 88, or c) an HCDR1 comprising the sequence of SEQ ID NO: 89, an HCDR2 comprising the sequence of SEQ ID NO: 90, and an HCDR3 comprising the sequence of SEQ ID NO: 91, and / or an LCDR1 comprising the sequence of SEQ ID NO: 92, an LCDR2 comprising the sequence of SEQ ID NO: 93, and an LCDR3 comprising the sequence of SEQ ID NO: 94, or d) an HCDR1 comprising the sequence of SEQ ID NO: 95, an HCDR2 comprising the sequence of SEQ ID NO: 96, and an HCDR3 comprising the sequence of SEQ ID NO: 97, and / or an LCDR1 comprising the sequence of SEQ ID NO: 98, an LCDR2 comprising the sequence of SEQ ID NO: 99, and an LCDR3 comprising the sequence of SEQ ID NO: 100, or e) HCDR1 comprising the sequence of SEQ ID NO: 101, HCDR2 comprising the sequence of SEQ ID NO: 102, and HCDR3 comprising the sequence of SEQ ID NO: 103, and / or LCDR1 comprising the sequence of SEQ ID NO: 104, LCDR2 comprising the sequence of SEQ ID NO: 105, and LCDR3 comprising the sequence of SEQ ID NO: 106. Includes.

[0197] [000268] In certain embodiments, the claudin 18.2 binding domain comprises HCDRs and LCDRs identical to an anti-claudin 18.2 antibody selected from the group consisting of hu26.H1L1, hu26.H1L2(S92A), hu28.H1L2, C10, C29, and C30; a) hu26.H1L1 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:65, and / or a light chain variable region comprising the sequence of SEQ ID NO:66; b) hu26.H1L2(S92A) comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 65 and / or a light chain variable region comprising the sequence of SEQ ID NO: 224; c) hu28.H1L2 comprises a heavy chain variable region comprising the sequence of SEQ ID NO:69 and / or a light chain variable region comprising the sequence of SEQ ID NO:70; d) C10 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 71 and / or a light chain variable region comprising the sequence of SEQ ID NO: 72; e) C29 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 73 and / or a light chain variable region comprising the sequence of SEQ ID NO: 74; and f) C30 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the sequence of SEQ ID NO: 76.

[0198] [000269] In certain embodiments, the claudin 18.2 binding domain of the multispecific molecules presented herein comprises any suitable framework region (FR) sequence, so long as the antigen binding domain is capable of specifically binding to claudin 18.2.

[0199] [000270] In certain embodiments, the claudin 18.2 binding domain of the multispecific molecules presented herein is humanized. A humanized antigen binding domain is desirable due to its reduced immunogenicity in humans. A humanized antigen binding domain is chimeric in its variable region since non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of an antigen binding domain can be substantially performed by substituting the corresponding human CDR genes for non-human (e.g., mouse, etc.) CDR genes in a human immunoglobulin gene (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536). As noted above, suitable human heavy and light chain variable domains may be selected to achieve this end using methods known in the art.

[0200] [000271] In certain embodiments, the humanized antigen-binding domains presented herein are composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable regions FR and the constant regions (if present) are derived completely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be derived from different human immunoglobulin genes, e.g., the FR sequences can be derived from one human antibody and the constant regions can be derived from another human antibody. In some embodiments, the humanized antigen-binding domain comprises human FR1-4.

[0201] [000272] In some embodiments, the FR region derived from a human may contain 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 replaced with the corresponding residues from the non-human parent antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more closely resemble the non-human parent antibody structure. In certain embodiments, the humanized claudin-18.2 binding domains presented herein contain 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 FRs of the heavy or light chain variable domain. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. Table 4.1 shows the FR sequences for humanized antibodies hu26.H1L1, hu26.H1L2, hu26.H3L1, hu26.H3L2, and hu28.H1L2.

[0202] [000273]

[0203] [Table 7-1]

[0204] [Table 7-2]

[0205] In the formula, X 18 is S or A, and X 19 is L or A, and X 23 is T or K, and X 24 is Y or F, and X 25 is Q or G, and X 26 is Q or K, and X 27 is P or A.

[0206] [000274] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises an amino acid sequence selected from the group consisting of EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167) and QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 168), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 is WVRQAPGKGLEWVX 18 (SEQ ID NO: 169) and WVRQAPGQGLEWMG (SEQ ID NO: 170), or a homologous sequence having at least 80% sequence identity thereto; c) The HFR3 sequence is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAX 23 (SEQ ID NO: 171) and RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 172), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises an amino acid sequence selected from the group consisting of DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174) and DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 175), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 is WYQQKPGX 26 X 27 PKX 19 LIY (SEQ ID NO: 176), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 is GVPSRFSGSGSGTEX 24 TLTISSLQPEDFATYYC (SEQ ID NO: 178) and GVPDRFSGSGSGTDFTLTISSLQAEDVAVYHC (SEQ ID NO: 179), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 is FGX 25 GTKLEIK (SEQ ID NO: 180) or a homologous sequence having at least 80% sequence identity thereto; In the formula, X 18 is S or A, and X 19 is L or A, and X 23 is T or K, and X 24 is Y or F, and X 25 is Q or G, and X 26 is Q or K, and X 27 is P or A.

[0207] [000275] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 is WVRQAPGKGLEWVX 18(SEQ ID NO: 169), or a homologous sequence having at least 80% sequence identity thereto; c) The HFR3 sequence is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAX 23 (SEQ ID NO: 171), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 is WYQQKPGKAPKX 19 LIY (SEQ ID NO: 206), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 is GVPSRFSGSGSGTEX 24 TLTISSLQPEDFATYYC (SEQ ID NO: 178), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 181), or a homologous sequence having at least 80% sequence identity thereto; In the formula, X 18 is S or A, and X 19 is L or A, and X 23 is T or K, and X 24 is Y or F.

[0208] [000276] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 comprises WVRQAPGKGLEWVS (SEQ ID NO: 199), or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 sequence comprises RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 200), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 comprises WYQQKPGKAPKLLIY (SEQ ID NO: 201), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 comprises GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 202), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 181), or a homologous sequence having at least 80% sequence identity thereto.

[0209] [000277] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 comprises WVRQAPGKGLEWVS (SEQ ID NO: 199), or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 sequence comprises RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 200), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 comprises WYQQKPGKAPKALIY (SEQ ID NO: 203), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 comprises GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC (SEQ ID NO: 204), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 181), or a homologous sequence having at least 80% sequence identity thereto.

[0210] [000278] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 comprises WVRQAPGKGLEWVA (SEQ ID NO: 213), or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 sequence comprises RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT (SEQ ID NO: 205), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 comprises WYQQKPGKAPKLLIY (SEQ ID NO: 201), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 comprises GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 202), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 181), or a homologous sequence having at least 80% sequence identity thereto.

[0211] [000279] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises EVQLLESGGGLVQPGGSLRLSCAASGFTLS (SEQ ID NO: 167), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 comprises WVRQAPGKGLEWVA (SEQ ID NO: 213), or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 sequence comprises RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT (SEQ ID NO: 205), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 174), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 comprises WYQQKPGKAPKALIY (SEQ ID NO: 203), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 comprises GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC (SEQ ID NO: 204), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 comprises FGQGTKLEIK (SEQ ID NO: 181), or a homologous sequence having at least 80% sequence identity thereto.

[0212] [000280] In some embodiments, the claudin 18.2 binding domain further comprises one or more of the heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of the light chains LFR1, LFR2, LFR3, and LFR4; a) HFR1 comprises QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 168), or a homologous sequence having at least 80% sequence identity thereto; b) HFR2 comprises WVRQAPGQGLEWMG (SEQ ID NO: 170), or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 sequence comprises RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR (SEQ ID NO: 172), or a homologous sequence having at least 80% sequence identity thereto; d) HFR4 comprises WGQGTLVTVSS (SEQ ID NO: 173), or a homologous sequence having at least 80% sequence identity thereto; e) LFR1 comprises DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 175), or a homologous sequence having at least 80% sequence identity thereto; f) LFR2 comprises WYQQKPGQPPKLLIY (SEQ ID NO: 177), or a homologous sequence having at least 80% sequence identity thereto; g) LFR3 comprises GVPDRFSGSGSGTDFTLTISSLQAEDVAVYHC (SEQ ID NO: 179), or a homologous sequence having at least 80% sequence identity thereto; and h) LFR4 comprises FGGGTKLEIK (SEQ ID NO: 182), or a homologous sequence having at least 80% sequence identity thereto.

[0213] [000281] In some embodiments, the claudin 18.2 binding domain comprises: a) a heavy chain variable region comprising the sequence of SEQ ID NO: 65 or 68, and / or a light chain variable region comprising the sequence of SEQ ID NO: 66 or 67 or 224, or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 69 and / or a light chain variable region comprising the sequence of SEQ ID NO: 70, or c) a heavy chain variable region comprising the sequence of SEQ ID NO: 71 and / or a light chain variable region comprising the sequence of SEQ ID NO: 72, or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 73, and / or a light chain variable region comprising the sequence of SEQ ID NO: 74, or e) a heavy chain variable region comprising the sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the sequence of SEQ ID NO: 76 Includes.

[0214] [000282] In some embodiments, the claudin 18.2 binding domain provided herein comprises all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the claudin 18.2 binding domain provided herein is a single domain antibody that is composed of all or part of the heavy chain variable domain provided herein. More information on such single domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0215] [000283] In certain embodiments, the claudin 18.2 binding domain further comprises one or more amino acid residue substitutions or modifications while still retaining specific binding to claudin 18.2. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or one or more of the non-CDR sequences of the heavy chain variable region or the light chain variable region. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of, for example, hu26.H1L2(S92A).

[0216] [000284] PD-L1 binding domain [000285] In certain embodiments, the tumor surface antigen is PD-L1. In certain embodiments, the PD-L1 binding domain has the ability to specifically bind to PD-L1 (such as, for example, human PD-L1).

[0217] [000286] In certain embodiments, the PD-L1 binding domain comprises one or more (e.g. one, two, or three) CDR sequences of an anti-PD-L1 antibody selected from the group consisting of: C71, C71v38, C239, C492, C570, C446, C2811, C1778, C1793, C2855, C2713, and C2719.

[0218] [000287] "C71," as used herein, refers to a humanized heavy chain antibody having a heavy chain variable region of SEQ ID NO:107. [000288] "C71v38," as used herein, refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:108.

[0219] [000289] "C239," as used herein, refers to a humanized heavy chain antibody having a heavy chain variable region of SEQ ID NO:109. [000290] "C492," as used herein, refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:110.

[0220] [000291] "C570," as used herein, refers to a humanized heavy chain antibody having a heavy chain variable region of SEQ ID NO:111. [000292] "570h3" as used herein refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:223.

[0221] [000293] "C446," as used herein, refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:112. [000294] "C2811," as used herein, refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:113.

[0222] [000295] "C1778" as used herein refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:114. [000296] "C1793," as used herein, refers to a humanized heavy chain antibody having a heavy chain variable region of SEQ ID NO:115.

[0223] [000297] "C2855," as used herein, refers to a humanized heavy chain antibody having a heavy chain variable region of SEQ ID NO:116. [000298] "C2713," as used herein, refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:117.

[0224] [000299] "C2719," as used herein, refers to a humanized heavy chain antibody having the heavy chain variable region of SEQ ID NO:118. [000300] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 119, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 120, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 121.

[0225] [000301] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 122, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 123, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 124.

[0226] [000302] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 125, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 126, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 127.

[0227] [000303] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 128, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 129, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 130.

[0228] [000304] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 131, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 132, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 133.

[0229] [000305] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 134, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 135, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 136.

[0230] [000306] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 137, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 138, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 139.

[0231] [000307] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 140, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 141, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 142.

[0232] [000308] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 143, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 144, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 145.

[0233] [000309] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 146, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 147, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 148.

[0234] [000310] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 149, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 150, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 151.

[0235] [000311] In certain embodiments, the PD-L1 binding domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 152, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 153, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 154.

[0236] [000312] In certain embodiments, the PD-L1 binding domain comprises HCDRs identical to an anti-PD-L1 antibody selected from the group consisting of: C71, C71v38, C239, C492, C570, 570h3, C446, C2811, C1778, C1793, C2855, C2713, and C2719.

[0237] [000313] In certain embodiments, the PD-L1 binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 107-118 and 223. [000314] Table 5 shows the heavy chain variable region sequences of anti-PD-L1 antibodies. The CDR sequences are also provided in Table 6 below.

[0238] [000315]

[0239] [Table 8-1]

[0240] [Table 8-2]

[0241] [000316]

[0242] [Table 9-1]

[0243] [Table 9-2]

[0244] [Table 9-3]

[0245] [000317] In certain embodiments, the PD-L1 binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 107-118 and 223. [000318] In certain embodiments, the PD-L1 binding domain further comprises one or more amino acid residue substitutions or modifications while still retaining specific binding to PD-L1. In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or one or more non-CDR sequences of the heavy chain or light chain variable region.

[0246] [000319] iv. Activating Receptor Binding Domain [000320] The multispecific molecule further comprises an activating receptor binding domain. As used herein, the term "activating receptor" refers to a receptor (e.g., FcγR) that is expressed on an immune effector cell (e.g., a phagocytic cell, such as a macrophage) and is involved in at least one effector function of the immune effector cell (e.g., a phagocytic cell, such as a macrophage) or a proinflammatory response, by being activated, e.g., by binding to an Fc domain. In certain embodiments, the immune effector cells provided herein co-express SIRP-alpha and an activating receptor.

[0247] [000321] In certain embodiments, the activating receptor is an FcγR and the activating receptor binding domain is an Fc domain. The Fc domain can activate Fc receptors (FcRs) on macrophages and drive a phosphorylation cascade propagated by the receptor's 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 (Igs). ITAMs can be characterized by a conserved amino acid sequence motif, consisting of paired YXXL / I motifs (where Y, L, and I refer to tyrosine, lysine, and isoleucine, respectively), separated by a defined interval of 6-8 amino acids. Phosphorylation of some residues within the ITAMs introduces several signaling molecules for phagocytosis activation. Thus, an activating receptor can be any receptor expressed on an immune effector cell that binds to and activates, and that can induce phagocytosis via an ITAM-containing intracellular phagocytosis signaling domain.

[0248] [000322] In other embodiments, the activating receptor is a receptor that is involved in different phagocytic signaling or mechanisms, such as Akt-mediated signaling cascades (via CD19, CD28, CSFR, or PDGFR receptors), clustering of receptors on immune effector cells (e.g., macrophages) that enhance phagocytosis (e.g., via integrins or selectins), or antigen-mediated cytotoxicity (via FcDR1 (CD89) receptor or CD206).

[0249] [000323] For example, activating receptors associated with effector functions, such as phagocytosis, include crystallizable gamma receptor fragments (FcγR), TREM2, lectins, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, CD68, CD205, CD206, FcDR1, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CD64, CD32a, CD16a, CD89, CD19, CD28, CSFR, PDGFR, and the like. , MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, Dectin 1, RAGE (SR-E1), LRP1, LRP2, ASGP, SR-PSOX, CXCL16, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor, or complement receptors such as CR1 and CR3. In certain embodiments, the activating receptor is an FcγR.

[0250] [000324] In certain embodiments, activating receptors capable of generating pro-inflammatory signals upon activation include, but are not limited to, PI3K, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, BAH.Tyro3, ​​Ax1, Traf6, Syk, MyD88, Zap70, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.

[0251] [000325] The term "activating receptor binding domain" as used herein refers to a domain (e.g., a portion of an antibody) that has the ability to specifically bind to an activating receptor on an immune effector cell, such binding triggering activation of the receptor and its downstream signaling (e.g., immune cell effector function or proinflammatory response). For example, the activating receptor binding domain comprises an antibody Fc domain or a variant thereof. In certain embodiments, the Fc domain may be derived from IgG1 or IgG4.

[0252] [000326] In certain embodiments, the activating receptor binding domain of the multispecific molecules presented herein is selected from the group consisting of crystallizable gamma receptor fragment (FcγR), TREM2, lectin, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, CD68, CD205, CD206, FcDR1, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CD64, CD32a, CD16a, CD89, CD19, CD28, CSFR, PDGFR, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, Dectin 1, RAGE (SR-E1), LRP1, LRP2, ASGP, SR-PSOX, It binds to and activates an activating receptor selected from the group consisting of 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, FcγR1, FcγR2A, FcγR2B2, FcγR2C, FcγR3A, BAH.Tyro3, ​​Ax1, Traf6, Syk, MyD88, Zap70, FcεR1, FcαR1, BAFF-R, DAP12, NFAM1, MRC1, ItgB5, MERTK, ELMO, and CD79b.

[0253] [000327] In certain embodiments, the activating receptor binding domain of the multispecific molecules presented herein comprises an Fc domain or a variant thereof (which activates an Fc receptor (FcR) on macrophages, such as FcγRII).

[0254] [000328] In certain embodiments, the activating receptor binding domain comprises the Fc domain of human IgG1 (hIgG1), or human IgG4 (hIgG4), whose heavy chain constant regions are shown below, respectively: a) Heavy chain constant region (hIgG4.S228P), SEQ ID NO: 155 (the sequence of the CH1 region (SEQ ID NO: 228) is in bold and wavy underlined, the sequence of the hinge region (SEQ ID NO: 229) is in italics, and the sequence of the Fc region (SEQ ID NO: 230) is underlined): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV ESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK b) Heavy chain constant region (hIgG1), SEQ ID NO: 156 (the sequence of the CH1 region (SEQ ID NO: 231) is in bold and wavy underlined, the sequence of the hinge region (SEQ ID NO: 232) is in italics, and the sequence of the Fc region (SEQ ID NO: 233) is underlined): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [000329] The multispecific molecules provided herein can also include human light chain constant regions (CL), such as kappa and lambda chains, the amino acid sequences of which are provided below: [000330] Light Chain Constant Region (Kappa), SEQ ID NO:157: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [000331] Light chain constant region (lambda), SEQ ID NO:158: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS [000332] v. Configuration of Multispecific Molecules [000333] In certain embodiments, the engagers provided herein are recombinant proteins comprising a plurality of binding domains as described throughout the present specification, each of which has a specific binding affinity for SIRP alpha, an activating receptor, or a target antigen, and which are connected to each other by one or more linkers. The one or more linkers may have cognate peptides that exhibit complementary binding properties that bind to each other. For example, the SIRP-alpha binding domain of the engagers provided herein is fused to a first of a pair of cognate peptides, and the target antigen binding domain of the engagers provided herein is fused to a second of a pair of cognate peptides; thus, the SIRP-alpha binding domain and the target antigen binding domain may be linked by a pair of cognate peptides through complementary bonds formed between each of the pairs of cognate peptides.

[0255] [000334] In certain embodiments, the cognate peptide pair comprises two heavy chains of an antibody or any complementary portions thereof, a pair of mutually complementary light and heavy chains of an antibody or any complementary portions thereof, synthetic peptides designed to specifically bind to each other via complementary leucine zipper domains that bind to each other (e.g., zipper sequences in the binding regions of c-Fos and c-June proteins), or synthetic connectors.

[0256] [000335] The SIRP-alpha binding domain, activating receptor binding domain, and target antigen binding domain of the engagers provided herein may also be linked via chemical bonds, such as crosslinking (e.g., BS2G crosslinker (bis[sulfosuccinimidyl]glutarate), BS3 crosslinker (bis[sulfosuccinimidyl]suberate), sulfo-DSS, DST crosslinker (disuccinimidyltartrate), BMPS (N-(B-maleimidopropyloxy) succinimide ester; MBS crosslinker (mmaleimidobenzoyl-N-hydroxysuccinimide ester); or PDPH crosslinker (3-[2-pyridyldithio]propionyl hydrazide)).

[0257] [000336] In certain embodiments, the multispecific molecules presented herein are configured such that the SIRP-alpha binding domain and the activating receptor binding domain are in close proximity to each other, allowing the multispecific molecules presented herein to bind to both SIRP-alpha and activating receptors co-expressed on the same immune effector cell. As used herein, the term "close proximity" between the SIRP-alpha binding domain and the activating receptor binding domain refers to the relative location between the two domains as seen from a three-dimensional perspective, allowing the two domains to bind to SIRP-alpha and activating receptors, respectively, co-expressed on the same immune effector cell. The specific binding of the SIRP-alpha binding domain and the activating receptor binding domain, respectively, to SIRP-alpha and activating receptors (co-expressed on the same immune effector cell) can be detected, for example, by confocal microscopy, fluorescence resonance energy transfer (FRET), or single molecule super-resolution microscopy.

[0258] [000337] The target antigen binding domain of the multispecific molecules presented herein may comprise an antibody domain or an antibody mimetic domain that binds to the target antigen. The terms "antibody domain" and "antibody mimetic domain" are defined above.

[0259] [000338] In certain embodiments, the target antigen-binding antibody domain (e.g., tumor antigen-binding antibody domain) is linked to the N-terminus of an activating receptor binding domain (e.g., Fc domain). In certain embodiments, the target antigen-binding antibody domain (e.g., tumor antigen-binding antibody domain) comprises a Fab domain, optionally comprising a heavy chain linked to one of the N-terminus of the activating receptor binding domain (e.g., Fc domain). In certain embodiments, the multispecific molecules presented herein comprise two target antigen-binding antibody domains, each of which comprises a Fab domain, optionally comprising a heavy chain linked to each of the N-terminus of the activating receptor binding domain (e.g., Fc domain).

[0260] [000339] In certain embodiments, the SIRP-alpha binding domain is linked to an activating receptor binding domain (e.g., an Fc domain) or a target antigen-binding antibody domain (e.g., a tumor antigen-binding antibody domain).

[0261] [000340] In certain embodiments, the SIRP-alpha binding domain is linked to the C-terminus of an activating receptor binding domain (e.g., an Fc domain). In such embodiments, the multispecific molecules provided herein comprise a target antigen-binding antibody, where the SIRP-alpha binding domain is linked to the C-terminus of the Fc region of the target antigen-binding antibody. Illustrative embodiments are found, for example, in Figures 2B, 6F, and 6G.

[0262] [000341] In certain embodiments, both the target antigen binding antibody domain (e.g., tumor antigen binding antibody domain) and the SIRP-alpha binding domain are linked to the N-terminus of an activating receptor binding domain (e.g., Fc domain), provided that the SIRP-alpha binding domain and the target antigen binding antibody domain (e.g., tumor antigen binding antibody domain) are not linked to the same N-terminus of the activating receptor binding domain (e.g., Fc domain). Illustrative embodiments are found, for example, in Figure 6E.

[0263] [000342] In certain embodiments, the SIRP-alpha binding domain is linked to the C-terminus of the light chain of the target antigen binding domain (e.g., a tumor antigen binding Fab domain). Illustrative embodiments are found, for example, in Figure 2A.

[0264] [000343] When the multispecific molecules presented herein comprise two target antigen-binding antibody domains, the SIRP-alpha binding domain may be linked to the C-terminus of the light chain of one of the two target antigen-binding domains (e.g., the tumor antigen-binding Fab domain), and the SIRP-alpha binding domain may also be linked to the C-terminus of the light chain of both of the two target antigen-binding domains (e.g., the tumor antigen-binding Fab domain).

[0265] [000344] In certain other embodiments, the SIRP-alpha binding antibody domain is linked to the N-terminus of an activating receptor binding domain (e.g., Fc domain). In certain embodiments, the SIRP-alpha binding antibody domain comprises a Fab domain, optionally comprising a heavy chain linked to one of the N-terminus of the activating receptor binding domain (e.g., Fc domain). In certain embodiments, the multispecific molecules presented herein comprise two SIRP-alpha binding antibody domains, each of which comprises a Fab domain, optionally comprising a heavy chain linked to a respective N-terminus of the activating receptor binding domain (e.g., Fc domain). In such embodiments, the multispecific molecules presented herein comprise a SIRP-alpha binding antibody. In any of these embodiments, the target antigen binding domain (e.g., tumor antigen binding domain) is linked to the activating receptor binding domain (e.g., Fc domain), or the SIRP-alpha binding antibody domain.

[0266] [000345] In certain embodiments, the multispecific molecules presented herein comprise a SIRP-alpha binding antibody, in which the target antigen binding domain is linked to the C-terminus of the Fc region of the SIRP-alpha binding antibody (see, e.g., Figures 2D, 6B) or to the C-terminus of the light chain of the SIRP-alpha binding antibody (see, e.g., Figures 2C and 6A).

[0267] [000346] In certain embodiments, the target antigen binding domain (e.g., tumor antigen binding domain) is linked to the C-terminus of the light chain of the SIRP-alpha binding Fab domain. Illustrative embodiments are found, for example, in Figure 6A. In certain embodiments, the target antigen binding domain (e.g., tumor antigen binding domain) is linked to the N-terminus of the heavy or light chain of the SIRP-alpha binding Fab domain. Illustrative embodiments are found, for example, in Figures 6C and 6D.

[0268] [000347] As used herein, the term "linked to" refers to a covalent or non-covalent interaction (e.g., hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds) between two components.

[0269] [000348] The configurations of the multispecific molecules presented herein are summarized in Table 7 below. [000349]

[0270] [Table 10-1]

[0271] [Table 10-2]

[0272] [Table 10-3]

[0273] [000350]B. Multispecific molecules [000351] In certain embodiments, the multispecific molecules presented herein comprise a SIRP-alpha binding domain presented herein, an activating receptor binding domain comprising an Fc domain presented herein, and a target antigen binding domain presented herein.

[0274] [000352] The multispecific molecules provided herein may take any suitable format. An illustrative example is provided as follows: In certain embodiments, the multispecific molecules provided herein comprise a target antigen-binding antibody comprising two heavy chains and two light chains, where the C-terminus of each of the light chains is fused to an anti-SIRPα scFv (i.e., a SIRPα-binding domain). The target antigen-binding antibody comprises a target antigen-binding domain and an Fc domain. An illustrative example is shown in Figure 2A.

[0275] [000353] In certain embodiments, the multispecific molecules provided herein comprise a target antigen-binding antibody comprising two heavy chains and two light chains, where the C-terminus of each heavy chain is fused to an anti-SIRPα scFv (i.e., a SIRPα binding domain). The target antigen-binding antibody comprises a target antigen-binding domain and an Fc domain. An illustrative example is shown in Figure 2B.

[0276] [000354] In certain embodiments, the multispecific molecules provided herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, where the C-terminus of each light chain is fused to an scFv (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 2C.

[0277] [000355] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, where the C-terminus of each heavy chain is fused to an scFv (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 2D.

[0278] [000356] In certain embodiments, the multispecific molecules provided herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, where the C-terminus of each of the light chains is fused to a single domain antibody (sdAb) (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 6A.

[0279] [000357] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, where the C-terminus of each heavy chain is fused to a single domain antibody (sdAb) (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 6B.

[0280] [000358] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, where the N-terminus of each heavy chain is fused to a single domain antibody (sdAb) (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 6C.

[0281] [000359] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα antibody comprising two heavy chains and two light chains, where the N-terminus of each of the light chains is fused to a single domain antibody (sdAb) (i.e., a target antigen binding domain) capable of binding to a target antigen. The anti-SIRPα antibody comprises a SIRPα binding domain and an Fc domain. An illustrative example is shown in Figure 6D.

[0282] [000360] In certain embodiments, the multispecific molecules presented herein comprise an anti-SIRPα binding domain (e.g., a Fab) and a single domain antibody (sdAb) capable of binding to a target antigen, each fused to the N-terminus of a polypeptide chain of an Fc domain. An illustrative example is shown in Figure 6E.

[0283] [000361] In certain embodiments, the multispecific molecules presented herein comprise two heavy chains, each comprising a single domain antibody (sdAb) capable of binding to a target antigen fused to the N-terminus of a polypeptide chain of an Fc domain, and further comprising at least one anti-SIRPα binding domain fused to the C-terminus of one of the polypeptide chains of the Fc domain. In certain embodiments, the multispecific molecules presented herein comprise one anti-SIRPα binding domain fused to the C-terminus of one of the polypeptide chains of the Fc domain. An illustrative example is shown in Figure 6F.

[0284] [000362] In certain embodiments, the multispecific molecules presented herein comprise two anti-SIRPα binding domains, each fused to the C-terminus of one of the polypeptide chains of the Fc domain. An illustrative example is shown in Figure 6G.

[0285] [000363] In any of these embodiments, the target binding domain is a claudin 18.2 binding domain or a PD-L1 binding domain. [000364] In certain embodiments, the multispecific molecules provided herein comprise a SIRPα binding domain comprising one or more (e.g., one, two, three, four, five, or six) CDR sequences of SEQ ID NOs: 23-64 (derived from C25, hu025.021, hu025.033, hu025.023, hu025.059, hu025.060, C15, C42, C59, and C73), one or more (e.g., one, two, three, four, five, or six) CDR sequences of SEQ ID NOs: 77-106 and 225 (hu26.H1L1, hu26.H1L2, hu26.H1L3, hu26.H1L4, hu26.H1L5, hu26.H1L6, hu26.H1L7, hu26.H1L8, hu26.H1L9, hu26.H1L10, hu26.H1L11, hu26.H1L12, hu26.H1L13, hu26.H1L14, hu26.H1L15, hu26.H1L16, hu26.H1L17, hu26.H1L18, hu26.H1L19, hu26.H1L19, hu26.H1L11, hu26.H1L12, hu26.H1L14, hu26.H1L15, hu26.H1L16, hu26.H1L17, hu26.H1L18, hu26.H1L19, hu26.H1L19, hu26.H1L19, hu26.H1 2, hu26.H1L2(S92A), hu26.H3L1, hu26.H3L2, hu28.H1L2, C10, C29, and C30), and / or a PD-L1 binding domain comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences in SEQ ID NOs: 119-154 and 223 (e.g., derived from C71, C71v38, C239, C492, C570, 570h3, C446, C2811, C1778, C1793, C2855, C2713, and C2719).

[0286] [000365] In certain embodiments, the SIRPα / claudin18.2 / PD-L1 binding domain comprises an antibody domain or an antibody mimetic domain. In certain embodiments, the antibody domain is selected from the group consisting of a Fab, a VHH, a single chain Fv (scFv), a diabody, a Fab', a F(ab')2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a F(ab)2, a scFv dimer (a bivalent diabody), a camelized single domain antibody (adAb), a nanobody, a tetrabody, a domain antibody, or a bivalent domain antibody. In certain embodiments, the antibody mimetic domain is selected from the group consisting of intrabodies (e.g., fibronectin domains), monobodies, linear peptides, the Z domain of protein A (affibody), gamma B crystallographic domain, ubiquitin domain, cystatin domain, Sac7d domain, triple helix coiled-coil domain, lipocalin domain, the A domain of a membrane receptor, ankyrin repeat motifs, the SH3 domain of Fyn, the Kunitz domain of a protease inhibitor, the type III domain of fibronectin (minibody), a DARPin domain, and carbohydrate binding module 32-2.

[0287] [000366] In certain embodiments, the multispecific molecules presented herein comprise: a) a SIRPα binding domain comprising a heavy chain CDR1 comprising the sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 24, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 25, and / or a light chain CDR1 comprising the sequence of SEQ ID NO: 26, a light chain CDR2 comprising the sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the sequence of SEQ ID NO: 28; b) i) a heavy chain CDR1 comprising the sequence of SEQ ID NO: 83, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 84, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 85, and / or a light chain CDR1 comprising the sequence of SEQ ID NO: 86, a light chain CDR2 comprising the sequence of SEQ ID NO: 87, and a light chain CDR3 comprising the sequence of SEQ ID NO: 88, or ii) a heavy chain CDR1 comprising the sequence of SEQ ID NO: 77, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 78, a heavy chain CDR3 comprising the sequence of SEQ ID NO: 79, and / or a light chain CDR1 comprising the sequence of SEQ ID NO: 80, a light chain CDR2 comprising the sequence of SEQ ID NO: 81, and a light chain CDR3 comprising the sequence of SEQ ID NO: 223 and / or c) i) a heavy chain CDR1 comprising the sequence of SEQ ID NO: 119, a heavy chain CDR2 comprising the sequence of SEQ ID NO: 120, and a heavy chain CDR3 comprising the sequence of SEQ ID NO: 121, or ii) heavy chain CDR1 comprising the sequence of SEQ ID NO: 131, heavy chain CDR2 comprising the sequence of SEQ ID NO: 132, heavy chain CDR3 comprising the sequence of SEQ ID NO: 133 PD-L1 binding domain containing Includes.

[0288] [000367] In certain embodiments, the SIRPα binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, or a homologous sequence thereof having at least 80% sequence identity while still retaining specific binding affinity to SIRPα (e.g., human SIRPα), and / or a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, or a homologous sequence thereof having at least 80% sequence identity while still retaining specific binding affinity to SIRPα (e.g., human SIRPα).

[0289] [000368] In certain embodiments, the SIRPα binding domain comprises an anti-SIRPα scFv. In certain embodiments, the scFv comprises a heavy chain variable region linked, N-terminus to C-terminus, optionally via a linker (e.g., a polypeptide linker), to a light chain variable region. Alternatively, in certain embodiments, the scFv comprises a light chain variable region linked, N-terminus to C-terminus, optionally via a linker (e.g., a polypeptide linker), to a heavy chain variable region. In certain embodiments, the anti-SIRPα scFv comprises the amino acid sequence of SEQ ID NO: 226.

[0290] [000369] In certain embodiments, the claudin 18.2 binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 65, 68, 69, 71, 73, and 75, or a homologous sequence thereof having at least 80% sequence identity while still retaining specific binding affinity to claudin 18.2 (e.g., human claudin 18.2), and / or a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 66, 67, 70, 72, 74, 76, and 224, or a homologous sequence thereof having at least 80% sequence identity while still retaining specific binding affinity to claudin 18.2 (e.g., human claudin 18.2).

[0291] [000370] In certain embodiments, the claudin 18.2 binding domain comprises an anti-claudin 18.2 scFv. In certain embodiments, the scFv comprises a heavy chain variable region linked from the N-terminus to the C-terminus, optionally via a linker (e.g., a polypeptide linker), to a light chain variable region. Alternatively, in certain embodiments, the scFv comprises a light chain variable region linked from the N-terminus to the C-terminus, optionally via a linker (e.g., a polypeptide linker), to a heavy chain variable region. In certain embodiments, the anti-claudin 18.2 scFv comprises the amino acid sequence of SEQ ID NO: 227.

[0292] [000371] In certain embodiments, the PD-L1 binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 107-118, and 223, or a homologous sequence thereof with at least 80% sequence identity while still retaining specific binding affinity to PD-L1 (e.g., human PD-L1).

[0293] [000372] A variety of techniques are available for the generation of such antigen-binding domains. Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), and screening from phage display libraries as discussed above (e.g., for ScFv). Other techniques for generating antibody fragments will be apparent to those of skill in the art.

[0294] [000373] In certain embodiments, the SIRPα binding domain and / or the claudin 18.2 binding domain are or include scFv or Fab. The generation of scFv is described, for example, in WO 93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458. The scFv can be fused to an effector protein at either the amino or carboxyl terminus to provide a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck). The scFv can comprise a VH linked to a VL directly or via a peptide linker. In certain embodiments, the VH can be located at the N-terminus of the scFv, and the VL can be located at the C-terminus. In certain embodiments, the VL can be located at the N-terminus of the scFv, and the VH can be located at the C-terminus.

[0295] [000374] In certain embodiments, the SIRP alpha binding domain, the target antigen binding domain (e.g., claudin 18.2 binding domain / PD-L1 binding domain), and the activating receptor binding domain are connected via a peptide linker. The peptide linker may comprise a single or repeated sequence composed of threonine / serine and glycine, such as TGGGG (SEQ ID NO: 183), GGGGS (SEQ ID NO: 184), GGGGSGGGGS (SEQ ID NO: 185), (Gly4Ser)3 (SEQ ID NO: 186), or SGGGG (SEQ ID NO: 187), or tandem repeats thereof (e.g., 2, 3, 4, or more repeats). In certain embodiments, the peptide linker comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34). In certain embodiments, the peptide linker comprises or is (Gly4Ser)3 (SEQ ID NO: 186).

[0296] [000375] C. Characterization of Multispecific Molecules [000376] In some embodiments, the multispecific molecules provided herein have the ability to specifically bind to both human SIRPalpha and a CD47-expressing cell surface marker (e.g., human claudin 18.2 or PD-L1 expressed on the surface of CD47-expressing cells). The multispecific molecules provided herein retain specific binding affinity for both human SIRPalpha and human claudin 18.2 / human PD-L1, and in certain embodiments, are at least comparable or even better than the parent antibodies in such aspects.

[0297] [000377] The binding of multispecific molecules is measured using the "50% effective concentration" (EC 50 ) value, which refers to the concentration of antibody at which 50% of its maximal effect (e.g., binding or inhibition, etc.) is observed. 50 Levels can be measured by methods known in the art, such as sandwich assays, such as ELISA, Western blots, flow cytometry assays, and other binding assays.

[0298] [000378] The binding affinity of the antigen-binding domains presented herein is K D The value (ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on It is also possible to express the antigen binding affinity (e.g., K D ) may be suitably determined using suitable methods known in the art, including, for example, flow cytometry assays. In some embodiments, the binding of the antigen-binding domain to different concentrations of antigen can be determined by flow cytometry, and the determined mean fluorescence intensity (MFI) can be first plotted against the concentration of the antigen-binding domain, and then the dependence of the specific binding fluorescence intensity (Y) and the concentration of the antibody (X) can be calculated using the one site saturation equation: Y=B max *X / (K D +X), K D It is possible to calculate the value, where B max represents the maximum specific binding of the antigen-binding domain tested to the antigen.

[0299] [000379] In certain embodiments, the multispecific molecules presented herein have a cell cycle length of 25 x 10 -8 M, 20×10 -8 M, 15×10 -8 M, 12×10 -8 M, 10x10 -8 M, 9×10 -8 M, 8×10 -8 M, 7×10 -8 M, 6×10 -8 M, 5×10 -8 M, 4×10 -8 M, 3×10 -8 M, 2×10 -8 M, or 1 x 10 -8 Binding affinity (K D ) and specifically binds to human SIRP alpha.

[0300] [000380] In certain embodiments, the multispecific molecules presented herein have a cell density of 1 x 10 -12 Binding affinity (K D ) and specifically binds to human claudin 18.2.

[0301] [000381] In certain embodiments, the multispecific molecules presented herein have a cytotoxicity of 70x10 as measured by the Octet assay. -8 M, 65×10 -8 M, 60×10 -8 M, 55×10 -8 M, 50×10 -8 M, 45×10 -8 M, 40×10 -8 M, 35×10 -8 M, 30×10 -8 M, 25×10 -8 M, 20×10 -8 M, 15×10 -8 M, 12×10 -8 M, 10x10 -8 M, 9×10 -8 M, 8×10 -8 M, 7×10 -8 M, 6×10 -8 M, 5×10 -8 M, 4×10 -8 M, 3×10 -8 M, 2×10 -8 M, or 1 x 10 -8 Binding affinity (K D ) and specifically binds to human PD-L1.

[0302] [000382] The blocking effect of the multispecific molecules presented herein on the interaction of CD47 with SIRPalpha or the interaction of PD-1 with PD-L1 can be measured by various techniques, such as luciferase reporter assays, competitive ELISA assays, and competitive FACS assays, and can be expressed as an IC50. The IC50 for blocking the interaction of CD47 with SIRPalpha represents the concentration of the multispecific molecules presented herein at which the binding of CD47 to SIRPalpha is reduced by 50% in the presence of the multispecific molecules of the present disclosure. The IC50 for blocking the interaction of PD-1 with PD-L1 represents the concentration of the multispecific molecules presented herein at which the binding of PD-1 to PD-L1 is reduced by 50% in the presence of the multispecific molecules of the present disclosure. In certain embodiments, the IC50 of the multispecific molecules presented herein for blocking the interaction of PD-1 with PD-L1 is comparable to the IC50 of anti-PD-1 C71.

[0303] [000383] In certain embodiments, the SIRP-alpha binding domains presented herein have IC50 values ​​of 6.0 nM, 5.0 nM, 4.0 nM, 3.80 nM, 3.60 nM, 3.0 nM, 2.90 nM, 2.88 nM, 2.86 nM, 2.80 nM, 2.78 nM, 2.76 nM, 2.74 nM, 2.70 nM, 2.64 nM, 2.40 nM, 2.20 nM, 2.0 nM, 1.0 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.18 nM, 0.16 nM, 0.14 nM, 0.12 nM or less for blocking the interaction of SIRP-alpha with CD47 as measured by competitive ELISA assay or competitive FACS assay. In certain embodiments, the SIRP-alpha binding domains provided herein have a blocking activity of 6.0 nM or less, 5.0 nM or less, 4.0 nM or less, 3.80 nM or less, 3.60 nM or less, 3.0 nM or less, 2.90 nM or less, 2.88 nM or less, 2.86 nM or less, 2.80 nM or less, 2.7 nM or less, or 3.6 nM or less, for blocking SHP-1 transduction as measured by competitive ELISA assay or competitive FACS assay. and / or an IC50 value of 8nM or less, 2.76nM or less, 2.74nM or less, 2.70nM or less, 2.64nM or less, 2.40nM or less, 2.20nM or less, 2.0nM or less, 1.0nM or less, 0.4nM or less, 0.3nM or less, 0.2nM or less, 0.18nM or less, 0.16nM or less, 0.14nM or less, 0.12nM or less, 0.10nM or less, 0.09nM or less, 0.08nM, or 0.07nM or less.

[0304] [000384] D. Variant [000385] The multispecific molecules presented herein also encompass various variants thereof. In certain embodiments, the variants comprise one or more modifications or substitutions within one or more CDR sequences as presented in Table 2, Table 4, and Table 6, within one or more variable region sequences (but not included in any of the CDR sequences) presented in Table 1, Table 3, and Table 5, and / or within the constant region (e.g., Fc region). Such variants retain specific binding affinity for SIRPα, claudin 18.2, and / or PD-L1 like their parent antibodies, but have one or more desirable properties conferred by the modification(s) or substitution(s). For example, variants may have improved antigen binding affinity, increased productivity, improved stability, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function(s), improved FcRn receptor binding, pharmacokinetic half-life, pH sensitivity, and / or increased suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0305] [000386] The parent antibody sequence can be screened to identify which residues are suitable or preferred for modification or substitution using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for interesting properties. If substitutions at a particular amino acid site show interesting functional changes, then that position can be identified as a promising residue for modification or substitution. Promising residues can be further evaluated by substituting different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0306] [000387] In certain embodiments, the SIRPα-binding domain, claudin 18.2-binding domain, and / or PD-L1-binding domain presented herein comprise one or more amino acid residue substitutions in one or more CDR sequences, and / or in one or more FR sequences, and / or in one or more variable region sequences. In certain embodiments, the variants comprise no more than a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences, and / or in the FR sequences, and / or in the variable region sequences.

[0307] [000388] In certain embodiments, the SIRPα binding domain comprises one, two, three, four, five, or six 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 one, two, three, four, five, or six sequences selected from SEQ ID NOs: 23-64 and 198, while retaining a binding affinity for SIRPα at a level similar to or greater than that of its parent antibody.

[0308] [000389] In certain embodiments, the SIRPα-binding domain comprises 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 one or more sequences selected from SEQ ID NOs: 1-22 while retaining a binding affinity for SIRPα at a similar or superior level to that of its parent antibody. In some embodiments, a total of 1-10 amino acids have been substituted, inserted, or deleted in the variable region sequence selected from SEQ ID NOs: 1-22. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs).

[0309] [000390] In certain embodiments, the claudin 18.2 binding domain comprises one, two, three, four, five, or six 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 one, two, three, four, five, or six sequences selected from SEQ ID NOs: 77-106 and 225, while retaining a binding affinity for SIRPα at a level similar to or greater than that of its parent antibody.

[0310] [000391] In certain embodiments, the claudin 18.2 binding domain comprises 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 one or more sequences selected from SEQ ID NOs: 65-76 and 224 while retaining a binding affinity for claudin 18.2 at a level similar to or superior to that of its parent antibody. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted in the variable region sequence selected from SEQ ID NOs: 65-76 and 224. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (e.g., in the FR).

[0311] [000392] In certain embodiments, the PD-L1 binding domain comprises one, two, or three CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to one, two, or three sequences selected from SEQ ID NOs: 119-154, while retaining a similar or better binding affinity for PD-L1 as its parent antibody.

[0312] [000393] In certain embodiments, the PD-L1 binding domain comprises one or more variable region sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to one or more sequences selected from SEQ ID NOs: 107-118 and 223, while retaining a similar or better level of binding affinity for PD-L1 as its parent antibody. In some embodiments, a total of 1-10 amino acids have been substituted, inserted or deleted in the variable region sequence selected from SEQ ID NOs: 107-118 and 223. In some embodiments, the substitutions, insertions or deletions occur within regions outside the CDRs (e.g., within the FRs).

[0313] [000394] i. Glycosylation variants [000395] The multispecific molecules presented herein also encompass glycosylation variants, which can be obtained by increasing or decreasing the extent of glycosylation on the antigen-binding domains or activating receptor domains of the multispecific molecules.

[0314] [000396] The multispecific molecules provided herein may comprise one or more amino acid residues having a side chain that may be linked to a carbohydrate moiety (e.g., an oligosaccharide structure). Glycosylation of the antigen-binding domain of an antibody is generally either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue, e.g., an asparagine residue in a tripeptide sequence, e.g., asparagine-X-serine or asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently achieved, for example, by altering the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can be created in an analogous manner by introducing such a tripeptide sequence, or a serine or threonine residue.

[0315] [000397] ii. Cysteine ​​Engineered Variants [000398] The multispecific molecules presented herein also encompass cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0316] [000399] A free cysteine ​​residue is a residue that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful, for example, for conjugation with cytotoxic and / or imaging compounds, labels, or especially radioisotopes at the site of engineered cysteine, for example, through maleimide or haloacetyl. Methods for engineering antibody polypeptides to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0317] [000400] iii. Fc variants [000401] The multispecific molecules presented herein also encompass Fc variants, which comprise modifications or substitutions of one or more amino acid residues in the Fc region and / or hinge region, e.g., to alter effector function, such as ADCC, ADCP, and CDC. Methods for altering ADCC activity by antibody engineering have been described in the art, e.g., 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 See Ther. 2008, 7(8):2517-27; Shields R Let al, J. Biol. Chem, 2002, 277:26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278:3466-3473.

[0318] [000402] The CDC activity of the antibodies provided herein can also be altered, for example, by improving or reducing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821); and WO 94 / 29351 for other examples of Fc region variants. One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be substituted with a different amino acid residue to alter Clq binding and / or reduce or abolish complement dependent cytotoxicity (CDC) (see U.S. Pat. No. 6,194,551 to Idusogie et al.). One or more amino acid substitution(s) can also be introduced to alter the ability of the antibody to fix complement (see Bodmer et al., PCT Publication WO 94 / 29351).

[0319] [000403] The terms "antibody-dependent cellular phagocytosis" and "ADCP" refer to the process by which antibody-coated cells or particles are internalized, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind the immunoglobulin Fc region. Methods for altering the ADCP activity of antibodies by antibody engineering are known in the art, see, for example, Kellner C et al., Transfus Med Hemother, (2017) 44: 327-336 and Chung AW et al., AIDS, (2014) 28: 2523-2530. Examples of Fc variants are known in the art, see, for example, Wang et al., Protein Cell 2018, 9(1): 63-73 and Kang et al., Exp & Mol., Med. (2019) 51: 138, which are incorporated herein in their entirety.

[0320] [000404] i) Fc variants with enhanced effector function [000405] In certain embodiments, the Fc variants provided herein have increased ADCC and / or increased affinity for Fcγ receptors (e.g., FcγRI (CD64), FcγRII (CD32), and / or FcγRIII (CD16)) compared to a wild-type Fc (e.g., an IgG1 Fc). In certain embodiments, the Fc variants have an increased affinity for the following positions of the Fc region: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, and one or more amino acid substitution(s) at one or more of: 339, 340, 345, 360, 373, 376, 378, 382, ​​388, 389, 396, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, 439, and 440 (see WO 00 / 42072 by Presta, WO 2006 / 019447 by Lazar, and WO 2016 / 196228 by Lazar, which are incorporated herein in their entireties), where the numbering of residues in the Fc region is that of the EU index according to Kabat (Kabat See EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).Exemplary substitutions for increasing effector function include, but are not limited to, 234Y, 235Q, 236A, 236W, 239D, 239E, 239M, 243L, 247I, 268D, 267E, 268D, 268E, 268F, 270E, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 324T, 326A, 326D, 326W, 330L, 330M, 333S, 332D, 332E, 298A, 333A, 334A, 334E, 326A, 247I, 33 9D, 339Q, 345R, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, 396L, 430G, 440Y, or any combination thereof (e.g., 239D / 332E, 239D / 332E / 330L, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T, etc.) (WO 2016 / 196228; Richards (see, e.g., Moore et al. (2010) mAbs 2:181; and Strohl (2009) Current Opinion in Biotechnology 20:685-691).

[0321] [000406] Specific mutations at positions 256, 290, 298, 333, 334, and 339 were found to improve binding to FcγRIII. In addition, the following combination mutations were found to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, F243L / R292P / Y300L / V305I / P396L, S298A / E333A / K334A, and L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the opposing heavy chain (enhancing FcγRIII binding and ADCC activity). Other Fc variants with highly enhanced binding to FcγRIIIa include variants with S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and strong cytotoxic activity, as well as variants with L235V, F243L, R292P, Y300L, V305I, and P396L mutations, which showed enhanced FcγRIIIa binding with concomitant enhanced ADCC activity (Lazar et al. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005; Awan et al. (2010) Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res, Stavenhagen et al. (2007) Cancer Res. 67:8882). Modifications that increase binding to Clq can be introduced to enhance CDC activity. Exemplary modifications include K326 (e.g., K326W) and / or E333 modifications in IgG2, or S267E / H268F / S324T modifications, alone or in any combination, in IgG1 (see Idusogie et al. (2001) J. Immunol. 166:2571, Moore et al. (2010) mAbs 2:181).Other exemplary modifications include K326W / E333S, S267E / H268F / S324T, and E345R / E430G / S440Y.

[0322] [000407] ii) Fc with reduced effector function [000408] In certain embodiments, the Fc variants presented herein have reduced effector function compared to a wild-type Fc (e.g., an IgG1 Fc) and comprise one or more amino acid substitution(s) at positions selected from the group consisting of 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 in the Fc region (WO 2016 / 196228; Richards et al. (2008) Mol. Cancer Therap. 7:2517; Moore et al. (2010) mAbs 2:181; and Strohl (2009) Current Opinion in Biotechnology 20:685-691), where the numbering of residues in the Fc region is that of the EU index according to Kabat. Exemplary substitutions to reduce effector function include, but are not limited to, 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S, or any combination thereof (see WO 2016 / 196228 and Strohl (2009) Current Opinion in Biotechnology 20:685-691).

[0323] [000409] In certain embodiments, the Fc variants presented herein are of the IgG1 isotype and contain one or more amino acid substitution(s) selected from the group consisting of L234A, L234F, L234V, F234A, V234A, L235A, L235E, G237A, P238S, H268Q, H268A, N297A, N297Q, N297G, V309L, A330S, and P331S, or any combination thereof (e.g., L234A / L235A, etc.). In certain embodiments, the Fc variants presented herein are of the IgG2 isotype and contain one or more amino acid substitution(s) selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof. In certain embodiments, the Fc variants presented herein are of the IgG4 isotype and contain one or more amino acid substitution(s) selected from the group consisting of S228P, F234A, L235E, L235A, G237A, E318A, N297A, N297Q, N297G, and any combination thereof.

[0324] [000410] iii) Fc with altered binding to FcRn [000411] In certain embodiments, the Fc variant contains one or more amino acid substitution(s) that improve binding affinity to the neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4. Such a variant may have an extended pharmacokinetic half-life because it binds to FcRn at acidic pH, which allows it to escape degradation in lysosomes and then be translocated and released outside the cell. Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al, Structure, 6(1):63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70:3269-3277(2010); Hinton, P. et al, J. Immunology, 176:346-356(2006); Petkova et al.(2006) Int. Immunol.18:1759, Ball Acqua et al. Journal of Immunology 2002,169:5171-5180, Dall'Acqua WF.et al., J. Immunology, 176:346-356(2006). See Biol Chem. 281:23514-23524 (2006); Zalevsky J, et al, Nat Biotechnol.; 28:157-159 (2010); WO 2009 / 086320; U.S. Patent No. 6,277,375; U.S. Patent No. 6,821,505; WO 97 / 34631; and WO 2002 / 060919.

[0325] [000412] Non-limiting examples of Fc modifications that may result in an increase in the serum half-life of an antibody when administered include, for example, 234 (e.g., with F), 235 (e.g., with Q), 238 (e.g., with D), 250 (e.g., with E or Q), 252 (e.g., with L / Y / F / W, or T), 254 (e.g., with S or T), 256 (e.g., with S / R / Q / E / D, or T); 259 (e.g., with I); 272 (e.g., with A), 305 (e.g., with A), 307 (e.g., with A or P), 308 (e.g., with F, C, or P), 311 (e.g., with A or R), 312 (e.g., with A), 322 (e.g., Q), 328 ( For example, substitution(s) at one or more positions selected from E), 331 (e.g., with A), 378 (e.g., with A), 380 (e.g., with A), 382 (e.g., with A), 428 (e.g., with L or F), 432 (e.g., with C), 433 (e.g., with H / L / R / S / P / Q or K), 434 (e.g., with H / F or Y or S or A or W), 435 (e.g., with H), 436 (e.g., with L), and 437 (e.g., with C) (all positions according to EU numbering) (see WO2016049000A2; WO2020052692; WO2016196228). In some embodiments, the Fc variant comprises one or more amino acid substitution(s) selected from the group consisting of 234F, 235Q, 238D, 250Q, 252T, 252Y, 254T, 256E, 259I, 272A, 305A, 307A, 308F, 311A, 322Q, 328E, 331S, 380A, 428L, 432C, 433K, 433S, 434S, 434Y, 434F, 434W, 434A, 435H, 436L, 437C and any combination thereof.In some embodiments, the Fc modifications include: a) 428L (e.g., M428L) and 434S (e.g., N434S) substitutions; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) substitutions; b) 433K (e.g., H433K) and 434 (e.g., N434Y or N434F) substitutions; c) 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E) substitutions; d) 250Q and 428L substitutions (e.g., T250Q and M428L); e) 307A, 380A, and 434A substitutions (e.g., T307A, E380A, and N434A); f) P238D and L328E substitutions; g) L234F, L235Q, K322Q, M252T, S254T, and T256E substitutions; and h) L432C, H433S, N434W, Y436L, and T437C substitutions.

[0326] [000413] In some embodiments, hybrid IgG isotypes can be used to increase FcRn binding and half-life of the antibody. Hybrid Igs can be generated from two or more isotypes. For example, IgG1 / IgG3 hybrid variants can be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions that differ in the two isotypes. In some embodiments, hybrid Igs can include one or more modifications (e.g., substitutions) disclosed herein.

[0327] [000414] E. Conjugates [000415] In some embodiments, the multispecific molecules presented herein further comprise a conjugate moiety. The conjugate moiety may be linked to the multispecific molecule. The conjugate moiety is a non-protein moiety that can be linked to the multispecific molecule. It is contemplated that a variety of conjugate moieties may be linked to the multispecific molecules presented herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). Such conjugate moieties may be linked to the multispecific molecule by covalent binding, affinity binding, intercalation, coordinate binding, complex formation, association, mixing, or addition, among other methods.

[0328] [000416] In certain embodiments, the multispecific molecules disclosed herein can be engineered to contain specific sites outside the epitope-binding moiety that can be utilized to bind one or more conjugates. For example, such sites can include one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent binding to a conjugate.

[0329] [000417] In certain embodiments, the multispecific molecule may be indirectly linked to the conjugate moiety or through another conjugate moiety. For example, the multispecific molecule may be conjugated to biotin and then indirectly conjugated to a second conjugate moiety that is conjugated to avidin. The conjugate moiety may be a clearance modifier, a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), or a purification moiety.

[0330] [000418] A "toxin" can be any agent that is harmful to cells or can cause cell damage or death. Examples of toxins include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents. (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin-binding agents.

[0331] [000419] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luceriferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In,112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides), luminescent labels, chromogenic moieties, digoxigenin, biotin / avidin, DNA molecules for detection, or gold.

[0332] [000420] In certain embodiments, the conjugate moiety may be a clearance modifier that serves to increase the half-life of the multispecific molecule. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers linked to the multispecific molecule may vary, and when more than one polymer is linked, they may be the same or different molecules.

[0333] [000421] In certain embodiments, the conjugate moiety can be a purification moiety, such as a magnetic bead. [000422] In certain embodiments, the multispecific molecules presented herein are used as substrates for conjugates.

[0334] [000423] F. Polynucleotides and Recombinant Methods [000424] The present disclosure provides isolated polynucleotides encoding the multispecific molecules provided herein.

[0335] [000425] The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise limited, the term encompasses polynucleotides that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed group and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0336] [000426] Many vectors are available. The vector components typically 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 alpha), and a transcription termination sequence.

[0337] [000427] The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequences provided herein encoding the multispecific molecules, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequences, and at least one selectable marker. Exemplary vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, and the like. , pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0338] [000428] Vectors containing polynucleotide sequences encoding the multispecific molecules can be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and the like. marcescens, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0339] [000429] In addition to prokaryotes, eukaryotic pathogens such as filamentous fungi or yeasts are suitable cloning or expression hosts for the subject vectors. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, several other genera, species, and strains are also suitable, such as Schizosaccharomyces pombe. pombe, etc.; Kluyveromyces hosts, such as 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, etc.; Yarrowia (European Patent No. 402,226); Pichia pastoris pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are commonly available and useful herein.

[0340] [000430] Suitable host cells for expressing the glycosylated multispecific molecules presented herein are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx Mori. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx Mori NPV, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfecting Armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

[0341] [000431] However, interest has grown most 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 with SV40 (COS-7, ATCC CRL1651); 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 CCL10); 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 CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some preferred embodiments, the host cells are 293F cells.

[0342] [000432] To produce the multispecific molecules presented herein, host cells are transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, the multispecific molecules presented herein can be produced by homologous recombination methods known in the art.

[0343] [000433] The host cells used to produce the multispecific molecules presented herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum 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. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor, etc.), salts (e.g., sodium chloride, calcium, magnesium, and phosphate, etc.), buffers (e.g., HEPES, etc.), nucleotides (e.g., adenosine and thymidine, etc.), antibiotics (e.g., the drug Gentamicin™, etc.), 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 suitable concentrations known to those of skill in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0344] [000434] When using recombinant techniques, the multispecific molecules can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibodies are produced intracellularly, as a first step, 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) describes a procedure for isolating antibodies secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the multispecific molecules are secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the above steps to inhibit proteolysis, while antibiotics may also be included to prevent the growth of adventitious contaminants.

[0345] [000435] The multispecific molecules 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.

[0346] [000436] In certain embodiments, Protein A immobilized on a solid phase is used in the immunoaffinity purification of multispecific molecules. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domains present in the multispecific molecule. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (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 are feasible with agarose. Where the multispecific molecule contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography using anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and fractionation by ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0347] [000437] Following any preliminary purification step(s), the mixture containing the antibody molecules of interest and contaminants is subjected to low pH hydrophobic interaction chromatography using an elution buffer of about pH 2.5-4.5, preferably performed at a low salt concentration (e.g., about 0-0.25 M salt).

[0348] [000438] G. Pharmaceutical Compositions [000439] The disclosure further provides pharmaceutical compositions comprising the multispecific molecules and one or more pharma- ceutically acceptable carriers.

[0349] [000440] Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein can include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispensing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0350] [000441] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising multispecific molecules and conjugates as presented herein reduces oxidation of the multispecific molecules. This reduced oxidation prevents or inhibits loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided that include one or more multispecific molecules as disclosed herein and one or more antioxidants, such as methionine. Methods are further provided for preventing oxidation, extending shelf life, and / or improving efficacy of multispecific molecules as provided herein by combining the multispecific molecules with one or more antioxidants, such as methionine.

[0351] [000442] To further illustrate, pharma- ceutically acceptable carriers include, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonicity agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; and the like, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, 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 used as carriers may be added to the pharmaceutical compositions in multi-dose containers, and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0352] [000443] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0353] [000444] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion, etc., or a solid form suitable for producing a liquid solution, suspension, or emulsion. Preparations for injection can include sterile and / or non-pyrogenic solutions for immediate injection, sterile dry soluble products, such as freeze-dried powders (including hypodermic tablets) that are immediately mixed with a solvent immediately before use, sterile suspensions for immediate injection, sterile dry insoluble products that are immediately mixed with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. Solutions can be either aqueous or non-aqueous.

[0354] [000445] In certain embodiments, unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration should be sterile and nonpyrogenic, as known and practiced in the art.

[0355] [000446] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the multispecific molecule as disclosed herein in a suitable solvent. The solvent may contain excipients that improve stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art and near neutral pH in one embodiment. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single or multiple doses of the multispecific molecule or composition thereof. Overfilling the vial with a small amount (e.g., about 10%) beyond that required for a single or set dose is permissible to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0356] [000447] The lyophilized powder can be reconstituted with water for injection to obtain a formulation for use in parenteral administration. In one embodiment, when reconstituted, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder. The exact amount depends on the selected therapy to be performed and can be determined empirically.

[0357] [000448] H. How to use [000449] In another aspect, the present disclosure provides a method of use for the multispecific molecules provided herein. The multispecific molecules provided herein include a SIRP-alpha binding domain provided herein, an activating receptor binding domain provided herein, and a target antigen binding domain provided herein. The target antigen binding domain binds to a target antigen expressed on a target cell that co-expresses the target antigen and CD47. The multispecific molecules provided herein have the ability to selectively induce effector functions of immune effector cells in the presence of the target antigen, and the immune effector cells co-express SIRP-alpha and an activating receptor.

[0358] [000450] In one aspect, the present disclosure provides a method for inducing phagocytosis in vitro, comprising contacting target cells with a SIRPα-positive phagocytic cell sample in the presence of a multispecific molecule provided herein, thereby inducing phagocytosis of the target cells by the SIRPα-positive phagocytic cells.

[0359] [000451] In one aspect, the present disclosure provides a method of inducing phagocytosis of a target cell in a subject, comprising administering to the subject a multispecific molecule provided herein in a dose effective to induce phagocytosis of the target cell in the subject.

[0360] [000452] In one aspect, the present disclosure provides a method of redirecting tumor-associated monocytes or macrophages (TAMs) to anti-tumor macrophages to enhance phagocytosis of cancer cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multispecific molecule or engager provided herein. In addition, the engagers provided herein can activate macrophages and engage the activated macrophages with the tumor microenvironment to exert a phagocytic effect on cancer cells.

[0361] [000453] In another aspect, the present disclosure also provides a method of increasing the level of M1 macrophages in a tumor microenvironment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multispecific molecule or engager provided herein. As used herein, the term "tumor microenvironment" refers to the tissue(s), cells, and environment surrounding a cancer or tumor cell. The tumor microenvironment may include stromal cells, such as fibroblasts, pericytes, endothelial cells, adipocytes, and bone marrow mesenchymal stromal cells (MSCs). The tumor microenvironment may also include an extracellular matrix associated with the cancer cells or associated with the stromal cells surrounding the cancer cells. The extracellular matrix is ​​primarily composed of intercellular material (a porous, hydrated gel primarily composed of proteoglycan aggregates) and connective tissue fibers. Experimentally, the tumor microenvironment for a particular tumor can be obtained, for example, by dissecting and isolating tissue bearing a particular tumor.

[0362] [000454] The term "increase" or "increasing" as provided herein, when referring to levels of M1 macrophages, refers to an elevation in M1 macrophage count (normalized to total macrophage count) in a tumor microenvironment in the presence of a multispecific molecule or engager as provided herein compared to a tumor microenvironment in the absence of a multispecific molecule or engager as provided herein. The level of M1 macrophages or M1 macrophage count within a tumor microenvironment can be measured by conventional techniques known in the art, such as FACS assays, for the amount of macrophages with a surface marker profile of the M1 phenotype (CD80 high and CD206 midSee profile, Zhang M et al., Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo. PloS one 11, e0153550, 10.1371 / journal.pone.0153550 (2016)). The level of M1 macrophages or M1 macrophage count can also be measured by qPCR analysis of inducible nitric oxide synthase 1 (Nos1) mRNA expression levels (normalized to total mRNA expression levels - known to be elevated in M1 macrophages) (Zhang M et al., Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo. PloS one 11, e0153550, 10.1371 / journal.pone.0153550(2016)). Other methods that can count the number of macrophages with the M1 phenotype or determine the expression level of one or more characteristic markers for M1 macrophages are within the contemplation of this disclosure, such as confocal fluorescence microscopy or western blotting. In another aspect, the present disclosure also provides a method of treating a disease, disorder, or condition that may benefit from inducing phagocytosis of target cells in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule as provided herein.

[0363] [000455] In certain embodiments, the target cells co-express the target antigen and CD47. In some embodiments, the target cells include cancer cells, inflammatory cells, and / or chronically infected cells. In some embodiments, the target antigen is a tumor surface antigen, an inflammatory antigen, or an antigen of an infectious microorganism. In some embodiments, the target antigen can be a tumor antigen (e.g., a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), such as a neoantigen, etc.), or an antigen presented on an infected cell (e.g., Hepatitis B surface antigen (HBsAg)).

[0364] [000456] In certain embodiments, disorders or conditions that may benefit from inducing phagocytosis of target cells can include, for example, cancer (e.g., solid tumors, hematological malignancies), inflammatory diseases, infectious diseases (e.g., chronic infections), autoimmune diseases (e.g., multiple sclerosis), neurological diseases, brain injury, neurological damage, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction, and arthritis.

[0365] [000457] In another aspect, the present disclosure also provides a method of treating a disease, disorder, or condition associated with a target antigen in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein. For example, when the target antigen comprises a tumor antigen, the target antigen-associated disease can include tumor or cancer. For example, when the target antigen comprises an antigen presented on an infected cell, the target antigen-associated disease can include an associated infectious disease. In certain embodiments, the target antigen comprises PD-L1. In certain embodiments, the target antigen comprises Claudin 18.2.

[0366] [000458] In another aspect, the disclosure also provides a method of treating a disease, disorder, or condition associated with SIRPα in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule presented herein.

[0367] [000459] In another aspect, the present disclosure also provides a method of treating a CD47-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule presented herein.

[0368] [000460] In some embodiments, the subject is a human. In some embodiments, the subject is homozygous for SIRPαv1. In some embodiments, the subject is homozygous for SIRPαv2.

[0369] [000461] In some embodiments, the subject has been diagnosed with or is at risk for a disease, disorder, or condition selected from the group consisting of cancer (e.g., solid tumors, hematological malignancies), inflammatory disease, infectious disease (e.g., chronic infections), autoimmune disease (e.g., multiple sclerosis), neurological disease, brain injury, neurological injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction, and arthritis. In preferred embodiments, the subject has been diagnosed with or is at risk for one or more solid tumors.

[0370] [000462] In certain embodiments, the condition or disorder treatable by the methods presented herein can be an immune-related disease or disorder, tumors, and cancer, an autoimmune disease, or an infectious disease. In certain embodiments, the immune-related disease or disorder is selected from the group consisting of systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), vasculitis, myasthenia gravis, idiopathic pulmonary fibrosis, Crohn's disease, asthma, rheumatoid arthritis, graft-versus-host disease, spondyloarthritic disorders (e.g., ankylosing spondylitis, psoriatic arthritis, sporadic acute enteropathic arthritis associated with inflammatory bowel disease, reactive arthritis, Behcet's syndrome, undifferentiated spondyloarthropathy, anterior uveitis, and juvenile idiopathic arthritis), multiple sclerosis, endometriosis, glomerulonephritis, sepsis, diabetes, acute coronary syndrome, ischemia-reperfusion, psoriasis, progressive systemic sclerosis, atherosclerosis, Sjogren's syndrome, scleroderma, or inflammatory autoimmune myositis.

[0371] [000463] In certain embodiments, the conditions or disorders treatable by the methods provided herein include tumors and cancers. In certain embodiments, the conditions or disorders treatable by the methods provided herein include solid tumors and hematological malignancies. Examples of cancers and tumors include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematological malignancies, such as classical Hodgkin's lymphoma (CHL), primary mediastinal large cell carcinoma, and other hematological malignancies, such as primary mediastinal large cell carcinoma. Follicular B-cell lymphoma, T-cell / histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, etc., nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasms of the central nervous system (CNS), such as primary CNS lymphoma, spinal axis tumors, brain stem glioma, etc., anal cancer, appendix Cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary gland Cancer, vaginal cancer, thyroid cancer, pharyngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma, or metastases thereof. In a preferred embodiment, the condition or disorder treatable by the methods presented herein comprises one or more solid tumors.

[0372] [000464] In some embodiments, the cancer is a CD47 positive cancer. In some embodiments, the cancer is a CD47 positive and target antigen positive cancer. In some embodiments, the subject to be treated has been identified as having a CD47 positive cancer or a CD47 positive and target antigen positive cancer. "CD47 positive" cancer, as used herein, refers to a cancer characterized by expression of CD47 in the cancer cells or expression of CD47 in the cancer cells at a level significantly higher than expected in normal cells. "Target antigen positive" cancer, as used herein, refers to a cancer characterized by expression of a target antigen in the cancer cells or expression of a target antigen in the cancer cells at a level significantly higher than expected in normal cells.

[0373] [000465] The presence and / or amount of CD47 and target antigen in a biological sample of interest can be determined in a biological test sample from a subject using a variety of suitable methods. For example, the biological test sample can be exposed to an anti-CD47 antibody or anti-target antigen antibody, or an antigen-binding fragment thereof, that binds to and detects the expressed CD47 protein or target antigen protein. Alternatively, CD47 or target antigen protein can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, etc. In some embodiments, the test sample is derived from cancer cells or tissues or tumor-infiltrating immune cells. In certain embodiments, the presence or upregulated levels of CD47 or target antigen protein in the biological test sample indicates a possible response. The term "upregulated" as used herein refers to an overall increase of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more in the expression level of CD47 or target antigen protein contained in a test sample compared to a reference expression level of CD47 or target antigen. The reference level can be the expression level of CD47 or target antigen found in normal cells of the same tissue type, optionally normalized to the expression level of another gene (e.g., a housekeeping gene). Alternatively, the reference level can be the expression level of CD47 or target antigen found in a healthy subject. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample is obtained. For example, the reference sample can be a non-diseased sample adjacent to or located near the test sample (e.g., a tumor). In some embodiments, the reference material is tested and / or determined substantially simultaneously with the testing or determining step of interest, hi some embodiments, the reference material is a history of the reference material, optionally embodied in a tangible medium.Generally, as will be appreciated by one of skill in the art, a reference material is determined or characterized under conditions or circumstances comparable to those that are the subject of the assessment.

[0374] [000466] In certain embodiments, the tumors and cancers are metastatic tumors, particularly metastatic tumors that express CD47. [000467] In certain embodiments, the tumors and cancers are PD-L1 positive cancers. In certain embodiments, the PD-L1 positive cancers are selected from the group consisting of NSCLC, SCLC, melanoma, head and neck cancer, hepatocellular carcinoma, MSI-H or dMMR cancer, cervical cancer, breast cancer, gastric cancer, classical Hodgkin lymphoma, pancreatic cancer, and urothelial cancer.

[0375] [000468] In certain embodiments, the tumors and cancers are claudin 18.2 positive cancers. In some embodiments, the claudin 18.2 positive cancers are cancers of epithelial origin. In some embodiments, the cancers are gastric cancer, pancreatic cancer, lung cancer, esophageal cancer, ovarian cancer, and metastases thereof.

[0376] [000469] In certain embodiments, conditions or disorders treatable by the methods presented herein include autoimmune diseases. Autoimmune diseases include acquired immune deficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac sprue-herpetiform dermatitis; chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic hematologic malignancies, and chronic inflammatory demyelinating polyneuropathy (CIPD). Thrombocytopenic 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, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, leukemia, and psoriatic arthritis. These include, but are not limited to, Inault phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also known as progressive systemic sclerosis (PSS) or systemic sclerosis (SS)), Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0377] [000470] In certain embodiments, the conditions or disorders treatable by the methods provided herein include infectious diseases, such as chronic viral infections, such as fungal infections, parasitic / protozoan infections, or chronic viral infections, such as malaria, coccidioidomycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candida albicans, erythrocyte sedimentation, and erythrocyte sedimentation. albicans, paracoccidioidomycosis, microsporidiosis, acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, raccoon ascariasis, Chagas' disease, clonorchiasis, cochliomiiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, intestinal enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenosteosis, isosporosis, Katayama fever, leishmaniasis, Lyme disease, yokogawa fluke disease, myiasis, onchocerciasis, lice Infections include parasitism, scabies, schistosomiasis, sleeping sickness, strongyliasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis, helminth infections, and infections with the following viruses: hepatitis B (HBV), hepatitis C (HCV), herpes virus, Epstein-Barr virus, HIV-1, HIV-2, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papillomavirus, adenovirus, Kaposi-West sarcoma-associated herpes virus epidemic, thin ring virus (torque teno virus), human T-cell lymphotropic virus I, human T-cell lymphotropic virus II, Varicella Zoster, JC virus, or BK virus.

[0378] [000471] The therapeutically effective amount of a multispecific molecule as provided herein will depend on a variety of factors known in the art, such as weight, age, medical history, current drug treatments, the subject's health status and potential cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and extent of disease manifestations, etc. Dosages may be proportionally reduced or increased by one skilled in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0379] [000472] In certain embodiments, the multispecific molecules provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In any of these embodiments, the multispecific molecules are administered at a dosage of about 50 mg / kg or less, and in any 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 administered dosage may vary over the course of treatment. For example, in certain embodiments, the initial administered dosage may be greater than subsequent administered dosages. In certain embodiments, the administered dosage may vary over the course of treatment depending on the subject's response.

[0380] [000473] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be administered or several divided doses can be administered chronically.

[0381] [000474] The multispecific molecules disclosed herein may be administered by any route known in the art, such as 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.

[0382] [000475] In some embodiments, the multispecific molecules disclosed herein can be administered alone or in combination with one or more additional therapeutic procedures or agents. For example, the multispecific molecules disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anti-cancer drug.

[0383] [000476] In any of these embodiments, a multispecific molecule 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 any of these embodiments, the multispecific molecule and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, a multispecific molecule that is administered "in combination with" another therapeutic agent need not be administered at the same time as the agent or in the same composition as the agent. A multispecific molecule that is administered before or after another agent is considered to be administered "in combination with" that agent, even if the multispecific molecule and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with the multispecific molecules disclosed herein will be administered according to the schedule published in the product information sheet of the additional therapeutic agent, or according to protocols known in the art, such as those described in the Physicians' Desk Reference, 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)).

[0384] [000477] The disclosure further provides methods of using the multispecific molecules. [000478] In some embodiments, the present disclosure also provides for the use of the multispecific molecules provided herein in the manufacture of a medicament for treating a disease, disorder, or condition that may benefit from inducing phagocytosis of target cells in a subject.

[0385] [000479] The present disclosure is based on the surprising discovery of multispecific molecules (e.g., multispecific antibodies) consisting of anti-SIRP-alpha antibodies or antigen-binding fragments thereof that have special properties, such as, for example, substantially or completely blocking the interaction between SIRP-alpha and CD47; substantially or completely blocking SHP-1 transduction downstream of the interaction between SIRP-alpha and CD47; not inducing phagocytosis of specific target cells when used alone; and / or having the ability to bind to epitopes outside the IgV domain of SIRPα. Such multispecific molecules may be in special configurations as described above, but in addition, the resulting multispecific molecules show unexpectedly high selectivity for inducing an immune response (e.g., phagocytosis) against undesirable cells to eliminate undesirable cells (e.g., cancerous or infected cells).

[0386] [000480] Accordingly, the present disclosure also provides the use of such multispecific molecules as provided herein in the manufacture of a medicament for treating CD47-associated conditions and disorders.

[0387] [000481] The following examples are presented to better illustrate the claimed invention and are not to be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below, whether in whole or in part, fall within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention, but rather merely illustrate specific embodiments falling within the scope of the invention. Those 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 is understood that many variations can be made in the procedures described herein while still remaining within the boundaries of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.

[0388] [000482] EXAMPLES

[0389] [000483] Example 1: Construction and expression of anti-claudin 18.2 / SIRPα bispecific antibody [000484] The principle of SIRPα-based bispecific macrophage engager (BiME) antibodies is shown in Figure 1. BiMEs can specifically kill macrophages by physically cross-linking them with cancer cells, block CD47-SIRPα interaction, remove SHP-1 / 2 inhibition, and engage Fc receptors with macrophages to activate phagocytosis.

[0390] [000485] Anti-claudin 18.2 / SIRPα bispecific antibodies are constructed as anti-claudin 18.2 antibody hu28.H1L2 fused to anti-SIRPα C25 single chain variable fragment (scFv) at the C-terminus of the heavy or light chain (Figures 2A-2B). A flexible (Gly4Ser)3 linker was genetically linked to the N-terminus of the anti-SIRPα scFv. Anti-claudin 18.2 / SIRPα bispecific antibodies are constructed as anti-SIRPα antibody fused to anti-claudin 18.2 single chain variable fragment (scFv) at the C-terminus of the heavy or light chain (Figures 2C-2D). A flexible (Gly4Ser)3 linker was genetically linked to the N-terminus of the anti-claudin 18.2 scFv.

[0391] [000486] Bispecific protein ES028-001 containing one anti-claudin 18.2 antibody and two anti-SIRPα scFvs at the C-terminus of its light chain (Figure 2A, see also Table 7).

[0392] [000487] Bispecific protein ES028-005, comprising one anti-claudin 18.2 antibody and two anti-SIRPα scFvs at the C-terminus of its heavy chain (Figure 2B, see also Table 7).

[0393] [000488] Bispecific protein ES028-009 containing one anti-SIRPα antibody and two anti-claudin 18.2 scFvs at the C-terminus of its light chain (Figure 2C, see also Table 7).

[0394] [000489] Bispecific protein ES028-013 containing one anti-SIRPα antibody and two anti-claudin 18.2 scFvs at the C-terminus of its heavy chain (Figure 2D, see also Table 7).

[0395] [000490] For expression, DNA encoding the light and heavy chains in the same or separate expression vectors was used for transfection into HEK293 cells. The culture medium was harvested and the fusion protein was purified by protein A sepharose column.

[0396] [000491] Example 2 Binding affinity of anti-claudin 18.2 / SIRPα bispecific antibodies [000492] Anti-claudin 18.2 / SIRPα bispecific proteins were characterized for their binding affinity to human claudin 18.2 or SIRPα individually using the Octet assay (ForeBio) according to the manufacturer's manual. Briefly, the antibodies were coupled onto the sensor, and then the sensor was immersed in a claudin 18.2 or SIRPα protein gradient (starting at 200 nM, diluting 2-fold, total of 8 doses). The binding response was measured in real time, and the results were approximated overall. The affinity data of the tested antibodies are summarized in Tables 8 and 9.

[0397] [000493]

[0398] [Table 11]

[0399] [000494]

[0400] [Table 12]

[0401] [000495] Example 3 Binding of anti-claudin 18.2 / SIRPα bispecific antibody to claudin 18.2 and SIRPα by FACS [000496] Approximately 100,000 Raji lymphoma cells overexpressing human claudin 18.2 were washed with wash buffer and incubated with 100 μl of serial dilutions of claudin 18.2 / SIRPα bispecific protein on ice for 30 min. The cells were then washed twice with wash buffer and incubated with 100 μl of anti-human Fc-PE on ice for 30 min. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). As shown in FIG. 3A, anti-claudin 18.2 / SIRPα bispecific antibody bound to Raji / h claudin 18.2 cells in a dose-dependent manner. Bispecific antibodies ES028-001, ES028-005, and ES028-013 bound to Raji / h-claudin-18.2 similarly to the anti-claudin-18.2 monoclonal antibody hu28H1L2, whereas ES028-009 bound to Raji / h-claudin-18.2 but to a lesser extent than anti-claudin-18.2, hu28H1L2.

[0402] [000497] CHO-K1 cells overexpressing human SIRPα were washed with wash buffer and incubated with 100 μl of serial dilutions of claudin18.2 / SIRPα bispecific protein on ice for 30 min. The cells were then washed twice with wash buffer and incubated with 100 μl of anti-human Fc-PE on ice for 30 min. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). As shown in Figure 3B, anti-claudin18.2 / SIRPα bispecific antibodies bound to CHO-K1 / SIRPα cells in a dose-dependent manner. ES028-001 and ES028-005 bound to CHO-K1 / hSIRPα but to a lesser extent compared to anti-SIRPα,C25.

[0403] [000498] Example 4 Anti-claudin 18.2 / SIRPα bispecific antibody enhances macrophage phagocytosis of claudin 18.2+ cancer cells in vitro [000499] Mouse MC38 colon tumor cells expressing human CD47 and human claudin 18.2 were labeled with the fluorescent dye CFSE and incubated with mouse bone marrow derived macrophages (BMDM) prepared from C57BL6 / hCD47 / hSIRPα knock-in mice in the presence of either an isotype control, an anti-claudin 18.2 antibody, an anti-SIRPα antibody, a combination of anti-claudin 18.2 and anti-SIRPα antibodies, or an anti-claudin 18.2 / SIRPα bispecific antibody. After 2 hours, macrophages were harvested, stained with a fluorescently labeled anti-mouse macrophage antibody, and analyzed by flow cytometry. CD11b+CFSE+ double positive events identify macrophages that have phagocytosed CFSE-labeled tumor cells. Each sample is represented by a different color. Phagocytosis index is shown for three different samples.

[0404] [000500] As shown in Figure 4A, anti-claudin 18.2 antibody hu28H1L2 induced about 25% phagocytosis by antibody-dependent cellular phagocytosis (ADCP), while anti-SIRPα antibody C25 induced only a small amount of phagocytosis. The combination of anti-claudin 18.2 and anti-SIRPα antibody C25 significantly improved phagocytosis. Anti-claudin 18.2 / SIRPα bispecific antibodies ES028-001, ES028-005, ES028-009 induced stronger phagocytosis in a dose-dependent manner compared to the combination, and ES028-005 showed the best phagocytosis effect, while ES028-013 showed no synergistic phagocytosis effect. In the case of MC38 cells, which do not express claudin 18.2, the combination of anti-claudin 18.2 with the anti-SIRPα antibody C25 did not significantly improve phagocytosis on non-claudin 18.2-expressing cells (Figure 4B). Neither the combination of anti-claudin 18.2 / SIRPα bispecific antibody nor the combination of anti-claudin 18.2 with anti-SIRP alpha antibody induced an improvement in phagocytosis on cells that do not express claudin 18.2 (Figure 4B).

[0405] [000501] Example 5 Active IgG1 Fc of anti-claudin18.2 / SIRPα bispecific antibody activates FcγR-dependent ADCP and provides stronger efficacy than IgG4 or IgG1LALA isotypes.

[0406] [000502] In another mouse BMDM phagocytosis assay of MC38 / hCD47 / hClaudin18.2, anti-Claudin18.2 antibodies with different isotypes, IgG1, IgG4, and IgG1LALA, were tested as single agent treatments or in combination with anti-SIRPα antibodies. Similarly, anti-Claudin18.2 / SIRPα bispecific antibodies ES028-001, ES028-005, and ES028-009, with IgG1, IgG4, and IgG1LALA, were also tested for phagocytosis.

[0407] [000503] After 2 hours of co-culture, macrophages were harvested, stained with fluorescently labeled anti-human macrophage antibody, and analyzed by flow cytometry. CD11b+CFSE+ double positive events identify macrophages that have phagocytosed CFSE-labeled tumor cells. Each sample is represented by a different color. Phagocytosis index is shown for three different samples.

[0408] [000504] As shown in Figure 5, anti-claudin 18.2 IgG1 showed the best phagocytosis compared to anti-claudin 18.2 IgG4 and anti-claudin 18.2 IgG1 LALA mutant, both as single agent treatment or in combination with anti-SIRPα antibody. The anti-claudin 18.2 / SIRPα bispecific antibodies ES028-001 IgG1, ES028-005 IgG1, and ES028-009 IgG1 induced a stronger phagocytosis effect compared to ES028-001 IgG4, ES028-005 IgG4, and ES028-009 IgG4, while no phagocytosis efficacy was observed for the anti-claudin 18.2 / SIRPα bispecific antibodies ES028-001 IgG1 LALA, ES028-005 IgG1 LALA, and ES028-009 IgG 1LALA. The data showed that the anti-claudin 18.2 / SIRPα bispecific antibody IgG1 activated FcγR-dependent ADCP and provided stronger efficacy than the IgG4 or IgG1 LALA isotypes.

[0409] [000505] Example 6 Construction and expression of anti-PDL1 / SIRPα bispecific antibodies [000506] Anti-PDL1 / SIRPα bispecific antibodies were constructed as anti-SIRPα antibody C25 fused to anti-PDL1 single domain antibody (sdAb) C71 or C570 at the N-terminus or C-terminus of the heavy or light chain (see also Figures 6A-6D, Table 7). Anti-PDL1 / SIRPα bispecific antibodies can also be constructed as anti-SIRPα Fab in one arm and two copies of anti-PDL1 sdAb in the other arm linked by knob-in-hole (KiH) in the Fc region (see also Figure 6E, Table 7). Anti-PDL1 / SIRPα bispecific antibodies can also be constructed as anti-PDL1 sdAb at the N-terminus of Fc and anti-SIRPα Fab fused to the C-terminus of Fc (see also Figures 6F-6G, Table 7). A flexible (Gly4Ser)3 linker was genetically linked to the N-terminus of the anti-SIRPα Fab.

[0410] [000507] Bispecific protein ES019-020 containing one anti-SIRPα antibody and two anti-PDL1 sdAbs at the C-terminus of its light chain (Figure 6A, see also Table 7). [000508] Bispecific protein ES019-024 containing one anti-SIRPα antibody and two anti-PDL1 sdAbs at the C-terminus of its heavy chain (Figure 6B, see also Table 7).

[0411] [000509] Bispecific protein ES019-025 containing one anti-SIRPα antibody and two anti-PDL1 sdAbs at the N-terminus of its heavy chain (Figure 6C, see also Table 7). [000510] Bispecific protein ES019-026 containing one anti-SIRPα antibody and two anti-PDL1 sdAbs at the N-terminus of its light chain (Figure 6D, see also Table 7).

[0412] [000511] Bispecific protein ES019-029 containing a heterodimer of one anti-SIRPα Fab arm and two anti-PDL1 sdAb arms formed by knob-in-hole mutations in the Fc region (Figure 6E, see also Table 7).

[0413] [000512] Bispecific protein ES019-072 containing two anti-PDL1 sdAbs at the N-terminus of the Fc and one copy of an anti-SIRPα Fab at the C-terminus of the Fc region. The asymmetric heterodimer is formed by knob-in-hole mutations in the Fc region (Figure 6F, see also Table 7).

[0414] [000513] Bispecific proteins ES019-073 or ES019-079 containing two anti-PDL1 sdAbs at the N-terminus of the Fc and two anti-SIRPα Fabs at the C-terminus of the Fc region (Figure 6G, see also Table 7).

[0415] [000514] Example 7: Binding affinity of anti-PDL1 / SIRPα bispecific antibodies [000515] Anti-PDL1 / SIRPα bispecific proteins were individually characterized for binding affinity to human PDL1 using the Octet assay (ForeBio) according to the manufacturer's manual. Briefly, the antibodies were coupled onto the sensor, and then the sensor was immersed in a PDL1 protein gradient (starting at 200 nM, with 2-fold dilutions, for a total of 8 doses). The binding response was measured in real time, and the results were approximated overall. The affinity data of the tested antibodies are summarized in Table 9.1.

[0416] [Table 13]

[0417] [000516] Example 8: Binding of anti-PDL1 / SIRPα bispecific antibodies to PDL1 and SIRPα by FACS [000517] Approximately 100,000 Raji cells overexpressing human PDL1 were washed with wash buffer and incubated with 100 μl of serial dilutions of PDL1 / SIRPα bispecific protein on ice for 30 min. The cells were then washed twice with wash buffer and incubated with 100 μl of anti-human Fc-PE on ice for 30 min. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). As shown in FIG. 7A, anti-PDL1 / SIRPα bispecific antibody bound to Raji / hPDL1 cells in a dose-dependent manner. The bispecific antibodies ES019-025 and ES019-026 bound to Raji / hPDL1 similarly to the anti-PDL1 monoclonal antibody C71, whereas ES019-020 and ES019-024 bound to Raji / hPDL1, but to a lower extent than anti-PDL1 and C71.

[0418] [000518] CHO-K1 cells overexpressing human SIRPα were washed with wash buffer and incubated with 100 μl of serial dilutions of PDL1 / SIRPα bispecific protein on ice for 30 min. The cells were then washed twice with wash buffer and incubated with 100 μl of anti-human Fc-PE on ice for 30 min. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). As shown in FIG. 7B, anti-PDL1 / SIRPα bispecific antibodies bound to CHO-K1 / SIRPα cells in a dose-dependent manner. ES019-020, ES019-024, ES019-025, and ES019-026 bound to CHO-K1 / hSIRPα similarly to anti-SIRPα,C25.

[0419] [000519] Example 9: PDL1 blocking activity of anti-PDL1 / SIRPα bispecific antibodies by Jurkat / PD1 reporter cell assay [000520] PD1-PDL1 blockade reporter assay was performed using the PD1 blockade reporter assay developed by GenScript (using two cell lines based on CHO cells expressing PDL1 and anti-CD3 scFv, and Jurkat / NFAT-RE reporter cell line overexpressing PD1). Cells growing in logarithmic growth phase were harvested and resuspended in RPMI1640 20 containing 1% heat-inactivated FBS at a concentration of 2x106 cells / ml for CHO and 4x106 cells / ml for Jurkat / PD1 NFAT-RE cells. Then, serial dilutions of test antibodies in assay medium (RPMI1640 containing 1% FBS) were added to each well. Plates were incubated at 37°C, 5% CO2, 95% relative humidity for 6 hours. Luciferase (Bio-Glo Luciferase Assay System), 40 μl, was added the next day and the amount of luciferase activity was measured using a BioTek multi-mode microplate reader.

[0420] [000521] As shown in Figure 8, bispecific antibodies ES019-024, ES019-025, ES019-026 activate Jurkat / PD1 reporter cells similarly to anti-PDL1 monoclonal antibody C71, while ES019-020 activates Jurkat / PD1 reporter cells but to a lesser extent compared to anti-PDL1, C71.

[0421] [000522] Example 10: Anti-PDL1 / SIRPα bispecific antibodies enhance macrophage phagocytosis of PDL1+ cancer cells in vitro [000523] Human K562 leukemia tumor cells lacking expression of human PDL1 or K562 expressing human PDL1 were labeled with the fluorescent dye CFSE and incubated with human macrophage-colony stimulating factor (M-CSF)-treated monocyte-derived macrophages in the presence of either an isotype control, an anti-PDL1 antibody, an anti-SIRPα antibody, a combination of anti-PDL1 and anti-SIRPα antibodies, or an anti-PDL1 / SIRPα bispecific antibody. After 2 hours, macrophages were harvested, stained with fluorescently labeled anti-human macrophage antibody, and analyzed by flow cytometry. CD11b+CFSE+ double positive events identify macrophages that have phagocytosed CFSE-labeled tumor cells. Each sample is represented by a different color. Phagocytosis index is shown for three different samples.

[0422] [000524] As shown in Figure 9A, anti-PDL1 antibody C71 induced approximately 20% phagocytosis by antibody-dependent cellular phagocytosis (ADCP), while anti-SIRPα antibody C25 induced only slight phagocytosis. The combination of anti-PDL1 and anti-SIRPα antibody C25 significantly improved phagocytosis. Anti-PDL1 / SIRPα bispecific antibodies ES019-020, ES019-029 induced potent phagocytosis in a dose-dependent manner, as in the combination, while ES019-024, ES019-025, and ES019-026 did not show synergistic phagocytosis effect.

[0423] [000525] As shown in Figure 9B, anti-PDL1 antibody C71 monotherapy or in combination with anti-SIRPα antibody C25 induces negligible phagocytosis in K562 parental cells lacking PDL1 expression. Anti-PDL1 / SIRPα bispecific antibodies ES019-020, ES019-024, ES019-025, ES019-026, ES019-029 showed no phagocytic effect on K562 cells (PDL1 negative).

[0424] [000526] As shown in Figure 9C, anti-PDL1 antibody C71 monotherapy or in combination with anti-SIRPα antibody C25 induces negligible phagocytosis on the human Jurkat T cell line expressing human SIRPγ. Anti-PDL1 / SIRPα bispecific antibodies ES019-020, ES019-024, ES019-025, ES019-026, ES019-029 showed no phagocytic effect on Jurkat T cells (SIRPγ positive).

[0425] [000527] Example 11: Anti-claudin18.2 / SIRPα bispecific antibody enhances macrophage phagocytosis of PDL1+ cancer cells in vitro [000528] Human SIRPa / CD47 double KI mice were inoculated with hCD47 / hCLDN18.2-overexpressing MC38 cells. The mean tumor volume was approximately 70-100 mm. 3 When the tumor volume reached 10 mpk, the mice were grouped into 7 groups based on tumor volume. Mice were administered ip with the same molar concentration of 10 mpk isotype antibody, 10 mpk CLDN18.2 mAb, 10 mpk SIRPa mAb, 10 mpk CLDN18.2 mAb + 10 mpk SIRPa mAb, or 14 mpk ES028-001, ES028-005, ES009. The administration schedule was BIW, 5 doses. Tumor volumes were measured twice per week. Mice were sacrificed 3 days after the 5th dose and tumors were weighed. Statistics were performed by 2-way ANOVA comparing the mean tumor volumes of the different treatment groups with those of the isotype control group.

[0426] [000529] The relative tumor inhibition rate (TGI) (%) was calculated as follows: TGI%=(1-T / C)×100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treated and control groups, respectively, at a given time point. T / C% = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the vehicle control group; RTV=Vt / V0, where V0 is the tumor volume of the animals at the time of grouping and Vt is the tumor volume of the animals after treatment); T / C can be calculated based on tumor weight as follows: T / C%=TTW / CTW×100% (TTW: mean tumor weight of the treatment group at the final time point; CTW: mean tumor weight of the vehicle control group at the final time point).

[0427] [000530] As shown in Figure 10, anti-claudin 18.2 antibody hu28H1L2 and anti-SIRPα antibody C25 monotherapy do not inhibit tumor growth as well as isotype treatment. The combination of anti-claudin 18.2 and anti-SIRPα antibody C25 significantly reduces tumor growth. Anti-claudin 18.2 / SIRPα bispecific antibody ES028-005 induces strong tumor growth inhibition similar to the combination; ES028-001 showed weaker tumor inhibition, and ES028-009 did not inhibit tumor growth.

[0428] [000531] Example 12: BiME based on anti-SIRPα complete blocking antibody provides the best synergistic phagocytosis effect while being selective for tumor cells [000532] Characterization of chimeric antibodies [000533] 1.1 Detection of binding specificity [000534] The binding activity of the purified chimeric antibodies against human SIRPα variants was detected by FACS assay using CHOK1 cells or 293F cells stably expressing human SIRPα v1, and CHOK1 cells stably expressing human SIRPα v2. As shown in Figure 12, all the tested antibodies strongly bind to cell surface human SIRPα v1 and cell surface human SIRPα v2. EC 50 and maximum signal was calculated using GraphPad Prism 9.0.

[0429] [000535] 1.2 Detection of CD47 / SIRPα interaction blocking activity [000536] A competitive ELISA assay was used to determine whether the purified chimeric antibodies could block the interaction of CD47 with SIRPα. Briefly, the antibodies and mFc-tagged human CD47 ECD recombinant protein were co-incubated with human SIRPα v1 ECD or human SIRPα v2 ECD recombinant protein coated on an ELISA microplate. The concentration of soluble ECD of CD47 is 25 nM and the concentration of soluble ECD of SIRPα is 20 nM. After washing, HRP-labeled anti-mouse Fc secondary antibody (Sigma) was added and incubated at 37°C for 1 hour. Then, TMB solution (Biotechnology) was added at 100 μl / well. After incubation at room temperature for 15 minutes, the reaction was stopped by adding 50 μl of 1N HCl. OD450nm was read. The blocking ratio was determined by the blocking rate of binding of human SIRPα ECD recombinant protein to human CD47 ECD recombinant protein coated on an ELISA microplate. IC was calculated using GraphPad Prism 9.0. 50 and the highest blocking rate (%) are summarized in Figure 13. All tested antibodies are able to block the interaction between human CD47 and different human SIRPα variants.

[0430] [000537] 1.2.1 Epitope Analysis [000538] A competitive ELISA assay was used for epitope binning of purified chimeric antibodies. Briefly, excess competing antibodies and mFc-tagged human SIRPα v1 ECD recombinant protein were co-incubated with antibodies coated on ELISA microplates. After washing, HRP-labeled anti-mouse Fc secondary antibody (Sigma) was added and incubated at 37°C for 1 hour. Then, TMB solution (Biotechnology) was added at 100 μl / well. After incubation at room temperature for 15 minutes, the reaction was stopped by adding 50 μl of 1N HCl. OD450nm was read. The competitive ratio was calculated. Antibodies that can compete with each other for binding to SIRPα may have related binding epitopes. As shown in Table B, 025c did not show any competition with 042c, 073c, and hu1H9G4 for binding to human SIRPα, suggesting that it may bind to a different epitope. Competition between 042c, 073c, and hu1H9G4 was not bidirectional, suggesting that their binding epitopes may be related but not completely identical.

[0431] [000539] Epitope mapping of 025c, 042c, 073c, HEFLB, and hu1H9G4 was further performed using hydrogen deuterium exchange mass spectrometry (HDX-MS). As shown in Figure 23A, the binding of 025c resulted in a lower hydrogen deuterium exchange ratio in the region of YNQKEGHFPRVTTVSDL (SEQ ID NO: 218) of His-tagged human SIRPα v1 ECD, suggesting that these amino acids may be crucial for 025c binding. As shown in Figure 23B, the binding of 042c resulted in a lower hydrogen deuterium exchange ratio in two regions of SGAGTEL (SEQ ID NO: 219) and TNVDPVGESVS (SEQ ID NO: 220) of His-tagged human SIRPα v1 ECD, suggesting that these amino acids may be crucial for 042c binding. As shown in Figure 23C, the binding of 073c resulted in a lower hydrogen deuterium exchange ratio in the region of TNVDPVGESVSY (SEQ ID NO: 221) of His-tagged human SIRPα v1 ECD, suggesting that these amino acids may be crucial for 073c binding. Notably, these three regions are not located in the IgV domain of SIRPα ECD where CD47 binds, suggesting that 042c and 073c may act as allosteric antibodies and block the interaction of CD47 with SIRPα, or that the blocking activity of 042c and 073c is a steric hindrance effect. As shown in Figure 23D, the binding of hu1H9G4 resulted in a lower hydrogen deuterium exchange ratio in the region of YNQKEGHFPRVTTVSDL (SEQ ID NO: 218) of His-tagged human SIRPα v1 ECD, suggesting that these amino acids may be crucial for hu1H9G4 binding. As shown in Figure 23E, HEFLB binding resulted in a lower hydrogen-deuterium exchange ratio in the region of VGPIQW (sequence number 222) of his-tagged human SIRPα v1 ECD, suggesting that these amino acids may be crucial for HEFLB binding.

[0432] [000540] Considering the competitive ELISA data and HDX-MS data together, it is concluded that 025c, 042c, and 073c may have different binding epitopes (also different from the reference antibodies hu1H9G4 and HEFLB). In addition, the results indicate that the epitopes of 025c, 042c, and 073c may be outside the IgV domain. The IgV domain is involved in the binding of the extracellular Ig domain of CD47. The sequence information of the reference antibodies is shown in Table A below.

[0433] [000541]

[0434] [Table 14]

[0435] [000542]

[0436] [Table 15]

[0437] [000543] 1.3 SHP-1 transduction assay [000544] The efficacy of purified chimeric antibodies in blocking CD47 / SIRPα-mediated "don't eat me" signaling was assessed by a cell-based SHP-1 transduction assay. Full-length human SIRPα v1 was engineered with a small β-gal fragment (ED) fused to its C-terminus, and the SH2-domain of SHP-1 was engineered with a complementary β-gal fragment (EA). These constructs were stably expressed in K562 cells. Ligand engagement through co-culture with human CD47-expressing cells results in phosphorylation of the SIRPα-ED fusion protein, leading to transduction of SHP-1-EA, which generates the active β-gal enzyme. This active enzyme hydrolyzes the substrate and produces chemiluminescence as an indicator of reporter activity. IC 50and maximum blocking rate (%) were calculated using GraphPad Prism 9.0. As summarized in Figure 14, all tested antibodies can disrupt CD47 / SIRPα-mediated "don't eat me" signaling at different levels.

[0438] [000545] 1.4 In vitro phagocytosis assay [000546] The functional efficacy of purified chimeric antibodies was evaluated by a flow cytometry-based phagocytosis assay. Briefly, M0 non-polarized or M1 polarized human monocyte-derived macrophages with different SIRPA genotypes were co-cultured with CellTrace Violet (Life technologies) labeled CD47 expressing cancer cells in the presence of the antibody being tested. Phagocytosis was assayed by determining the percentage of macrophages positive for CellTrace Violet dye. For non-polarized macrophages, peripheral blood mononuclear cells were seeded in 10 cm tissue culture plates in 1640 medium supplemented with 10% FBS and 50 ng / ml M-CSF for 7-9 days. Adherent cells were harvested as M0 non-polarized macrophages. For M1 polarized macrophages, peripheral blood mononuclear cells were seeded in 10 cm tissue culture plates in 1640 medium supplemented with 10% FBS and 50 ng / ml GM-CSF for 5 days. 50 μg / ml IFNγ and 100 μg / ml LPS were added to the cultures for an additional 2-4 days. Adherent cells were harvested as M1 polarized macrophages.

[0439] [000547] As shown in Figure 20A, 015c, 025c, 042c, 059c, and 073c did not show single agent activity in enhancing tumor cell uptake of Raji cells by M0 macrophages obtained from SIRPA heterozygous v1 / v2 individuals. However, in the presence of rituximab (anti-CD20 antibody), all other purified chimeric antibodies tested, except for 059c, which has weaker activity in blocking the interaction between human CD47 and human SIRPα v2, enhanced macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) of Raji cells.

[0440] [000548] Combinations of SIRPα and PD-L1 antibodies were tested in phagocytosis assays using M0 macrophages obtained from SIRPA homozygous v1 / v1 (Figure 20B) and v2 / v2 (Figure 20C) individuals. In the presence of PD-L1 antibodies, 025c, 042c, and 073c effectively enhanced macrophage-mediated ADCP of Raji cells stably expressing PD-L1.

[0441] [000549] Characterization of humanized antibodies [000550] 1.5 Detection of binding specificity [000551] All humanized antibodies tested were confirmed to retain similar activity as the C25 parent antibody and bind to SIRP family members. EC calculated using GraphPad Prism 9.0 50 and maximum signals are summarized in Figure 15. Thus, all humanized antibodies presented herein (e.g., hu025.021, hu025.033, hu025.023, hu025.059, hu025.060, hu26.H1L1, hu26.H1L2, hu26.H1L2(S92A), C71, C71v38, C239, C492, C570, 570h3, C446, C2811, C1778, C1793, C2855, C2713, and C2719) can be expected to have similar activity, respectively, to the corresponding parent antibody.

[0442] [000552] 1.5.1 Affinity Detection [000553] The humanized antibodies were characterized for their binding affinity to human SIRPα v1, human SIRPα v2 using surface plasmon resonance technology (Biacore system). The association and dissociation curves were fitted to a 1:1 binding model, and the Ka / Kd / KD values ​​for each antibody were calculated. The affinity data of Ka / Kd / KD values ​​for each antibody are summarized in Figure 16.

[0443] [000554] 1.7 Detection of CD47 / SIRPα interaction blocking activity [000555] The humanized antibodies were tested for their ability to block the interaction of CD47 with SIRPα using a competitive ELISA assay (see methods above). As shown in Figure 17, all humanized antibodies tested were confirmed to retain similar activity to the C25 parent antibody and to block the interaction between human CD47 and several different human SIRPα variants. IC calculated using GraphPad Prism 9.0 50 and the maximum blocking rate (%) are summarized in FIG.

[0444] [000556] To further compare the blocking activity of the humanized antibody and some known anti-SIRPα antibodies, a competitive FACS assay was also performed. Briefly, the antibody and mFc-tagged human CD47ECD recombinant protein were co-incubated with CHOK1 cells stably expressing human SIRPα v1 or human SIRPα v2. After washing, a dye-labeled anti-mouse Fc secondary antibody (Sigma) was added and incubated at 37°C for 1 hour. The fluorescence intensity was detected. The blocking ratio was determined by the blocking rate of the binding of human CD47ECD recombinant protein to SIRPα expressed by CHOK1 cells. The IC was calculated using GraphPad Prism 9.0. 50 and the maximum blocking rate (%) are summarized in FIG.

[0445] [000557] 1.8 SHP-1 transduction assay [000558] The efficacy of the humanized antibodies to block CD47 / SIRPα-mediated "don't eat me" signaling was evaluated using a cell-based SHP-1 transduction assay (see methods above). All humanized antibodies tested were found to retain activity similar to the C25 parent antibody and to block CD47-mediated SHP-1 transduction to the SIRPα intracellular tail. IC calculated using GraphPad Prism 9.0 50 and the maximum blocking rate (%) are summarized in FIG.

[0446] [000559] 1.9 In vitro phagocytosis assay [000560] For in vitro functional validation, combinations of SIRPα antibodies with PD-L1 antibodies or rituximab were tested in phagocytosis assays using M0 macrophages obtained from SIRPA homozygous v1 / v1 (Figures 21A and 21B) and v2 / v2 (Figures 21C and 21D) individuals. All humanized antibodies tested were found to retain activity similar to the 025c parent antibody and enhance macrophage-mediated ADCP of Raji cells stably expressing PD-L1 in the presence of PD-L1 antibodies or rituximab.

[0447] [000561] 2. Categorization of anti-SIRPα antibodies into full blockers, partial blockers, and non-blockers [000562] A competitive ELISA assay was used to determine whether the purified chimeric antibody could block the interaction of CD47 with SIRPα. Briefly, the antibody and mFc-tagged human CD47 ECD recombinant protein were co-incubated with human SIRPα v1 ECD recombinant protein coated on an ELISA microplate. After washing, HRP-labeled anti-mouse Fc secondary antibody (Sigma) was added and incubated at 37°C for 1 hour. Then, TMB solution (Biotechnology) was added at 100 μl / well. After incubation at room temperature for 15 minutes, the reaction was stopped by adding 50 μl of 1N HCl. OD450nm was read. The blocking ratio was determined by the blocking rate of binding of human SIRPα ECD recombinant protein to human CD47 ECD recombinant protein coated on an ELISA microplate. FIG. 22 shows that 025 or C25 has a maximum blocking percentage (%) greater than 90% and is defined as a full blocker, 050 or C50 has a maximum blocking percentage (%) close to zero and is defined as a non-blocker, and 035 or C35 has an intermediate maximum blocking percentage (%) and is defined as a partial blocker (Table 10).

[0448] [000563]

[0449] [Table 16]

[0450] [000564] 3. Combination therapy and bispecific molecules using full blockers C25, C15, C42, C59, and C73, partial blocker C35, and non-blocker C50 [000565] Different CD47 and SIRPα blocking antibodies were tested on human Raji lymphoma cells lacking human PDL1 expression or Raji expressing human PDL1, labeled with the fluorescent dye CFSE and incubated with monocyte-derived macrophages treated with human macrophage-colony stimulating factor (M-CSF) in the presence of either an isotype control, an anti-PDL1 antibody, anti-SIRPα antibodies C25, C35, C50, a combination of anti-PDL1 and SIRPα antibodies, or an anti-PDL1 / SIRPα bispecific antibody based on C25, C35, C50. After 2 hours, macrophages were harvested, stained with fluorescently labeled anti-human macrophage antibodies, and analyzed by flow cytometry. CD11b+CFSE+ double positive events identify macrophages that have phagocytosed CFSE-labeled tumor cells. Each sample is represented by a different color. Phagocytosis index is shown for three different samples.

[0451] [000566] As shown in Figure 11A, anti-SIRPα partial blocking antibody C35 and non-blocking antibody C50 induced about 40% phagocytosis in Raji / PDL1 cells by single treatment; single treatment with anti-SIRPα complete antibody C25 induced only slight phagocytosis. BiME based on the combination of anti-PDL1 and anti-SIRPα antibody C25 or C25 significantly improved phagocytosis. The combination of anti-PDL1 and anti-SIRPα antibody C35, C50, or BiME based thereon did not show synergistic phagocytosis effect.

[0452] [000567] In the phagocytosis assay for Raji (PDL1 negative) cells (Figure 11B), single-agent treatment with anti-SIRPα partial blocking antibody C35 and non-blocking antibody C50 can still induce phagocytosis for Raji (PDL1 negative) cells; single-agent treatment with anti-SIRPα complete antibody C25 induces phagocytosis only slightly. PDL1 / SIRPα bispecific antibodies based on the combination of anti-PDL1 and anti-SIRPα antibody C25 or C25 did not induce phagocytosis due to lack of PDL1 expression on tumor cells. BiME based on the combination of anti-PDL1 and anti-SIRPα antibody C35 or C50 showed a phagocytosis effect similar to that of single-agent treatment with C35 or C50 antibody.

[0453] [000568] The results revealed that monotherapy, combination therapy, or bispecific antibodies with anti-SIRPα antibodies C35 or C50 can induce tumor cell phagocytosis independent of target antigen expression compared to C25 antibody. The properties of bispecific antibodies based on anti-SIRPα antibody C25 provide more specific and safety properties.

[0454] [000569] The experimental data is summarized in Table 11 below. [000570]

[0455] [Table 17-1]

[0456] [Table 17-2]

[0457] [000571] As shown in Example 4, BiME based on a combination of anti-claudin 18.2 and anti-SIRPα antibody C25 or C25 also significantly and selectively improves phagocytosis of claudin 18.2-expressing cells over non-claudin 18.2-expressing cells.

[0458] [000572] Additionally, as shown in Figures 20A and 21A, the combination of C15, C42, C59, or C73 with an antibody targeting a target antigen (e.g., PD-L1, claudin 18.2) is expected to have selective phagocytosis of cells expressing the target antigen (e.g., PD-L1, claudin 18.2) over cells not expressing the target antigen. The full blockers C15, C42, C59, and C73 can also be used to generate multispecific molecules provided in the form of, for example, ES028-001, ES028-005, ES028-009, ES028-013, ES019-020, ES019-024, ES019-025, ES019-026, ES019-029, ES019-072, ES019-073, and ES019-079, and would be expected to have similar selective phagocytosis towards cells expressing the target antigen (e.g., PD-L1, claudin 18.2) over cells that do not express the target antigen.

[0459]

[0004] Although macrophages are exemplified throughout this specification, the multispecific molecules, compositions, and methods described herein are applicable to cells of myeloid lineage, such as dendritic cells, etc. Minor optimizations and modifications are contemplated on a cell-by-cell basis, as known to those of skill in the art, and are considered to be within the scope of this disclosure.

Claims

1. (a) an SIRP-alpha binding domain, (b) an activating receptor binding domain, (c) a target antigen binding domain that binds to a target antigen expressed on a target cell that co-expresses a target antigen and CD47, A multispecific molecule comprising: The activating receptor binding domain comprises an Fc domain, and optionally, the Fc domain is derived from IgG1 or IgG4, The target antigen binding domain comprises a target antigen binding antibody domain, The SIRP-alpha binding domain comprises an SIRP-alpha binding antibody domain, The multispecific molecule selectively induces an effector function of an immune effector cell in the presence of a target antigen and co-expresses SIRP-alpha and an activating receptor, The above multispecific molecule.

2. The multispecific molecule according to claim 1, wherein the target antigen binding antibody domain is linked to the N-terminus of the Fc domain.

3. The multispecific molecule according to claim 2, wherein the target antigen binding antibody domain comprises a Fab domain or a VHH domain, and optionally, the Fab domain or the VHH domain is linked to one of the N-termini of the Fc domain.

4. The multispecific molecule according to claim 2, wherein the multispecific molecule comprises two target antigen binding antibody domains, each of which comprises a Fab domain or a VHH domain, and optionally, each of the Fab domains or VHH domains comprises a heavy chain linked to each N-terminus of the Fc domain.

5. The multispecific molecule according to claim 1, wherein the SIRP-alpha binding antibody domain is linked to the Fc domain or the target antigen binding antibody domain.

6. (a) The SIRP-alpha binding antibody domain is linked to the C-terminus of the Fc domain, and optionally, the SIRP-alpha binding antibody domain comprises a Fab domain; (b) The SIRP-alpha binding antibody domain is linked to the N-terminus of the Fc domain, provided that the SIRP-alpha binding domain and the target antigen binding antibody domain are not linked to the same N-terminus of the Fc domain; or (c) The SIRP-alpha binding domain is linked to the C-terminus of the light chain of the target antigen binding Fab domain; The multispecific molecule according to claim 5.

7. The multispecific molecule according to claim 1, wherein the SIRP-alpha binding antibody domain is linked to the N-terminus of the Fc domain.

8. The multispecific molecule according to claim 1, wherein the SIRP-alpha binding antibody domain comprises a Fab domain, and optionally, the Fab domain is linked to one of the N-termini of the Fc domain.

9. The multispecific molecule according to claim 1, wherein the antibody comprises two SIRP-alpha binding antibody domains, each of which comprises a Fab domain, and optionally, each of the Fab domains comprises a heavy chain linked to each N-terminus of the Fc domain.

10. The multispecific molecule according to claim 1, wherein the target antigen binding domain is linked to the Fc domain or the SIRP-alpha binding antibody domain.

11. a) The target antigen binding domain is linked to the N-terminus of the Fc domain, provided that the target antigen binding domain and the SIRP-alpha binding domain are not linked to the same N-terminus of the Fc domain; or b) The target antigen binding antibody domain is linked to the C-terminus of the light chain of the SIRP-alpha binding Fab domain; The multispecific molecule according to claim 10.

12. The SIRP-alpha binding antibody domain comprises a) X １ HCDR1, RIDPEDX containing the YYMH sequence (SEQ ID NO: 161) ２ EX ３ HCDR2 containing the KYAPKFQG sequence (SEQ ID NO: 162), and GX １５ X ４ X ５ HCDR3 containing the Y sequence (SEQ ID NO: 163), and / or LCDR1 containing the SASSSSVSSSYLY sequence (SEQ ID NO: 26), LCDR2 containing the STSNLAS sequence (SEQ ID NO: 27), and X ６ LCDR3 containing the QWSSYPYT sequence (SEQ ID NO: 164), or b) HCDR1 containing the sequence of TYGMSS (SEQ ID NO: 35), WINTYSGVX ７ TX ８ HCDR2 containing the sequence of ADDFKGS (SEQ ID NO: 165), and DPHX ９ YGX １０ SPAWFX １１ HCDR3 containing the sequence of Y (SEQ ID NO: 166), and / or X １２ ASQX １３ VGIX １４ LCDR1 containing the sequence of VA (SEQ ID NO: 188), LCDR2 containing the sequence of SASNRYT (SEQ ID NO: 39), and QQYSX １６ YPX １７ LCDR3 containing the sequence of T (SEQ ID NO: 189), or c) HCDR1 comprising the sequence of EYVLS (SEQ ID NO: 41), HCDR2 comprising the sequence of EIYPGTITTYYNEEKFKGG (SEQ ID NO: 42), and HCDR3 comprising the sequence of FYDYDGGWFAY (SEQ ID NO: 43), and / or LCDR1 comprising the sequence of SASSSSVSSSDLH (SEQ ID NO: 44), LCDR2 comprising the sequence of GTSNLAS (SEQ ID NO: 45), and LCDR3 comprising the sequence of QQWSGYPW T (SEQ ID NO: 46) comprising, wherein X １ is A or D, X ２ is G or A, X ３ is T or S, X ４ is L or Y, X ５ is E or A, X ６ is Y or H, X ７ is S or P, X ８ is Y or C, X ９ is Y or S, X １０ is N or S, X １１ is P or V, X １２ is E or K, X １３ is N or I, X １４ is S or A, X １５ is S or does not exist, X １６ is S or A, X １７ is F or L The multispecific molecule according to claim 1.

13. The SIRP-alpha binding antibody domain comprises a) HCDR1 comprising the sequence of SEQ ID NO: 23, HCDR2 comprising the sequence of SEQ ID NO: 24 or SEQ ID NO: 198, and HCDR3 comprising the sequence of SEQ ID NO: 25, and / or LCDR1 comprising the sequence of SEQ ID NO: 26, LCDR2 comprising the sequence of SEQ ID NO: 27, and LCDR3 comprising the sequence of SEQ ID NO: 28, or b) HCDR1 comprising the sequence of SEQ ID NO: 29, HCDR2 comprising the sequence of SEQ ID NO: 30, and HCDR3 comprising the sequence of SEQ ID NO: 31, and / or LCDR1 comprising the sequence of SEQ ID NO: 32, LCDR2 comprising the sequence of SEQ ID NO: 33, and LCDR3 comprising the sequence of SEQ ID NO: 34, or c) an HCDR1 comprising the sequence of SEQ ID NO: 35, an HCDR2 comprising the sequence of SEQ ID NO: 36, and an HCDR3 comprising the sequence of SEQ ID NO: 37, and / or an LCDR1 comprising the sequence of SEQ ID NO: 38, an LCDR2 comprising the sequence of SEQ ID NO: 39, and an LCDR3 comprising the sequence of SEQ ID NO: 40, or d) an HCDR1 comprising the sequence of SEQ ID NO: 47, an HCDR2 comprising the sequence of SEQ ID NO: 48, and an HCDR3 comprising the sequence of SEQ ID NO: 49, and / or an LCDR1 comprising the sequence of SEQ ID NO: 50, an LCDR2 comprising the sequence of SEQ ID NO: 51, and an LCDR3 comprising the sequence of SEQ ID NO: 52 The multispecific molecule according to claim 12, comprising

14. The SIRP-alpha binding antibody domain comprises the same HCDRs and LCDRs as an anti-SIRP-alpha antibody selected from the group consisting of C25, C15, C42, C59, and C73, a) C25 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 1 and / or a light chain variable region comprising the sequence of SEQ ID NO: 2, b) C15 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 11 and / or a light chain variable region comprising the sequence of SEQ ID NO: 12, c) C42 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the sequence of SEQ ID NO: 14, d) C59 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 15 and / or a light chain variable region comprising the sequence of SEQ ID NO: 16, and e) C73 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and / or a light chain variable region comprising the sequence of SEQ ID NO: 18, The multispecific molecule according to claim 1.

15. The SIRP-alpha binding antibody domain is a) a heavy chain variable region comprising the sequence of SEQ ID NO: 1 and / or a light chain variable region comprising the sequence of SEQ ID NO: 2, or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 3 and / or a light chain variable region comprising the sequence of SEQ ID NO: 4, or c) a heavy chain variable region comprising the sequence of SEQ ID NO: 5 and / or a light chain variable region comprising the sequence of SEQ ID NO: 6, or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and / or a light chain variable region comprising the sequence of SEQ ID NO: 8, or e) a heavy chain variable region comprising the sequence of SEQ ID NO: 9 and / or a light chain variable region comprising the sequence of SEQ ID NO: 10, or f) a heavy chain variable region comprising the sequence of SEQ ID NO: 11 and / or a light chain variable region comprising the sequence of SEQ ID NO: 12, or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and / or a light chain variable region comprising the sequence of SEQ ID NO: 14, or h) A heavy chain variable region containing the sequence of SEQ ID NO: 15, and / or a light chain variable region containing the sequence of SEQ ID NO: 16, or i) A heavy chain variable region containing the sequence of SEQ ID NO: 17, and / or a light chain variable region containing the sequence of SEQ ID NO: 18, or j) A heavy chain variable region containing the sequence of SEQ ID NO: 159, and / or a light chain variable region containing the sequence of SEQ ID NO: 160 The multispecific molecule according to claim 1, comprising the same.

16. The multispecific molecule according to claim 1, wherein the target antigen binding domain comprises a Claudin 18.2 binding domain.

17. The Claudin 18.2 binding domain is a) An HCDR1 containing the sequence of SEQ ID NO: 77, an HCDR2 containing the sequence of SEQ ID NO: 78, and an HCDR3 containing the sequence of SEQ ID NO: 79, and / or an LCDR1 containing the sequence of SEQ ID NO: 80, an LCDR2 containing the sequence of SEQ ID NO: 81, and an LCDR3 containing the sequence of SEQ ID NO: 82 or SEQ ID NO: 225, or b) An HCDR1 containing the sequence of SEQ ID NO: 83, an HCDR2 containing the sequence of SEQ ID NO: 84, and an HCDR3 containing the sequence of SEQ ID NO: 85, and / or an LCDR1 containing the sequence of SEQ ID NO: 86, an LCDR2 containing the sequence of SEQ ID NO: 87, and an LCDR3 containing the sequence of SEQ ID NO: 88, or c) An HCDR1 containing the sequence of SEQ ID NO: 89, an HCDR2 containing the sequence of SEQ ID NO: 90, and an HCDR3 containing the sequence of SEQ ID NO: 91, and / or an LCDR1 containing the sequence of SEQ ID NO: 92, an LCDR2 containing the sequence of SEQ ID NO: 93, and an LCDR3 containing the sequence of SEQ ID NO: 94, or d) An HCDR1 containing the sequence of SEQ ID NO: 95, an HCDR2 containing the sequence of SEQ ID NO: 96, and an HCDR3 containing the sequence of SEQ ID NO: 97, and / or an LCDR1 containing the sequence of SEQ ID NO: 98, an LCDR2 containing the sequence of SEQ ID NO: 99, and an LCDR3 containing the sequence of SEQ ID NO: 100, or e) An HCDR1 containing the sequence of SEQ ID NO: 101, an HCDR2 containing the sequence of SEQ ID NO: 102, and an HCDR3 containing the sequence of SEQ ID NO: 103, and / or an LCDR1 containing the sequence of SEQ ID NO: 104, an LCDR2 containing the sequence of SEQ ID NO: 105, and an LCDR3 containing the sequence of SEQ ID NO: 106 The multispecific molecule according to claim 16, comprising the same.

18. The Claudin 18.2 binding domain comprises the same HCDRs and LCDRs as an anti-Claudin 18.2 antibody selected from the group consisting of hu26.H1L1, hu26.H1L2(S92A), hu28.H1L2, C10, C29, and C30, a) hu26.H1L1 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 65 and / or a light chain variable region comprising the sequence of SEQ ID NO: 66, b) hu26.H1L2(S92A) comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 65 and / or a light chain variable region comprising the sequence of SEQ ID NO: 224, c) hu28.H1L2 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 69 and / or a light chain variable region comprising the sequence of SEQ ID NO: 70, d) C10 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 71 and / or a light chain variable region comprising the sequence of SEQ ID NO: 72, e) C29 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 73 and / or a light chain variable region comprising the sequence of SEQ ID NO: 74, and f) C30 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the sequence of SEQ ID NO: 76, The multispecific molecule according to claim 16.

19. The Claudin 18.2 binding domain is a) a heavy chain variable region comprising the sequence of SEQ ID NO: 65 or 68 and / or a light chain variable region comprising the sequence of SEQ ID NO: 66 or 67 or 224, or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 69 and / or a light chain variable region comprising the sequence of SEQ ID NO: 70, or c) a heavy chain variable region comprising the sequence of SEQ ID NO: 71 and / or a light chain variable region comprising the sequence of SEQ ID NO: 72, or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 73 and / or a light chain variable region comprising the sequence of SEQ ID NO: 74, or e) a heavy chain variable region comprising the sequence of SEQ ID NO: 75 and / or a light chain variable region comprising the sequence of SEQ ID NO: 76 The multispecific molecule according to claim 16.

20. The multispecific molecule according to claim 1, wherein the target antigen binding domain comprises a PD-L1 binding domain.

21. The PD-L1 binding domain is a) HCDR1 comprising the sequence of SEQ ID NO: 119, HCDR2 comprising the sequence of SEQ ID NO: 120, and HCDR3 comprising the sequence of SEQ ID NO: 121, or b) HCDR1 comprising the sequence of SEQ ID NO: 122, HCDR2 comprising the sequence of SEQ ID NO: 123, and HCDR3 comprising the sequence of SEQ ID NO: 124, or c) an HCDR1 comprising the sequence of SEQ ID NO: 125, an HCDR2 comprising the sequence of SEQ ID NO: 126, and an HCDR3 comprising the sequence of SEQ ID NO: 127, or d) an HCDR1 comprising the sequence of SEQ ID NO: 128, an HCDR2 comprising the sequence of SEQ ID NO: 129, and an HCDR3 comprising the sequence of SEQ ID NO: 130, or e) an HCDR1 comprising the sequence of SEQ ID NO: 131, an HCDR2 comprising the sequence of SEQ ID NO: 132, and an HCDR3 comprising the sequence of SEQ ID NO: 133, or f) an HCDR1 comprising the sequence of SEQ ID NO: 134, an HCDR2 comprising the sequence of SEQ ID NO: 135, and an HCDR3 comprising the sequence of SEQ ID NO: 136, or g) an HCDR1 comprising the sequence of SEQ ID NO: 137, an HCDR2 comprising the sequence of SEQ ID NO: 138, and an HCDR3 comprising the sequence of SEQ ID NO: 139, or h) an HCDR1 comprising the sequence of SEQ ID NO: 140, an HCDR2 comprising the sequence of SEQ ID NO: 141, and an HCDR3 comprising the sequence of SEQ ID NO: 142, or i) an HCDR1 comprising the sequence of SEQ ID NO: 143, an HCDR2 comprising the sequence of SEQ ID NO: 144, and an HCDR3 comprising the sequence of SEQ ID NO: 145, or j) an HCDR1 comprising the sequence of SEQ ID NO: 146, an HCDR2 comprising the sequence of SEQ ID NO: 147, and an HCDR3 comprising the sequence of SEQ ID NO: 148, or k) an HCDR1 comprising the sequence of SEQ ID NO: 149, an HCDR2 comprising the sequence of SEQ ID NO: 150, and an HCDR3 comprising the sequence of SEQ ID NO: 151, or an HCDR1 comprising the sequence of SEQ ID NO: 152, an HCDR2 comprising the sequence of SEQ ID NO: 153, and an HCDR3 comprising the sequence of SEQ ID NO: 154 The multispecific molecule according to claim 20, comprising

22. The PD-L1 binding domain comprises the same HCDRs as an anti-PD-L1 antibody selected from the group consisting of C71, C71v38, C239, C492, C570, 570h3, C446, C2811, C1778, C1793, C2855, C2713, and C2719, a) C71 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 107, b) C71v38 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 108, c) C239 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 109, d) C492 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 110, e) C570 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 111, f) 570h3 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 223, g) C446 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 112, h) C2811 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 113, i) C1778 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 114, j) C1793 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 115, k) C2855 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 116, l) C2713 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 117, and m) C2719 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 118, The multispecific molecule according to claim 20.

23. The multispecific molecule according to claim 20, wherein the PD-L1 binding domain comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 107 to 118, and 223.

24. The multispecific molecule according to claim 1, which is humanized or linked to one or more conjugate moieties.

25. A pharmaceutical composition comprising the multispecific molecule according to any one of claims 1 to 24 and one or more pharmaceutically acceptable carriers.

26. An isolated polynucleotide encoding the multispecific molecule according to any one of claims 1 to 24.

27. Use of the multispecific molecule according to any one of claims 1 to 24 in the manufacture of a medicament for treating a disease, disorder, or condition that can benefit from inducing phagocytosis of target cells in a subject, a disease, disorder, or condition related to a target antigen in a subject, a disease, disorder, or condition related to SIRPα in a subject, or a disease, disorder, or condition related to CD47 in a subject.

28. The use according to claim 27, further comprising the step of administering a therapeutically effective amount of a second therapeutic agent.

29. The use according to claim 28, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, radiotherapy agents, immunotherapy agents, anti-angiogenesis agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, anti-viral agents, antibiotics, analgesics, antioxidants, metal chelating agents, and cytokines.

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