Anti-b7-h3 antibodies and methods of use thereof

Abstract

The present disclosure provides anti-B7-H3 antibody variants with enhanced antigen binding affinity. The present disclosure also provides methods for using the anti-B7-H3 antibody for treating a cancer or modulating the immune system in a subject.

Description

PATENT Attorney Docket No.: 115631-000120PC-1507527 ANTI-B7-H3 ANTIBODIES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 650,125, filed May 21, 2024, and U.S. Provisional Application No. 63 / 777,935, filed March 26, 2025, the disclosures of which are herein incorporated by reference in their entirety for all purposes. BACKGROUND

[0002] B7-H3 (CD276), a member of the B7 family that plays an immunoregulatory role in the T cell response, has been highlighted as a novel potential target for cancer immunotherapy. B7-H3 has been shown to play an inhibitory role in T cell activation and proliferation, participate in tumor immune evasion and influence both the immune response and tumor behavior through different signaling pathways. B7-H3 expression has been found to be aberrantly upregulated in many different cancer types. Targeting B7-H3 might provide a novel and promising option for cancer therapy. However, there remains a need for therapeutic agents that target B7-H3 activity or expression levels, especially in cancer cells. SUMMARY

[0003] In one aspect, the present disclosure provides an isolated antibody that specifically binds to a human B7H3 (B7-H3, CD276) protein, wherein the antibody comprises: a heavy chain complementarity determining region 1 (HCDR1) sequence comprising the sequence of GYX1FTX2X3X4 (SEQ ID NO: 7), wherein: X1is T, D, N, S, or E; X2is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3 is Y, E, F, or R; and X4is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6 is S or R; and a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein: X7 is T, E, R, D, Y, W, N, Q, S, or H; X8 is T or Y;X9 is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10 is T, Y, W, R, N, H, or F; and X11is W, Y, or F; or a light chain complementarity determining region 1 (LCDR1) sequence comprising the sequence of X14X15X16X17X18X19(SEQ ID NO: 19), wherein: X14is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17is S, D, E, N, M, G, or Y; X18 is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein: X20is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22 is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23is F, W, or Y; X24is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25 is L, W, Y, F, R, K, E, N, H, or S; wherein the HCDR1 sequence is not GYTFTNYD (SEQ ID NO: 10), the HCDR2 sequence is not IFPGDGST (SEQ ID NO: 11) or IFPGDDST (SEQ ID NO: 12), or the HCDR3 sequence is not ARQTTATWFAY (SEQ ID NO: 13) or ARQTTGTWFAY (SEQ ID NO: 14).

[0004] In some embodiments, the antibody comprises: a heavy chain complementarity determining region 1 (HCDR1) sequence comprising the sequence of GYX1FTX2X3X4 (SEQ ID NO: 7), wherein: X1is T, D, N, S, or E; X2is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3 is Y, E, F, or R; and X4is D, W, R, Y, E, T, I, or H;a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6 is S or R; and a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein: X7 is T, E, R, D, Y, W, N, Q, S, or H; X8is T or Y; X9is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10 is T, Y, W, R, N, H, or F; and X11is W, Y, or F; and a light chain complementarity determining region 1 (LCDR1) sequence comprising the sequence of X14X15X16X17X18X19 (SEQ ID NO: 19), wherein: X14is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17is S, D, E, N, M, G, or Y; X18is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein: X20is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22 is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23 is F, W, or Y; X24is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25 is L, W, Y, F, R, K, E, N, H, or S.

[0005] In some embodiments, the LCDR1 sequence is not QSISDY (SEQ ID NO: 21), or the LCDR3 sequence is not QNGHSFPLT (SEQ ID NO: 22).

[0006] In some embodiments, the antibody further comprises: a heavy chain framework region 1 (HFR1) sequence comprising the sequence of QVQLQQSGAELVKPGASVKLSCKX12S (SEQ ID NO: 15), wherein X12 is A or T;a HFR2 sequence comprising the sequence of INWVRQRPX13QGLEWIGW (SEQ ID NO: 16), wherein X13 is E or G; a HFR3 sequence comprising the sequence of X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFC (SEQ ID NO: 17), where X31 is Q or Y; and a HFR4 sequence comprising the sequence of WGQGTLVTVSA (SEQ ID NO: 18).

[0007] In some embodiments, the antibody further comprises: a light chain framework region 1 (LFR1) sequence comprising the sequence of X26X27VMTQSPATLSVTPGDRVX28LSCRAS (SEQ ID NO: 23), wherein: X26 is D, Q, E, or N; X27 is I, W, E, Y, F, T, N, R, K, or Q; and X28is S or T; a LFR2 sequence comprising the sequence of LX29WYQQKSHESPRLLIK (SEQ ID NO: 24), wherein X29 is H, Y, N, or S; a LFR3 sequence comprising the sequence of QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID NO: 25), wherein X30 is S, D, E, A, N, Q, T, P, or V; and a LFR4 sequence comprising the sequence of FGAGTKLELK (SEQ ID NO: 26).

[0008] In some embodiments, X2 is L, X4 is W, X5 is R, X6 is Y, X7 is E, X8 is Y, X9 is W, X10is Y, X18is N or S, X19is W, X20is W, X21is W, X23is W, X24is Y, X25is W, and / or X27is W.

[0009] In some embodiments, the HCDR1 comprises the sequence of GYTFTNYW (SEQ ID NO: 27), the HCDR2 comprises the sequence of IFPGDDRT (SEQ ID NO: 28), the HCDR3 comprises the sequence of ARQTTWTWFAY (SEQ ID NO: 29), the LFR1 comprises the sequence of DWVMTQSPATLSVTPGDRVSLSCRAS (SEQ ID NO: 30), and / or the LCDR1 comprises the sequence of QSISNY (SEQ ID NO: 31).

[0010] In some embodiments, the antibody comprises a VH sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 32-41. In some embodiments, the antibodycomprises a VL sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 42-54. In some embodiments, the antibody comprises a VH sequence of any one of SEQ ID NOs: 32-41 and a VL sequence of any one of SEQ ID NOs: 42-54.

[0011] In some embodiments, the antibody is a Fab, a F(ab’)2, a scFv, or a bivalent scFv. In some embodiments, the antibody is a scFv comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 56-69 and 92-105.

[0012] In some embodiments, the antibody comprises a HC sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 70-75. In some embodiments, the antibody comprises a LC sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 76-80. In some embodiments, the antibody comprises a HC sequence of any one of SEQ ID NOs: 70-75 and a LC sequence of any one of SEQ ID NOs: 76-80.

[0013] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a fully human antibody.

[0014] In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody binds to both CD276 and CD3. In some embodiments, the CD3-binding portion of the bispecific antibody comprises a VH sequence of SEQ ID NO: 81 and a VL sequence of SEQ ID NO: 82. In some embodiments, the CD3-binding portion is an scFv. In some embodiments, the scFv comprises a sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises a first scFv binding to CD276 and a second scFv binding to CD3. In some embodiments, the first scFv binding to CD276 comprises a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 56-69 and 92-105. In some embodiments, the bispecific antibody comprises a sequence of any one of SEQ ID NOs: 88-91 and 106-119. In some embodiments, the antibody is conjugated to a therapeutic agent or detection agent.

[0015] In some embodiments, the therapeutic agent is an antitumor agent, a radioisotope, a drug agent, a nanoparticle, an immune toxin, or a combination thereof. In some embodiments, the detection agent is a diagnostic or imaging agent, or both.

[0016] The present disclosure also provides a pharmaceutical composition comprising the isolated antibody described herein, and a pharmaceutically acceptable carrier. The presentdisclosure further provides a kit comprising: the isolated antibody or the pharmaceutical composition described herein, and instructions for use thereof.

[0017] In another aspect, the present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject the isolated antibody or the pharmaceutical composition described herein. In some embodiments, the cancer comprises B7-H3-positive tumor cells. In some embodiments, the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, a chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.

[0018] In another aspect, the present disclosure provides a method of modulating the immune system of a subject, comprising administering to the subject the subject the isolated antibody or the pharmaceutical composition described herein.

[0019] In another aspect, the present disclosure provides a method of enhancing T-cell mediated cytotoxicity in a subject, comprising administering to the subject the subject the isolated antibody or the pharmaceutical composition described herein.

[0020] In another aspect, the present disclosure provides a method of detecting B7-H3 in a biological sample, comprising contacting a biological sample with the subject the isolated antibody or the pharmaceutical composition described herein.

[0021] In yet another aspect, the present disclosure provides a method of diagnosing a subject suffering from a cancer, comprising contacting a biological sample from the subject with the subject the isolated antibody or the pharmaceutical composition described herein, wherein detecting the presence of B7-H3 or the expression level of B7-H3 in the sample determines the presence of the disorder. In some embodiments, the disorder is a cancer. In some embodiments, the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, a chronic leukemia, a medulloblastoma, a multiplemyeloma, and a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma..

[0022] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes with the isolated antibody described herein for binding to the human B7-H3 (CD276) protein.

[0023] In one aspect, the present disclosure provides an isolated antibody that specifically binds to a human B7-H3 (CD276) protein, wherein the antibody comprises a heavy chain complementarity determining region 1 (HCDR1) sequence comprising the sequence of GYTFTNYD (SEQ ID NO: 10); a HCDR2 sequence comprising the sequence of IFPGDDST (SEQ ID NO: 12); a HCDR3 sequence comprising the sequence of ARQTTGTWFAY (SEQ ID NO: 14); a light chain complementarity determining region 1 (LCDR1) sequence comprising the sequence of QSISNY (SEQ ID NO: 31); a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNGHSFPLT (SEQ ID NO: 22).

[0024] In some embodiments, the antibody comprises a heavy chain framework region 1 (HFR1) sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121 or 122; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125 or 126; and / or a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127 or 128.

[0025] In some embodiments, the antibody comprises a HFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121 or 122; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125 or 126; and a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127 or 128.

[0026] In some embodiments, the antibody comprises a light chain framework region 1 (LFR1) sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 135-138; and / or a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139 or 140.

[0027] In some embodiments, the antibody comprises a LFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 135-138; and a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139 or 140.

[0028] In some embodiments, the antibody comprises a VH sequence that has at least 85% sequence identity to SEQ ID NO: 141 or 142. In some embodiments, the antibody comprises a VL sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 143-146. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 141 or 142 and the VL sequence of any one of SEQ ID NOs: 143-146.

[0029] In some embodiments, the antibody is a full-length monoclonal antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a chimeric antibody, a camelized single-domain antibody, a Fab, a F(ab’)2, a Fab′, a F(ab)2, a Fv fragment, a scFv, a di-scFv, a BiTE, a dAb, or a bivalent scFv. In some embodiments, the antibody is a scFv comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148.

[0030] In some embodiments, the antibody comprises a heavy chain (HC) comprising a sequence that has at least 85% sequence identity to SEQ ID NO: 149. In some embodiments, the antibody comprises a light chain (LC) comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 150-152. In some embodiments, the antibody comprises a HC comprising the sequence of SEQ ID NO: 149 and a LC comprising the sequence of any one of SEQ ID NOs: 150-152.

[0031] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a fully human antibody.

[0032] In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody binds to both B7-H3 and CD3. In some embodiments, the CD3-binding portion of the bispecific antibody comprises a VH sequence that has at least 85% sequence identity to SEQ ID NO: 81 and a VL sequence that has at least 85% sequence identity to SEQ ID NO: 82. In some embodiments, the CD3-binding portion is an scFv. In some embodiments, the scFv comprises the sequence of SEQ ID NO: 87.

[0033] In some embodiments, the B7-H3-binding portion of the bispecific antibody comprises a VH sequence that has at least 85% sequence identity to SEQ ID NO: 141 or 142 and a VL sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 143- 146. In some embodiments, the B7-H3-binding portion is an scFv comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148.

[0034] In some embodiments, the antibody is conjugated to a therapeutic agent or detection agent. In some embodiments, the therapeutic agent is an antitumor agent, a radioisotope, a drug agent, a nanoparticle, an immune toxin, or a combination thereof. In some embodiments, the detection agent is a diagnostic or imaging agent, or both.

[0035] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated antibody described herein, and a pharmaceutically acceptable carrier.

[0036] In another aspect, the present disclosure provides a kit comprising: the isolated antibody the isolated antibody or the pharmaceutical composition described herein, and instructions for use thereof.

[0037] In another aspect, the present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject the isolated antibody or the pharmaceutical composition described herein. In some embodiments, the cancer comprises B7-H3-positive tumor cells. In some embodiments, the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, a chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.

[0038] In another aspect, the present disclosure provides a method of modulating the immune system of a subject, comprising administering to the subject the isolated antibody or the pharmaceutical composition described herein.

[0039] In another aspect, the present disclosure provides a method of enhancing T-cell mediated cytotoxicity in a subject, comprising administering to the subject the isolated antibody or the pharmaceutical composition described herein.

[0040] In another aspect, the present disclosure provides a method of detecting B7-H3 in a biological sample, comprising contacting a biological sample with the isolated antibody or the pharmaceutical composition described herein.

[0041] In another aspect, the present disclosure provides a method of diagnosing a subject suffering from a cancer, comprising contacting a biological sample from the subject with the isolated antibody, or the pharmaceutical composition described herein, wherein detecting the presence of B7-H3 or the expression level of B7-H3 in the sample determines the presence of the disorder. In some embodiments, the disorder is a cancer. In some embodiments, the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, a chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.

[0042] In yet another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes with the isolated antibody described herein for binding to the human B7-H3 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 depicts murine and human B7-H3 amino acid sequences with their V-like and C-like immunoglobulin (Ig) domains indicated.

[0044] Figure 2 depicts amino acid sequence alignment of the outer and inner V-like and C- like Ig domains of human B7-H3. The sequences differ by 6 mismatches as shown in bold / shaded.

[0045] Figure 3 depicts amino acid sequence alignment of murine 2Ig-B7-H3 and the outer V-like + C-like Ig domain of human B7-H3. The sequences differ by 17 mismatches as shown in bold / shaded.

[0046] Figure 4 depicts amino acid sequence alignment of murine 2Ig-B7-H3 and the inner V-like + C-like Ig domain of human B7-H3. The sequences differ by 13 mismatches as shown in bold / shaded.

[0047] Figure 5 depicts amino acid sequence alignment of human B7-H3 (the SWISS- MODEL output sequence) and murine B7-H3. The first 34 residues of murine B7-H3 are not represented in the human B7-H3 sequence.

[0048] Figures 6A-6B depict the ch8H9[3mut]-huB7H3 model in3cl6 visualized in iCn3D (NCBI, https: / / www.ncbi.nlm.nih.gov / Structure / icn3d / full.html) [10-11]. (A) Zoomed-out view and (B) Zoomed-in view illustrate various non-covalent bonds between the ch8H9[3mut] Fab antibody and the protein human B7-H3 (huB7H3). “R” represents R127; “D” represents D128; “H” represents the heavy chain of the antibody; and “L” represents the light chain of the antibody.

[0049] Figures 7A-7D depict non-covalent interactions between the ch8H9[3mut] Fab antibody and huB7H3 (residues 35-240 of the Uniprot Q5ZPR3 sequence) in the in3cl6 model as identified in iCn3D (NCBI) in hydrogen bonds (A), pi-cation pairs (B), salt bridge / ionic interaction (C), and pi-stacking pair (D), respectively. The ch8H9[3mut] antibody chains and residue numbers are reflected as “A:X” or “B:X”, with “A” representing the heavy chain and “B” the light chain, and “X” representing a residue number: 1-220 for the heavy chain “A” and 1-213 (reflected as 301-513) for the light chain “B”. The atoms involved in the interactions are shown after the @ sign.

[0050] Figure 8 depicts non-covalent interactions between the ch8H9[3mut] Fab antibody and huB7H3 (residues 35-240 of the Uniprot Q5ZPR3 sequence) in the in3cl6 model as identified by iCn3D (NCBI) in contacts. The antibody ch8H9[3mut] chains and residue numbers are reflected as “A:X” and “B:X”, with “A” representing the heavy chain and “B” the light chain, and “X” representing a residue number: 1-220 for the heavy chain “A” and 1-213 (reflected as 301-513) for the light chain “B”. The atoms involved in the interactions are shown after the @ sign.

[0051] Figure 9 depicts verification of predicted affinity changes by modeling individual amino acid mutations in the ch8H9[3mut] antibody modeled as a Fab fragment. “Mutations” refers to the mutations modeled, “Energy-min. [No. Steps]” refers to the number of energy minimization steps, “Mutagenesis” refers to the method applied to initially predict an affinity change (visual or saturation mutagenesis), “H-huB7H3 [kJ / mol]”, “L-huB7H3 [kJ / mol]”, and “H+L-huB7H3 [kJ / mol]” refer to the total binding energy predicted using PPCheck [7] for binding of the light (L) chain, the heavy (H) chain, or the L + H chain, respectively, to huB7H3. “Rank” indicates the relative rank of the H+L-huB7H3 binding energies (a higher rank meansa higher predicted affinity). WT refers to wild-type ch8H9[3mut]. VH-G8A serves like the WT construct as a reference construct but contains a VH-G8A mutation predicted to not affect B7- H3 binding.

[0052] Figures 10A-10G depict predicted affinity changes caused by single amino acid mutations in the ch8H9[3mut] antibody modeled as a Fab fragment. (A) depicts a barplot showing total stabilizing energy (TSE) values for each amino acid mutation modeled in Figure 9, with 250 energy minimization steps. Each TSE value represents a combined TSE value from both the heavy (H) and light (L) chains of one construct (sum of H TSE and L TSE). The reference constructs (ch8H9[3mut] WT and VH-G8A) were energy-minimized with both 250 (third and fourth bars) and 1500 steps (first and second bars). Using a cutoff at the lowest reference TSE value (VH-G8A, 250 steps), 18 amino acid mutations were predicted with enhanced affinities for huB7H3. (B-G) each shows the non-covalent bonds between huB7H8 and a mutated construct, in comparison to the two references (ch8H9[3mut] and VH-G8A). Images were generated using iCn3D (NCBI). The amino acid mutations VH-G102W (B), VL- D31N (C), VH-D33W (D), VL-D31S (E), VH-T101Y (F), and VL-I2W (G) were predicted as having the highest binding affinities for the antigen huB7H8.

[0053] Figure 11A-11B depicts the heavy chain (A) and light chain (B) of the ch8H9[3mut] antibody shown as a Fab fragment with predicted substitutions to increase binding affinities for the human B7-H3. The CDRs in the heavy chain (VH-CH1) and light chain (V-KAPPA- C-KAPPA) are defined according to the IMGT definition. The substitutions shown above the sequences were newly predicted in this disclosure, through either a visual inspection of the ch8H9-based 3D model, or in silico saturation mutagenesis using the reference antibody ch8H9[3mut]. VH-S57R and VH-Q59Y mutations were discovered through visual inspection, and all other substitutions were predicted through in silico saturation mutagenesis. The 6 mutations: VH-A24T, VH-E42G, VH-G56D, VH-A102G in (A), and VL-S20T, VL-H34Y in (B), shown underneath the sequences, were identified by Ahmed et al., 2015 using yeast display [1].

[0054] Figure 12 depicts verification of predicted affinity changes by modeling the mutations in combinations in the ch8H9[3mut] antibody modeled as a Fab fragment. “Mutations” refers to the mutations modeled, “Energy-min. [No. Steps]” refers to the number of energy minimization steps, “Mutagenesis” refers to the method applied to initially predict an affinity change (visual or saturation mutagenesis), “H-huB7H3 [kJ / mol]”, “L-huB7H3[kJ / mol]”, and “H+L-huB7H3 [kJ / mol]” refer to the total binding energy predicted using PPCheck for binding of the light chain, the heavy chain, or the light + heavy chain, respectively, to huB7H3. “Rank” indicates the relative rank of the H+L-huB7H3 binding energies (a higher rank means a higher predicted affinity). WT refers to wild-type ch8H9[3mut], VH-G8A is based on ch8H9[3mut] with a G8A mutation which does not affect B7-H3 binding.

[0055] Figure 13 depict affinity changes caused by single, dual, triple, quadruple, or quintuple of the top-5 amino acid mutations in the ch8H9[3mut] antibody modeled as a Fab fragment. The top-5 mutations are VH-D33W, VH-S57R, VH-G102W, VL-I2W, and VL- D31N. The barplot shows the total stabilizing energy (TSE) values of the single or multi- mutation constructs as listed in Figure 12, each with 250 energy minimization steps. Each TSE value represents a combined TSE value from both the heavy (H) and light (L) chains of one construct (sum of H TSE and L TSE). Using a cutoff at the lowest reference TSE value (VH- G8A), all 31 constructs were predicted as having enhanced affinities for huB7H3. The quintuple mutation constructs were expected to have the highest binding affinities for the antigen.

[0056] Figure 14 depicts affinity changes caused by single mutations in the ch8H9[3mut] antibody modeled as a Fab fragment in an alternative model, in3cl0. The barplot shows the total stabilizing energy (TSE) values for the four single-mutation antibodies predicted through in silico saturation mutagenesis to have an increased binding affinity for huB7H3 (Table 7), after energy minimization with 250 steps. Each TSE value represents a combined TSE value from both the H and L chains of one construct (sum of H TSE and L TSE). The antibodies ch8H9[3mut] (WT) and VH-G8A were the reference constructs. Using a cutoff at the lowest reference TSE value (VH-G8A), all four mutations on the ch8H9[3mut] antibody were again predicted as having enhanced binding affinity for huB7H3.

[0057] Figure 15 depicts affinity changes caused by single mutations in the ch8H9[3mut] antibody modeled as a Fab fragment in an alternative model, in4cl0. The barplot shows the total stabilizing energy (TSE) values for the five single-mutation antibodies predicted through in silico saturation mutagenesis to have an increased binding affinity for huB7H3 (Table 8), after energy minimization with 250 steps. Each TSE value represents a combined TSE value from both the H and L chains of one construct (sum of H TSE and L TSE). The antibodies ch8H9[3mut] (WT) and VH-G8A were the reference constructs. Using a cutoff at the lowestreference TSE value (WT), four of the five mutations on the ch8H9[3mut] antibody were again predicted as having enhanced binding affinity for huB7H3.

[0058] Figure 16 depicts multiple amino acid sequence alignment of hu8H9[3mut] VH sequences obtained through 1-5 iterations (“rounds”) of an in silico humanization process using BioPhi (https: / / biophi.dichlab.org / )

[0014] . The alignment was conducted through Clustal Omega (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo)

[0015] . Irrespective of the iteration rounds (1- 5 rounds) or the prevalence stringency settings (>=10% or >= 90%), an identical humanized VH sequence was obtained. A24T and E42G refer to the “6m” mutations identified by Ahmed et al. [1]. Note that the in silico humanization employed here already yielded a Gly (G) at position 42.

[0059] Figure 17 depicts multiple amino acid sequence alignment of hu8H9[3mut] VL sequences. obtained through 1-5 iterations (“rounds”) of an in silico humanization process. The alignment was conducted using Clustal Omega. No matter the prevalence stringency setting is >=10% or >= 90%, the given round (X) of iterations, indicated as “_Xrd” in the sequence names, yielded identical sequences. For example, hu8H9_VL_1rd_10 shares the same sequence as hu8H9_VL_1rd_90. Three humanized VL sequences were obtained through the process. The differences among the three hu8H9[3mut] VL sequences are indicated in bold / shade. S20T refers to a “6m” mutation identified by Ahmed et al. [1]. Note that the in silico humanization employed here already yielded a Thr (T) at position 20.

[0060] Figures 18A-18C depict in silico prediction of affinity changes caused by mutations in hu8H9[3mut]. BioPhi was employed for in silico humanization of the VH and VL sequences of ch8H9[3mut], yielding hu8H9[3mut_Xrd] fragment variable (Fv) in silico constructs, whereby “X” refers to the number of iteration rounds employed. Modeling of the top-5 mutations: VH-D33W, VH-S57R, VH-G102W, VL-I2W, and VL-D31N, with 250 energy- minimization steps, were conducted with 1 (A), 2 (B), or 3-5 (C) iterations of humanization. The non-human fraction increased from 38% (A) after one iteration of humanization to 42% (B and C) with 2-5 iterations, suggesting that one iteration yielded a sequence with the lowest immunogenicity. However, the VL-D31N mutation (B), along with the frameworks obtained after 2 iterations, showed the highest antigen-binding affinity (the lowest TSE value) across all the mutations and all the iterations of humanization.

[0061] Figure 19 depicts affinity changes caused by the top-5 mutations in combination with the “6m” mutations identified by Ahmed et al. [1] in ch8H9 Fab constructs modeled usingSWISS-MODEL [28, 29] with the in3cl6 structural model containing the ch8H9[3mut] Fab as template and the heavy and light chain Fab sequences as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. The barplot shows the TSE values of the antibody variants for both the heavy (H) and light (L) chains combined (sum of H TSE and L TSE). In the labels, “ch8H9” refers to the chimeric anti- B7-H3 monoclonal Fab antibody described by Ahmed et al. [1], “ch8H9[3mut]” to a ch8H9 variant carrying the 3 CDR mutations of the “6m” mutations described by Ahmed et al. (heavy chain: G56D, A102G; light chain: H34Y, CDRs by Kabat definition), and “ch8H9-6m” to a ch8H9 variant carrying the “6m” mutations identified by Ahmed et al. i.e., the 3 CDR mutations of ch8H9[3mut] and 3 framework mutations (heavy chain: A24T, E42G; light chain: S20T; CDRs by Kabat definition). The “ch8H9[3mut]_VH-G8A” is based on ch8H9[3mut]. It includes a VH-G8A mutation predicted to not affect B7-H3 binding affinity. The “ch8H9_VH- D33W”, “ch8H9_VH-S57R”, “ch8H9_VH-G102W”, “ch8H9_VL-I2W, and “ch8H9_VL- D31N” are based on ch8H9 with one of the top-5 mutations, respectively. The “ch8H9_Top1- 5” is based on ch8H9 with all top-5 mutations combined. The data suggests that from the top- 5 mutations, VH-D33W, VH-G102W, VL-D31N, and all 5 mutations in combination, and potentially VL-I2W, could yield higher binding affinities for huB7H3 than any of the ch8H9- based reference Fab antibody constructs (ch8H9, ch8H9-6m, ch8H9[3mut], ch8H9[3mut]_VH-G8A).

[0062] Figure 20 depicts affinity changes caused by the top-5 mutations in combination with the “6m” mutations identified by Ahmed et al. [1] in hu8H9 fragment variable (Fv) (i.e., VH+VL only) constructs modeled using SWISS-MODEL with the in3cl6 structural model containing the ch8H9[3mut] Fab as template and the heavy and light chain variable sequences as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. The barplot shows the TSE values of the antibody variants for both the variable heavy (VH) and variable light (VL) chains combined (sum of VH TSE and VL TSE). In the labels, “hu[JBC]8H9” refers to a humanized anti-B7-H3 Fv with the VL and VH sequences described by Ahmed et al. [1], “hu[JBC]8H9-6m” to the hu[JBC]8H9 Fv with the inclusion of the “6m” mutations identified by Ahmed et al. (heavy chain: A24T, E42G, G56D, A102G; light chain: S20T, H34Y), and “VH-D33W”, “VH-S57R”, “VH-G102W”, “VL-I2W, and “VL-D31N” indicate the presence of the corresponding “top-5” mutations in the Fv. “Top1-5” refers to the presence of all 5 “top-5” mutations in the Fv, and “[1rd]”, “[2rd]”, or “[3rd]” refers to 1, 2, or 3 rounds / iterations, respectively, of in silico humanization. The datasuggests that the top-5 mutations, alone and in combination, yield higher binding affinities for huB7H3 than the humanized Fv antibodies (hu[JBC]8H9 and hu[JBC]8H9-6m) with sequences as shown by Ahmed et al. [1].

[0063] Figure 21 depicts affinity changes caused by the top-5 mutations in combination with the “6m” mutations identified by Ahmed et al. [1] in ch8H9 Fab constructs modeled using SWISS-MODEL with the in3cl6 structural model containing the ch8H9 Fab as template (previously modeled with the in3cl6 model containing the ch8H9[3mut] Fab as template) and the heavy (H) and light (L) chain Fab sequences as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. The barplot shows the TSE values of the antibody variants for both the heavy and light chains combined (sum of H TSE and L TSE). The labels above the bars include the following information: [3mut] refers to the 3 CDR mutations identified by Ahmed et al. [1] (heavy chain: G56D, A102G; light chain: H34Y; CDRs by Kabat definition); “6m” refers to these 3 CDR mutations plus 3 framework mutations (heavy chain: A24T, E42G; light chain: S20T); VH-G8A refers to a G8A mutation in the heavy chain not expected to affect B7-H3 binding; VH-D33W, VH- S57R, VH-G102W, VL-I2W; VL-D31N: individual mutations predicted to increase the binding affinity for huB7H3; Top1-5: inclusion of all of the 5 mutations. Using the ch8H9 Fab as part of the modeling template, the data suggests that from the top-5 mutations (Table 9), VH-D33W, VH-S57R, VH-G102W, and VL-D31N alone, and all the 5 mutations in combination, yield higher binding affinities for huB7H3 than the ch8H9 Fab antibody with the “6m” mutations.

[0064] Figure 22 depicts affinity changes caused by the top-5 mutations in combination with the “6m” mutations identified by Ahmed et al. [1] in hu8H9 fragment variable (Fv) (i.e., VH+VL only) constructs modeled using SWISS-MODEL with the in3cl6 structural model containing the ch8H9 Fab as template and the heavy and light chain variable sequences as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. The barplot shows the TSE values of the antibody variants for both the variable heavy (VH) and variable light (VL) chains combined (sum of VH TSE and VL TSE). In the labels, “hu[JBC]8H9” refers to a humanized anti-B7-H3 Fv with the VL and VH sequences as described by Ahmed et al. [1], “hu[JBC]8H9-6m” to the hu[JBC]8H9 Fv with the inclusion of the “6m” mutations identified by Ahmed et al. [1] (heavy chain: A24T, E42G, G56D, A102G; light chain: S20T, H34Y), and “VH-D33W”, “VH-S57R”, “VH-G102W”, “VL-I2W, and “VL-D31N” indicate the presence of the corresponding “top-5” mutations inthe Fv. “Top1-5” refers to the presence of all 5 “top-5” mutations in the Fv, and “[1rd]”, “[2rd]”, or “[3rd]” refers to 1, 2, or 3 rounds / iterations, respectively, of in silico humanization. The data suggests that the top-5 mutations, either alone or in combination, yield higher binding affinities for huB7H3 than the humanized antibodies hu[JBC]8H9 and hu[JBC]8H9-6m described by Ahmed et al. [1], especially for the antibodies in silico humanized with one round / iteration.

[0065] Figure 23 depicts TSE values of ch8H9[3mut] Fab antibody variants modeled to carry single or multiple amino acid mutations. To assess whether a combination of multiple (two or more) mutations can provide more favorable (i.e., lower) TSE values than individual mutations or other mutation combinations, TSE values were compared for each antibody variant accordingly. A combination of multiple (two or more) mutations yielding a TSE value lower than the combined TSE values of all its components indicates a cooperative effect (”C”). A combination of multiple (two or more) mutations yielding a TSE value higher than at least one TSE value of its components indicates an antagonistic effect (“A”). The data shows all but two combinations of mutations are predicted to act cooperatively to enhance the antigen- binding affinities.

[0066] Figure 24 depicts the predicted total stabilizing energy (TSE) values from PPCheck (https: / / caps.ncbs.res.in / ppcheck / ) for hu[1rd]8H9-6m fragment variable (Fv) constructs with the 17 single and VH-S57R + VH-Q59Y double mutations predicted to increase the affinity for human B7-H3 when modeled as ch8H9[3mut] Fab constructs (shown in Figure 10A). All but the VH-T100E mutation are predicted to increase the affinity (lower TSE values) for human B7-H3 compared to the wild-type or VH-G8A reference hu[1rd]8H9-6m Fv constructs.

[0067] Figure 25 depicts binding sensor-grams of ligand to analyte for scFv binding to human B7-H3 (Biacore 8K, Cytiva) in Surface Plasmon Resonance #1 (SPR #1) experiment. To assess the binding strengths between the scFvs indicated and human B7-H3, antigen (Fc- tagged B7-H3) was injected onto Series S Sensor Chip Protein A (Cytiva) as the capture molecule. Antibodies (scFvs) were diluted and injected over the surface of flow cell 1 and 2 during the association phase, followed by the injection of running buffer during the dissociation phase. Data was processed using the Biacore 8K Evaluation Software version 4.0. Flow cell 1 and blank injections of running buffer in each cycle were used as double reference for Resonance Units subtraction. Binding was observed for ch8H9-6m, hu[JBC]8H9, hu[JBC]8H9-6m, hu[1rd]8H9-6m, and hu[1rd]8H9-6m_VL-D31N, whereby “JBC” refers to VH and VL sequences as published by Ahmed et al. [1].

[0068] Figure 26 depicts a bar plot of the KD values corresponding to the sensor-grams of Figure 25 and the numeric binding data of Table 10. Binding was observed for ch8H9-6m, hu[JBC]8H9, hu[JBC]8H9-6m, hu[1rd]8H9-6m, and hu[1rd]8H9-6m_VL-D31N.

[0069] Figure 27 depicts binding sensor-grams of ligand to analyte for scFv binding to human B7-H3 in comparison to B7-H1, B7-H2, and B7-H4 (Biacore T200, Cytiva) in SPR #2 experiment. To assess the binding strengths between the scFvs and human B7-H1, B7-H2, B7- H3, or B7-H4, the antigens (Fc-tagged B7 proteins) were injected onto Series S Sensor Chip Protein A (Cytiva) as distinct capture molecules. The antibodies (scFvs) were diluted and injected over the surface of flow cell 1 and 2 during the association phase, followed by injection of running buffer during the dissociation phase. Data was processed using the Biacore T200 Evaluation Software version 3.2.1. Flow cell 1 and blank injections of running buffer in each cycle were used as double reference for Resonance Units subtraction. Binding was only observed with B7-H3 and tested for hu[1rd]8H9-6m, hu[1rd]8H9-6m_VL-D31N, and hu[JBC]8H9-6m.

[0070] Figure 28 depicts a bar plot of the KD values corresponding to the sensor-grams of Figure 27 and the numeric binding data of Table 11. Binding was only observed with B7-H3 and not with the other antigens.

[0071] Figure 29 shows predicted effects of the VL-D31N mutation in hu[1rd]8H9-6m Fv constructs. Shown are total stabilizing energy (TSE) values from PPCheck (https: / / caps.ncbs.res.in / ppcheck / ) for both the variable heavy (VH) and light (VL) chains combined (sum of VH TSE and VL TSE). PPCheck input structured were modeled using SWISS-MODEL with the ch8H9[3mut] Fab structure as template then energy-minimized using Open Babel (obabel, minimize) with 250 steps. Using a cutoff at the lowest reference TSE value (among VH-G8A and WT), the VL-D31N mutation yielded a TSE value smaller than the cutoff given by the references.

[0072] Figure 30 depicts binding sensor-grams of ligand to analyte for scFv binding to human B7-H3 for hu[JBC]8H9-6m (A), hu[1rd]8H9-6m (B, C), and hu[1rd]8H9-6m_VL- D31N (D, E) scFvs as assessed by SPR in a third SPR experiment. Two sets of scFvs were tested, generated in different production rounds (Production Round B, Production Round C).

[0073] Figure 31 depicts bar plots of the KD values corresponding to the sensor-grams of Figure 30 and the numeric binding data of Table 12.

[0074] Figure 32 depicts a statistical assessment of the affinity increase by hu[1rd]8H9- 6m_VL-D31N relative to hu[JBC]8H9-6m. Three SPR experiments (#1-3) were conducted with scFvs generated in 2-3 production rounds (referred to as production rounds A, B, or C). A) shows KD values in nanomoles (nM). B) shows the purity of the scFvs as assessed by SDS- PAGE and SEC-HPLC. C) shows arithmetic means of the KD values from the three experiments. For hu[1rd]8H9-6m and hu[1rd]8H9-6m_VL-D31N in SPR #3, the average KD values for production rounds B and C were used as the SPR #3 contribution to the means. For hu[JBC]8H9-6m, the mean was directly calculated with the 3 values shown. Error bars denote standard error of the mean (SEM) values. For hu[1rd]8H9-6m_VL-D31N, compared to hu[JBC]8H9-6m, there is a trend for higher affinity for human B7-H3 (lower KD values; p = 0.081; one-tailed t-test). D) shows arithmetic means of the KD values from the three SPR experiments after normalizing (dividing) each value with the hu[JBC]8H9-6m KD value from the same experiment. This approach corrects for potential systemic effects equally affecting all scFvs within an experiment. For SPR #3, the KD values prior to normalization were handled as for panel C. Error bars denote SEM values. The affinity for human B7-H3 is significantly higher (lower KD value) for hu[1rd]8H9-6m_VL-D31N than for hu[JBC]8H9-6m (p = 0.020; one-sample, one-tailed t-test).

[0075] Figure 33 depicts purity data of the hu[JBC]8H9-6m scFv generated in production round A. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition.

[0076] Figure 34 depicts purity data of the hu[JBC]8H9-6m scFv generated in production round B. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition..

[0077] Figure 35 depicts purity data of the hu[1rd]8H9-6m scFv generated in production round A. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition.

[0078] Figure 36 depicts purity data of the hu[1rd]8H9-6m scFv generated in production round B. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blotwas THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition.

[0079] Figure 37 depicts purity data of the hu[1rd]8H9-6m scFv generated in production round C. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition.

[0080] Figure 38 depicts purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round A. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition.

[0081] Figure 39 depicts purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round B. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition.

[0082] Figure 40 depicts SEC-HPLC purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round B undiluted (A) and diluted to 0.5 mg / ml (B).

[0083] Figure 41 depicts purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round C. A) SDS-PAGE gel. B) Western Blot. The primary antibody used in the Western Blot was THE™ His Tag Antibody, mAb, Mouse (GenScript, Cat. No. A00186-100). “M” refers to protein marker, “R” to reducing condition, and “NR” to non-reducing condition. DETAILED DESCRIPTION I. Introduction

[0084] Human B7-H3 (B7H3, or B7 Homolog 3, or Cluster of Differentiation 276), is a 316 amino acid-long type I transmembrane protein, existing in at least two isoforms determined by its extracellular domain. In mice, the extracellular domain consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans it consists of one pair (2Ig-B7-H3) or two identical pairs (4Ig-B7-H3) due to exon duplication. B7-H3 protein has a very limited expression on normal tissues because of its post-transcriptional regulation by microRNAs. However, B7-H3 protein is expressed athigh frequency in many different cancer types (60% of all cancers), including breast, prostate, lung, ovarian, brain, gastric, pancreatic, osteosarcoma, and nasopharyngeal carcinoma. Presence of B7-H3 correlates with worsened prognosis, poor survival, and higher recurrence rates. Objective responses (tumor regressions) were reported in the clinic in multiple tumor types following treatment with B7-H3 targeting agents. The 4Ig-B7-H3 isoform is predominant in cancer.

[0085] B7-H3 was initially discovered as a T cell co-stimulatory molecule but was thereafter found to predominantly act as a T cell inhibitory molecule. T cell inhibition is mediated via the FG loop of the Ig-like V domain of B7-H3. In non-malignant tissues, B7-H3 has a predominantly inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. In malignant tissues, B7-H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7-H3 also possesses non-immunological pro- tumorigenic functions such as promoting migration, invasion, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism. It has been demonstrated that B7-H3 contributes to tumorigenesis, metastasis and malignant behaviors through various mechanisms.

[0086] As disclosed herein, antibodies have been generated that specifically bind to human B7-H3 (huB7H3). In particular, the antibodies disclosed herein have enhanced antigen-binding affinities in comparison to the known anti-huB7H3 antibodies, such as the ch8H9 and its derivatives developed by Ahmed et al. and Nai-Kong et al. [1, 2]. In one aspect, the present disclosure provides anti-B7H3 antibodies based on the antibody ch8H9[3mut] (ch8H9 with 3 affinity-enhancing CDR mutations identified by Ahmed et al. and Nai-Kong et al.) with various amino acid substitutions. The one or more amino acid substitutions of the antibodies discovered herein substantially enhance binding affinities for the antigen B7-H3.

[0087] In some embodiments, the anti-B7H3 antibodies can be used to treat cancers. In some embodiments, the anti-B7H3 antibodies can be used to modulate the immune system, e.g., enhance T-cell mediated cytotoxicity, enhance NK cell activity, or suppress the inhibitory effect of B7-H3 on T cells or NK cells. In some embodiments, the anti-B7-H3 antibodies can be used to detect B7-H3 expression in biological samples. II. Definitions

[0088] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to whichthis disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0089] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0090] The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0091] The terms “identity,” “substantial identity,” “similarity,” “substantial similarity,” “homology” and the related terms and expressions used in the context of describing amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A “comparison window” includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection.

[0092] Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993).

[0093] The term “antibody” refers, in the broadest sense, to any protein / peptide / fragment with an immunoglobulin fold that specifically binds to an antigen via its variable region(s). The term specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies containing two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies. The term “antibody,” as used herein, also includes antigen-binding fragments that retain binding specificity via its variable regions, including but not limited to Fab, Fab′, F(ab)2, Fv fragments, F(ab′)2, scFv, di-scFv, BiTE, and / or dAb.

[0094] The term “monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, a cluster in which several antibodies are the same, except for a few natural mutants that may exist. The monoclonal antibody is generally highly specific for a single antigen site. Moreover, unlike conventional polyclonal antibody preparations (which generally comprise different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.

[0095] The term “chimeric antibody” generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Generally, the variable region is derived from an antibody (“parent antibody”) in an experimental animal such as a rodent, and the constant region is derived from a human antibody, such that the possibility of causing an adverse immune response in an individual human by the resulting chimeric antibody is reduced as compared with the parental (for example, mouse-derived) antibody.

[0096] The term “humanized antibody” generally refers to an antibody in which some or all of the amino acids outside the CDR of a non-human antibody (such as a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. In the CDR, small additions, deletions, insertions, substitutions, or modifications to the amino acids may also be allowed, as long as they still retain the capability of the antibody to bind to a specific antigen. The humanized antibody may optionally comprise at least a portion of a constant region of a human immunoglobulin. The “humanized antibody” reserves the antigen specificity similar to that of the original antibody. The “humanized” form of a non-human (for example, mouse antibody) antibody may minimally comprise a chimeric antibody derived from a non-human immunoglobulin sequence. In some cases, CDR residues in a human immunoglobulin (receptor antibody) may be replaced with CDR residues from a non-human species (donor antibody) (such as a mouse, a rat, a rabbit, or a non-human primate) with thedesired properties, affinity, and / or capability. In some cases, FR residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise an amino acid modification that is not present in the receptor antibody or in the donor antibody. These modifications may be made to further improve the properties such as binding affinity of the antibody.

[0097] The term “fully human antibody” generally refers to an antibody that is obtained by transferring a human antibody-encoding gene into a genetically engineered antibody gene- deficient animal to allow the animal to express it. All portions of the antibody (comprising the variable and constant regions of the antibody) are encoded by genes originating from humans. The fully human antibody can greatly reduce the side immune effects caused by heterologous antibodies on the human body. Methods for obtaining the fully human antibody in the art may include the phage display technology, the transgenic mouse technology, the ribosome display technology, the RNA-polypeptide technology, etc.

[0098] The term “Fab” generally refers to a fragment comprising a heavy-chain variable domain and a light-chain variable domain, and also comprising a light-chain constant domain and a heavy-chain first constant domain (CH1). The term “Fab′” generally refers to a fragment that is different from Fab by the addition of a few residues (comprising one or more cysteines from the hinge region of an antibody) to a carboxyl terminus of the heavy-chain CH1. The term “F(ab′)2” generally refers to a dimer of Fab′, comprising an antibody fragment in which two Fab fragments are linked by a disulfide bridge on the hinge region. The term “Fv fragment” generally refers to the smallest antibody fragment that comprises a complete antigen recognition and binding site. In some cases, this fragment may consist of a dimer in which one heavy-chain variable region and one light-chain variable region are tightly non-covalently bound; and the term “dsFv” generally refers to a disulfide-stabilized Fv fragment, with a disulfide bond between a single light-chain variable region and a single heavy-chain variable region. The term “dAb fragment” generally refers to an antibody fragment consisting of a VH domain. In the present application, the term “scFv” generally refers to a monovalent molecule produced by covalently linking and pairing one heavy-chain variable domain with one light- chain variable domain of an antibody by means of a flexible peptide linker. Such scFv molecule may have the following general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL- COOH. The term “BiTE” or “Bispecific T-Cell Engager” generally refers to a bispecific antibody comprising two antibody binding sites, wherein one site binds to a specific protein (i.e. CD19) found primarily on a cancer or diseased cell, and the other site binds simultaneouslyto a receptor / protein (i.e. CD3) on the surface of a T cell, thus linking the T cell to the cancer / diseased cell and killing the cancer / diseased cell.

[0099] The antibody may generally comprise a protein in which at least two heavy chains (HC) and two light chains (LC) are linked to each other by disulfide bonds, or an antigen- binding fragment thereof. Each heavy chain comprises a heavy-chain variable region (VH) and a heavy-chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, a heavy-chain constant region comprises three domains, namely, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light-chain variable region (VL) and a light-chain constant region. The light-chain constant region comprises one domain, i.e., CL. The regions VH and VL may be further subdivided into hyper-denatured regions, called complementarity determining regions (CDR), which alternate with conserved regions called framework regions (FR). Each VH or VL comprises three CDRs and four FRs, which are arranged in the following order from an amino terminus to a carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the naturally occurring heavy and light chains each comprise four FRs (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), most of which have a β-pleated configuration and which are linked by three CDRs to form a loop linkage, and to form a portion of a β-pleated structure in some cases. The CDRs in each chain are closely brought together by the FR, and form, together with the CDRs from another chain, an antigen-binding site of the antibody. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells (for example, effector cells) of the immune system and the first component (Clq) of the classical complement system.

[0100] In the present application, the term “variable” generally refers to the fact that some portions of the sequence of the variable domain of an antibody change drastically, which contributes to the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not uniformly distributed throughout the variable region of the antibody. It is concentrated in three segments, known as complementarity determining regions (CDRs) or hypervariable regions (HVRs), in the light-chain and heavy-chain variable regions. A more highly conserved portion in the variable domain is called a framework (FR). In the art, the CDR of an antibody may be defined by a variety of methods, for example, the Kabat definition rules based on sequence variability (see, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)), the Chothia definition rules based on the location of the structural loop regions (see,Al-Lazikani et al., J Mol Biol 273: 927-48, 1997), and the IMGT definition rules based on the concepts in IMGT-ONTOLOGY and IMGT Scientific chart rules. IMGT refers to the ImMunoGeneTics information system, which is a global reference database for immunogenetics and immunoinformatics (http: / / www.imgt.org). IMGT specializes in immunoglobulins (IGs) or antibodies from humans and other vertebrates, T cell receptors (TRs), major histocompatibility (MH), and immunoglobulin superfamily (IgSF) from vertebrates and non-vertebrates, MH superfamily (MhSF), and immune system related proteins (RPIs).

[0101] In the present application, the term “binding”, “specific binding”, or “specific to” generally refers to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, whereby the existence of a target may be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody binds to an epitope by means of its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to the target with greater affinity, avidity, easiness, and / or duration than it binds to other targets. When an antibody binds to an epitope by means of its antigen binding domain more easily than its binding to a random, unrelated epitope, the antibody is referred to as “specifically binding” to that antigen. “Epitope” refers to a specific atomic group (for example, a sugar side chain, a phosphoryl group, a sulfonyl group) or amino acid, that binds to an antibody on an antigen.

[0102] The term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0103] In the present application, the term “isolated” antibody generally refers to an antibody that has been identified, isolated, and / or recovered from (for example, native or recombinant) components of the environment in which it is produced. Contaminant components of the environment in which it is produced are generally substances that interfere with its investigational, diagnostic or therapeutic use, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. An isolated antibody is generally prepared by at least one purification step. The isolated antibody of the present application generally does not bind to antigens that are non-B7H3 antigens.

[0104] In the present application, the term “isolated nucleic acid molecule” or “isolated polynucleotide” generally refers to a genome, an mRNA, a cDNA, or a synthetic-origin DNA or RNA or a certain combination thereof. It is not associated with all or some of polynucleotides found in nature, or is linked to polynucleotides to which it is not linked in nature.

[0105] In the present application, the term “vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host. It transfers an inserted nucleic acid molecule into and / or between host cells. The vector may include a vector mainly for inserting DNA or RNA into cells, a vector mainly for replicating DNA or RNA, and a vector mainly for expressing DNA or RNA transcription and / or translation. Said vector also includes the vector having a variety of the functions defined above. The vector may be a polynucleotide that may be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector may produce a desired expression product by culturing a suitable host cell containing the vector.

[0106] In the present application, the term “cell” generally refers to an individual cell, a cell line or a cell culture, which may contain or already contain a plasmid or vector comprising a nucleic acid molecule of the present application, or which is capable of expressing the antibody or antigen-binding fragment thereof in the present application. The cell may include a progeny of a single host cell. Due to natural, accidental or deliberate mutations, progeny cells and original parent cells may not be necessarily identical in terms of morphology or genome, as long as they are capable of expressing the antibody or antigen-binding fragment thereof in the present application. The cells may be obtained by transfecting cells in vitro using the vector of the present application. The cells may be prokaryotic cells (for example, Escherichia coli), or eukaryotic cells (for example, yeast cells, for example, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some cases, the cells may be mammalian cells. For example, the mammalian cells may be CHO-K1 cells. In the present application, the term “recombinant cell” generally refers to a cell into which a recombinant expression vector is introduced. A recombinant host cell comprises not only certain specific cells, but also the progenies of these cells.

[0107] As used herein, the term “administering” or “administration” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra- arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0108] The term “treating” or “treatment” refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. Therapeutic benefit can also mean to effect a cure of one or more diseases, conditions, or symptoms under treatment.

[0109] The term “effective amount” or “sufficient amount” refers to the amount of an antibody or other composition that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the physical delivery system in which it is carried.

[0110] For the purposes herein, an effective amount is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect in a subject suffering from cancer. The desired therapeutic effect may include, for example, amelioration of undesired symptoms associated with cancer, prevention of the manifestation of such symptoms before they occur, slowing down the progression of symptoms associated with cancer, slowing down or limiting any irreversible damage caused by cancer, lessening the severity of or curing a cancer, or improving the survival rate or providing more rapid recovery from a cancer.

[0111] The effective amount depends, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the distribution profile of a therapeutic agent (e.g., an anti-B7H3 antibody described herein) or composition within the body, the relationship between a variety of pharmacological parameters (e.g., half-life in the body) and undesired side effects, and other factors such as age and gender, etc.

[0112] The term “pharmaceutically acceptable carrier” refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject. “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in the compositions of the disclosure and that causes no significant adverse toxicological effect on the subject. Non- limiting examples of pharmaceutically acceptable carriers include water, sodium chloride, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may also be substances for providing the formulation with stability, sterility and isotonicity (e.g. antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like) or for providing the formulation with an edible flavor etc. In some instances, the carrier is an agent that facilitates the delivery of an antibody to a target cell or tissue. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present disclosure.

[0113] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0114] The term “cancer” is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, recurrent, soft tissue, or solid, and cancers of all stages and grades including advanced, pre- and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, gynecological cancers (e.g., ovarian, cervical, uterine, vaginal, and vulvar cancers); lung cancers (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, carcinoid tumors, lung adenocarcinoma); breast cancers (e.g., triple-negative breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, cribriform carcinoma, invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, Paget’s disease, Phyllodes tumors); digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors (GIST), gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and esophageal cancer; thyroid cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer (e.g., prostate adenocarcinoma); renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system (e.g., glioblastoma, neuroblastoma, medulloblastoma); skin cancer (e.g., melanoma); bone and soft tissue sarcomas (e.g., Ewing’s sarcoma); lymphomas; choriocarcinomas; urinary cancers (e.g., urothelial bladder cancer); head and neck cancers; and bone marrow and blood cancers (e.g., acute leukemia, chronic leukemia (e.g., chronic lymphocytic leukemia), lymphoma, multiple myeloma). As used herein, a “tumor” comprises one or more cancerous cells.

[0115] The term “survival” refers to a length of time following the diagnosis of a disease and / or beginning or completing a particular course of therapy for a disease (e.g., cancer). The term “overall survival” includes the clinical endpoint describing patients who are alive for a defined period of time after being diagnosed with or treated for a disease, such as cancer. The term “disease-free survival” includes the length of time after treatment for a specific disease (e.g., cancer) during which a patient survives with no sign of the disease (e.g., without known recurrence). In certain embodiments, disease-free survival is a clinical parameter used to evaluate the efficacy of a particular therapy, which is usually measured in units of 1 or 5 years. The term “progression-free survival” includes the length of time during and after treatment for a specific disease (e.g., cancer) in which a patient is living with the disease without additional symptoms of the disease. In some embodiments, survival is expressed as a median or mean value.III. Detailed Description of the Embodiments

[0116] The present disclosure relates to anti-B7H3 antibodies derived from the antibody ch8H9 and designed by using computer algorithms, carrying one or more residue substitutions relative to ch8H9 which can enhance the antigen-binding affinities of the antibodies. The present disclosure also provides the uses of the anti-B7H3 antibodies. A. Anti-B7H3 Antibodies

[0117] In one aspect, the present disclosure provides an isolated antibody that specifically binds to a B7-H3 (CD276) protein. In some embodiments, the anti-B7H3 antibody specifically binds to a human B7-H3 protein. Complementarity Determining Regions (CDRs)

[0118] In some embodiments, the anti-B7H3 antibody comprises one or more complementarity determining regions (CDRs) as disclosed herein. In some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (HCDR1) sequence comprising the sequence of GYX1FTX2X3X4 (SEQ ID NO: 7), wherein X1 is T, D, N, S, or E; X2 is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3 is Y, E, F, or R; and X4 is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6 is S or R; and / or a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein X7 is T, E, R, D, Y, W, N, Q, S, or H; X8is T or Y; X9is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10is T, Y, W, R, N, H, or F; and X11is W, Y, or F.

[0119] In some embodiments, the antibody comprises a light chain complementarity determining region 1 (LCDR1) sequence comprising the sequence of X14X15X16X17X18X19(SEQ ID NO: 19), wherein X14is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17 is S, D, E, N, M, G, or Y; X18 is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and / or a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein X20 is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21 is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22 is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23 is F, W, or Y; X24 is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25is L, W, Y, F, R, K, E, N, H, or S.

[0120] In some embodiments, the antibody comprises a HCDR1 sequence comprising the sequence of GYX1FTX2X3X4 (SEQ ID NO: 7), wherein X1 is T, D, N, S, or E; X2 is N, L, W,Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3 is Y, E, F, or R; and X4 is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6is S or R; and / or a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein X7 is T, E, R, D, Y, W, N, Q, S, or H; X8 is T or Y; X9 is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10 is T, Y, W, R, N, H, or F; and X11is W, Y, or F, and a LCDR1 sequence comprising the sequence of X14X15X16X17X18X19(SEQ ID NO: 19), wherein X14is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17 is S, D, E, N, M, G, or Y; X18is D, N, S, or E; and X19is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and / or a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein X20 is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21 is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22 is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23is F, W, or Y; X24is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25is L, W, Y, F, R, K, E, N, H, or S.

[0121] In some embodiments, the antibody comprises a HCDR1 sequence comprising the sequence of GYX1FTX2X3X4(SEQ ID NO: 7), wherein X1is T, D, N, S, or E; X2is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3is Y, E, F, or R; and X4is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6is S or R; and a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein X7is T, E, R, D, Y, W, N, Q, S, or H; X8is T or Y; X9 is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10 is T, Y, W, R, N, H, or F; and X11 is W, Y, or F, and a LCDR1 sequence comprising the sequence of X14X15X16X17X18X19(SEQ ID NO: 19), wherein X14is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17 is S, D, E, N, M, G, or Y; X18 is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein X20is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21 is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22 is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23is F, W, or Y; X24is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25is L, W, Y, F, R, K, E, N, H, or S.

[0122] In some embodiments, the HCDR1 sequence of the antibody is not GYTFTNYD (SEQ ID NO: 10), the HCDR2 sequence of the antibody is not IFPGDGST (SEQ ID NO: 11) or IFPGDDST (SEQ ID NO: 12), or the HCDR3 sequence of the antibody is notARQTTATWFAY (SEQ ID NO: 13) or ARQTTGTWFAY (SEQ ID NO: 14). In some embodiments, the LCDR1 sequence of the antibody is not QSISDY (SEQ ID NO: 21), or the LCDR3 sequence of the antibody is not QNGHSFPLT (SEQ ID NO: 22). Framework Regions (FRs)

[0123] In some embodiments, the anti-B7H3 antibody comprises one or more framework regions (FRs) as disclosed herein. In some embodiments, the antibody comprises a heavy chain framework region 1 (HFR1) sequence comprising the sequence of QVQLQQSGAELVKPGASVKLSCKX12S (SEQ ID NO: 15), wherein X12 is A or T; a HFR2 sequence comprising the sequence of INWVRQRPX13QGLEWIGW (SEQ ID NO: 16), wherein X13is E or G; a HFR3 sequence comprising the sequence of X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFC (SEQ ID NO: 17), where X31 is Q or Y; and / or a HFR4 sequence comprising the sequence of WGQGTLVTVSA (SEQ ID NO: 18).

[0124] In some embodiments, the antibody comprises a light chain framework region 1 (LFR1) sequence comprising the sequence of a light chain framework region 1 (LFR1) sequence comprising the sequence of X26X27VMTQSPATLSVTPGDRVX28LSCRAS (SEQ ID NO: 23), wherein X26 is D, Q, E, or N; X27 is I, W, E, Y, F, T, N, R, K, or Q; and X28 is S or T; a LFR2 sequence comprising the sequence of LX29WYQQKSHESPRLLIK (SEQ ID NO: 24), wherein X29is H, Y, N, or S; a LFR3 sequence comprising the sequence of QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID NO: 25), wherein X30is S, D, E, A, N, Q, T, P, or V; and / or a LFR4 sequence comprising the sequence of FGAGTKLELK (SEQ ID NO: 26).

[0125] In some embodiments, the antibody comprises a HFR1 sequence comprising the sequence of QVQLQQSGAELVKPGASVKLSCKX12S (SEQ ID NO: 15), wherein X12 is A or T; a HFR2 sequence comprising the sequence of INWVRQRPX13QGLEWIGW (SEQ ID NO: 16), wherein X13is E or G; a HFR3 sequence comprising the sequence of X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFC (SEQ ID NO: 17), where X31 is Q or Y; and / or a HFR4 sequence comprising the sequence of WGQGTLVTVSA (SEQ ID NO: 18), and a LFR1 sequence comprising the sequence of X26X27VMTQSPATLSVTPGDRVX28LSCRAS (SEQ ID NO: 23), wherein X26is D, Q, E, or N; X27 is I, W, E, Y, F, T, N, R, K, or Q; and X28 is S or T; a LFR2 sequence comprising the sequence of LX29WYQQKSHESPRLLIK (SEQ ID NO: 24), wherein X29is H, Y, N, or S; aLFR3 sequence comprising the sequence of QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID NO: 25), wherein X30 is S, D, E, A, N, Q, T, P, or V; and / or a LFR4 sequence comprising the sequence of FGAGTKLELK (SEQ ID NO: 26).

[0126] In some embodiments, the antibody comprises a HFR1 sequence comprising the sequence of QVQLQQSGAELVKPGASVKLSCKX12S (SEQ ID NO: 15), wherein X12is A or T; a HFR2 sequence comprising the sequence of INWVRQRPX13QGLEWIGW (SEQ ID NO: 16), wherein X13 is E or G; a HFR3 sequence comprising the sequence of X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFC (SEQ ID NO: 17), where X31 is Q or Y; and a HFR4 sequence comprising the sequence of WGQGTLVTVSA (SEQ ID NO: 18), and a LFR1 sequence comprising the sequence of X26X27VMTQSPATLSVTPGDRVX28LSCRAS (SEQ ID NO: 23), wherein X26 is D, Q, E, or N; X27is I, W, E, Y, F, T, N, R, K, or Q; and X28is S or T; a LFR2 sequence comprising the sequence of LX29WYQQKSHESPRLLIK (SEQ ID NO: 24), wherein X29 is H, Y, N, or S; a LFR3 sequence comprising the sequence of QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID NO: 25), wherein X30is S, D, E, A, N, Q, T, P, or V; and a LFR4 sequence comprising the sequence of FGAGTKLELK (SEQ ID NO: 26). CDRs and FRs

[0127] In some embodiments, the antibody comprises a HCDR1 sequence comprising the sequence of GYX1FTX2X3X4 (SEQ ID NO: 7), wherein X1 is T, D, N, S, or E; X2 is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3 is Y, E, F, or R; and X4 is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5is G or D; and X6 is S or R; and a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein X7 is T, E, R, D, Y, W, N, Q, S, or H; X8 is T or Y; X9is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10is T, Y, W, R, N, H, or F; and X11 is W, Y, or F, and a HFR1 sequence comprising the sequence of QVQLQQSGAELVKPGASVKLSCKX12S (SEQ ID NO: 15), wherein X12 is A or T; a HFR2 sequence comprising the sequence of INWVRQRPX13QGLEWIGW (SEQ ID NO: 16), wherein X13is E or G; a HFR3 sequence comprising the sequence of X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFC (SEQ ID NO: 17), where X31 is Q or Y; and a HFR4 sequence comprising the sequence of WGQGTLVTVSA (SEQ ID NO:18), and a LCDR1 sequence comprising the sequence of X14X15X16X17X18X19 (SEQ ID NO: 19), wherein X14 is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17is S, D, E, N, M, G, or Y; X18is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein X20is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23is F, W, or Y; X24is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25 is L, W, Y, F, R, K, E, N, H, or S, and a LFR1 sequence comprising the sequence of X26X27VMTQSPATLSVTPGDRVX28LSCRAS (SEQ ID NO: 23), wherein X26is D, Q, E, or N; X27is I, W, E, Y, F, T, N, R, K, or Q; and X28is S or T; a LFR2 sequence comprising the sequence of LX29WYQQKSHESPRLLIK (SEQ ID NO: 24), wherein X29 is H, Y, N, or S; a LFR3 sequence comprising the sequence of QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID NO: 25), wherein X30is S, D, E, A, N, Q, T, P, or V; and a LFR4 sequence comprising the sequence of FGAGTKLELK (SEQ ID NO: 26). In some embodiments, X2 is L, X4 is W, X5 is R, X6 is Y, X7 is E, X8 is Y, X9is W, X10is Y, X18is N or S, X19is W, X20is W, X21is W, X23is W, X24is Y, X25is W, and / or X27is W.

[0128] In some embodiments, the antibody comprises a HCDR1 sequence of GYTFTNYW (SEQ ID NO: 27). In some embodiments, the antibody comprises a HCDR2 sequence of IFPGDDRT (SEQ ID NO: 28). In some embodiments, the antibody comprises a HCDR3 sequence of ARQTTWTWFAY (SEQ ID NO: 29). In some embodiments, the antibody comprises a LFR1 sequence of DWVMTQSPATLSVTPGDRVSLSCRAS (SEQ ID NO: 30). In some embodiments, the antibody comprises a LCDR1 sequence of QSISNY (SEQ ID NO: 31). HV and LV regions

[0129] In some embodiments, the anti-B7H3 antibody comprises one or more heavy chain variable (HV) regions, and / or light chain variable (LV) regions as disclosed herein. In some embodiments, the antibody comprises a VH region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 32-41. In some embodiments, the antibody comprises a VL region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 42-54. In someembodiments, the antibody comprises a VH region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 32-41, and a VL region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 42-54.

[0130] In some embodiments, the antibody comprises a VH region comprising an amino acid sequence of any one of SEQ ID NOs: 32-41. In some embodiments, the antibody comprises a VL region comprising an amino acid sequence of any one of SEQ ID NOs: 42-54. In some embodiments, the antibody comprises a VH region comprising an amino acid sequence of any one of SEQ ID NOs: 32-41 and a VL region comprising an amino acid sequence of any one of SEQ ID NOs: 42-54. HCs and LCs

[0131] In some embodiments, the anti-B7H3 antibody comprises one or more heavy chain (HC), and / or light chains (LC) sequences as disclosed herein. In some embodiments, the antibody comprises a HC comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 70-75. In some embodiments, the antibody comprises a LC comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 76-80. In some embodiments, the antibody comprises a HC comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 70-75, and a LC comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 76-80.

[0132] In some embodiments, the antibody comprises a HC comprising an amino acid sequence of any one of SEQ ID NOs: 70-75. In some embodiments, the antibody comprises a LC comprising an amino acid sequence of any one of SEQ ID NOs: 76-80. In some embodiments, the antibody comprises a HC comprising an amino acid sequence of any one of SEQ ID NOs: 70-75 and a LC comprising an amino acid sequence of any one of SEQ ID NOs: 76-80.Antibodies

[0133] In some embodiments, the anti-B7H3 antibody is a full-length monoclonal antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a chimeric antibody, a camelized single-domain antibody, a Fab, a F(ab’)2, a Fab′, a F(ab)2, a Fv fragment, a scFv, a di-scFv, a BiTE, a dAb, or a bivalent scFv. In some embodiments, the antibody is an scFv. In some embodiments, the anti-B7H3 scFv comprises a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 56-69 and 92-105. In some embodiments, the anti-B7H3 scFv comprises a sequence of any one of SEQ ID NOs: 56-69 and 92-105.

[0134] In some embodiments, the anti-B7H3 antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises one or more complementarity determining regions (CDRs), framework regions (FRs), heavy chain variable (HV) regions, light chain variable (LV) regions, heavy chain (HC), and / or light chains (LC) sequences, as disclosed herein.

[0135] In some embodiments, the anti-B7H3 antibody is a chimeric antibody. In some embodiments, the chimeric antibody comprises one or more complementarity determining regions (CDRs), framework regions (FRs), heavy chain variable (HV) regions, light chain variable (LV) regions, heavy chain (HC), and / or light chains (LC) sequences, as disclosed herein.

[0136] In some embodiments, the anti-B7H3 antibody is a humanized antibody. In some embodiments, the humanized antibody comprises one or more complementarity determining regions (CDRs), framework regions (FRs), heavy chain variable (HV) regions, light chain variable (LV) regions, heavy chain (HC), and / or light chains (LC) sequences, as disclosed herein.

[0137] In some embodiments, the anti-B7H3 antibody is a fully human antibody. In some embodiments, the fully human antibody comprises one or more complementarity determining regions (CDRs), framework regions (FRs), heavy chain variable (HV) regions, light chain variable (LV) regions, heavy chain (HC), and / or light chains (LC) sequences, as disclosed herein. Anti-B7H3 Antibodies with VL-D31N Mutation

[0138] In one aspect, the present disclosure provides an anti-human B7-H3 antibody (anti- huB7H3 antibody comprising a HCDR1 sequence comprising the sequence of GYTFTNYD (SEQ ID NO: 10); a HCDR2 sequence comprising the sequence of IFPGDDST (SEQ ID NO: 12); a HCDR3 sequence comprising the sequence of ARQTTGTWFAY (SEQ ID NO: 14); a LCDR1 sequence comprising the sequence of QSISNY (SEQ ID NO: 31); a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNGHSFPLT (SEQ ID NO: 22).

[0139] In some embodiments, the antibody comprises a HFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121 or 122; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125 or 126; and / or a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127 or 128. In some embodiments, the HFR1 sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 121 or 122 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 121 or 122. In some embodiments, the HFR2 sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 123 or 124 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 123 or 124. In some embodiments, the HFR3 sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 125 or 126 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 125 or 126. In some embodiments, the HFR4 sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 127 or 128 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 127 or 128.

[0140] In some embodiments, the antibody comprises a HFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121 or 122; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125 or 126; and a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127 or 128. In some embodiments, the antibody comprises a HFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125; and a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127. In some embodiments, the antibody is a humanizedantibody. In some embodiments, the humanized antibody comprises a HFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 122; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 126; and a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 128.

[0141] In some embodiments, the antibody comprises a HFR1 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 121 or 122; a HFR2 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 125 or 126; and a HFR4 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 127 or 128. In some embodiments, the antibody comprises a HFR1 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 121; a HFR2 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 123; a HFR3 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 125; and a HFR4 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 127. In some embodiments, the antibody is a humanized antibody. In some embodiments, the humanized antibody comprises a HFR1 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 122; a HFR2 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 124; a HFR3 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 126; and a HFR4 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 128.

[0142] In some embodiments, the antibody comprises a HFR1 sequence comprising the sequence of SEQ ID NO: 121 or 122; a HFR2 sequence comprising the sequence of SEQ ID NO: 123 or 124; a HFR3 sequence comprising the sequence of SEQ ID NO: 125 or 126; and a HFR4 sequence comprising the sequence of SEQ ID NO: 127 or 128. In a particular embodiment, the antibody comprises a HFR1 sequence comprising the sequence of SEQ ID NO: 121; a HFR2 sequence comprising the sequence of SEQ ID NO: 123; a HFR3 sequence comprising the sequence of SEQ ID NO: 125; and a HFR4 sequence comprising the sequence of SEQ ID NO: 127. In another particular embodiment, the antibody comprises a HFR1 sequence comprising the sequence of SEQ ID NO: 122; a HFR2 sequence comprising the sequence of SEQ ID NO: 124; a HFR3 sequence comprising the sequence of SEQ ID NO: 126; and a HFR4 sequence comprising the sequence of SEQ ID NO: 128.

[0143] In some embodiments, the antibody comprises a LFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 135-138; and / or a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139 or 140. In some embodiments, the LFR1 sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 129 or 130 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 129 or 130. In some embodiments, the LFR2 sequence comprises a sequence having at least 80%, 85%, or 90% identity to any one of SEQ ID NOs: 131-134 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 131-134. In some embodiments, the LFR3 sequence comprises a sequence having at least 80%, 85%, or 90% identity to any one of SEQ ID NOs: 135-138 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 135-138. In some embodiments, the LFR4 sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 139 or 140 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 139 or 140.

[0144] In some embodiments, the antibody comprises a LFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 135-138; and a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139 or 140. In some embodiments, the antibody comprises a LFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129; a LFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 131; a LFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 135; and a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139. In some embodiments, the antibody is a humanized antibody. In some embodiments, the humanized antibody comprises a LFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 132- 134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 136-138; and a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 140.

[0145] In some embodiments, the antibody comprises a LFR1 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 135-138; and a LFR4 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 139 or 140. In some embodiments, the antibody comprises a LFR1 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 129; a LFR2 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 131; a LFR3 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 135; and a LFR4 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 139. In some embodiments, the antibody is a humanized antibody. In some embodiments, the humanized antibody comprises a LFR1 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 130; a LFR2 sequence comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 132- 134; a LFR3 sequence comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 136-138; and a LFR4 sequence comprising a sequence having at least 90% identity to SEQ ID NO: 140.

[0146] In some embodiments, the antibody comprises a LFR1 sequence comprising the sequence of SEQ ID NO: 129 or 130; a LFR2 sequence comprising the sequence of any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising the sequence of any one of SEQ ID NOs: 135-138; and a LFR4 sequence comprising the sequence of SEQ ID NO: 139 or 140. In some embodiments, the antibody comprises a LFR1 sequence comprising the sequence of SEQ ID NO: 129; a LFR2 sequence comprising the sequence of SEQ ID NO: 131; a LFR3 sequence comprising the sequence of SEQ ID NO: 135; and a LFR4 sequence comprising the sequence of SEQ ID NO: 139. In some embodiments, the antibody is a humanized antibody. In some embodiments, the humanized antibody comprises a LFR1 sequence comprising the sequence of SEQ ID NO: 130; a LFR2 sequence comprising the sequence of SEQ ID NO: 132, 133, or 134; a LFR3 sequence comprising the sequence of SEQ ID NO: 136, 137, or 138; and a LFR4 sequence comprising the sequence of SEQ ID NO: 140. In a particular embodiment, the humanized antibody comprises a LFR1 sequence comprising the sequence of SEQ ID NO: 130; a LFR2 sequence comprising the sequence of SEQ ID NO: 132; a LFR3 sequence comprising the sequence of SEQ ID NO: 136; and a LFR4 sequence comprising the sequence of SEQ ID NO: 140. In another particular embodiment, the humanized antibody comprises a LFR1 sequence comprising the sequence of SEQ ID NO: 130; a LFR2 sequence comprisingthe sequence of SEQ ID NO: 133; a LFR3 sequence comprising the sequence of SEQ ID NO: 137; and a LFR4 sequence comprising the sequence of SEQ ID NO: 140. In yet another particular embodiment, the humanized antibody comprises a LFR1 sequence comprising the sequence of SEQ ID NO: 130; a LFR2 sequence comprising the sequence of SEQ ID NO: 134; a LFR3 sequence comprising the sequence of SEQ ID NO: 138; and a LFR4 sequence comprising the sequence of SEQ ID NO: 140.

[0147] Table 1 below presents the complementarity determining regions (CDRs) and framework regions (FRs) of an anti-huB7H3 antibody. In some embodiments, the antibody comprises six amino acid substitutions: A24T, E42G, G56D, and A102G in the heavy chain and S20T and H34Y in the light chain. These substitutions are indicated in Table 1 as italicized and underlined. Additionally, in some embodiments, the antibody further comprises a D31N substitution in the light chain, which is denoted in bold in Table 1. In some embodiments, the antibody is a humanized variant that includes one or more humanization mutations located within the framework regions. As shown in Table 1, the italicized symbols reflect amino acids introduced by the in silico humanization process (italicized, not underscored) or indicate “6m” mutations (italicized and underscored). In the “Element” column, “hu” indicates that the Element is part of a humanized antibody and “1rd”, “2rd”, and “3-5rds” specifies that the humanization occurred over 1, 2, or 3-5, respectively, rounds of in silico humanization. No change in the variable sequences occurred with 4 or 5 rounds compared to 3. Table 1. hu8H9-6m w / VL-D31N elements

[0148] In some embodiments, the antibody comprises a variable heavy (VH) chain amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 141 or 142. In some embodiments, the VH sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 141 or 142, or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 141 or 142.

[0149] In some embodiments, the antibody comprises a variable light (VL) chain amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 143-146. In some embodiments, the VL sequence comprises a sequence having at least 80%, 85%, or 90% identity to any one of SEQ ID NOs: 143-146, or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 143-146.

[0150] In some embodiments, the antibody comprises a VH sequence that has at least 80% sequence identity to SEQ ID NO: 141 or 142, and a VL sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 143-146. In some embodiments, the antibody comprises a VH sequence that has at least 80% sequence identity to SEQ ID NO: 141, and a VL sequence that has at least 80% sequence identity to SEQ ID NO: 143. In some embodiments, the antibody comprises a VH sequence that has at least 80% sequence identity to SEQ ID NO: 142, and a VL sequence that has at least 80% sequence identity to SEQ ID NO: 144. In some embodiments, the antibody comprises a VH sequence that has at least 80% sequence identity to SEQ ID NO: 142, and a VL sequence that has at least 80% sequence identity to SEQ ID NO: 145. In some embodiments, the antibody comprises a VH sequence that has at least 80% sequence identity to SEQ ID NO: 142, and a VL sequence that has at least 80% sequence identity to SEQ ID NO: 146.

[0151] In some embodiments, the antibody comprises a VH sequence that has at least 90% sequence identity to SEQ ID NO: 141 or 142, and a VL sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 143-146. In some embodiments, the antibody comprisesa VH sequence that has at least 90% sequence identity to SEQ ID NO: 141, and a VL sequence that has at least 90% sequence identity to SEQ ID NO: 143. In some embodiments, the antibody comprises a VH sequence that has at least 90% sequence identity to SEQ ID NO: 142, and a VL sequence that has at least 90% sequence identity to SEQ ID NO: 144. In some embodiments, the antibody comprises a VH sequence that has at least 90% sequence identity to SEQ ID NO: 142, and a VL sequence that has at least 90% sequence identity to SEQ ID NO: 145. In some embodiments, the antibody comprises a VH sequence that has at least 90% sequence identity to SEQ ID NO: 142, and a VL sequence that has at least 90% sequence identity to SEQ ID NO: 146.

[0152] In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 141 or 142 and the VL sequence of any one of SEQ ID NOs: 143-146. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 143. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 142 and the VL sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 142 and the VL sequence of SEQ ID NO: 145. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 142 and the VL sequence of SEQ ID NO: 146.

[0153] Exemplary VH and VL sequences are provided in Table 2, where the CDRs are indicated by underlining, the D31N substitution in the VL sequence is shown in bold, and all other mutations, including the six substitutions by Ahmed et al. [1] (“6m” mutations) and the humanization mutations, are presented in italics. Table 2. hu8H9-6m w / VL-D31N VH&VLID ID

[0154] As disclosed herein, the antibody can be a full-length monoclonal antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a chimeric antibody, a camelized single-domain antibody, a Fab, a F(ab’)2, a Fab′, a F(ab)2, a Fv fragment, a scFv, a di-scFv, a BiTE, a dAb, or a bivalent scFv. In some embodiments, the antibody is conjugated with a payload, such as a therapeutic drug or diagnostic agent. In some embodiments, the antibody is a bispecific antibody targeting B7-H3 and another molecule / protein / antigen. In a particular embodiment, the bispecific antibody binds to both B7-H3 and CD3.

[0155] In certain embodiments, the antibody is provided in the form of a single-chain variable fragment (scFv). In some embodiments, the scFv has a VH-VL orientation from the 5’ to the 3’ end of the nucleic acid sequence encoding the scFv. In some embodiments, the scFv has a VL-VH orientation from the 5’ to the 3’ end.

[0156] In some embodiments, the scFv comprises a sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148. In some embodiments, the scFv comprises a sequence having at least 80%, 85%, or 90% identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148, or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148. In some embodiments, the scFv comprises a sequence selected from SEQ ID NOs: 60, 61, 104, 147 and 148. In some embodiments, the scFv comprises the six substitutions and the VL-D31N substitution. In some embodiments, the scFv comprises the sequence of SEQ ID NO: 147 or 148. Some exemplary scFv sequences are listed in Table 3, where the VH region and VL region are linked through a (GGGGS)3 linker. Table 3. hu8H9-6m w / VL-D31N scFv DD

[0157] In certain embodiments, the antibody is provided in the form of a full-length antibody comprising a heavy chain (HC) and a light chain (LC). In some embodiments, the HC comprises a VH-CH1 sequence that has at least 80% sequence identity to SEQ ID NO: 149. In some embodiments, the HC sequence comprises a VH-CH1 sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 149 or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 149. In some embodiments, the HC comprises the VH-CH1 sequence of SEQ ID NO: 149. In some embodiments, the LC comprises a VL-CL sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 150-152. In some embodiments, the LC sequence comprises a VL-CL sequence having at least 80%, 85%, or 90% identity to any one of SEQ ID NOs: 150-152 or comprises a VL-CL sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 150-152. In some embodiments, the LC comprises the VL-CL sequence of any one of SEQ ID NOs: 150-152.

[0158] In some embodiments, the antibody comprises a HC comprising the sequence of SEQ ID NO: 149 and an LC comprising the sequence of any one of SEQ ID NOs: 150-152. In a particular embodiment, the antibody comprises a HC comprising the sequence of SEQ ID NO: 149 and an LC comprising the sequence SEQ ID NO: 150. In another particular embodiment, the antibody comprises a HC comprising the sequence of SEQ ID NO: 149 and a LC comprising the sequence SEQ ID NO: 151. In yet another particular embodiment, the antibody comprises a HC comprising the sequence of SEQ ID NO: 149 and a LC comprising the sequence SEQ ID NO: 152. Table 4 provides some exemplary sequences of VH-CH1 of HC and VL-CL of LC of the anti-huB7H3 antibody.Table 4. hu8H9-6m w / VL-D31N HC&LC elements O: O: O: O:Bispecific Antibodies

[0159] In some embodiments, the anti-B7H3 antibody is a bispecific antibody. In some embodiments, the bispecific antibody comprises one or more complementarity determining regions (CDRs), framework regions (FRs), heavy chain variable (HV) regions, light chainvariable (LV) regions, heavy chain (HC), and / or light chains (LC) sequences, as disclosed herein.

[0160] In some embodiments, the bispecific antibody binds to both B7-H3 and CD3. In some embodiments, the CD3-binding portion of the bispecific antibody comprises a VH region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 81 and / or a VL region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 82. In some embodiments, the CD3-binding portion of the bispecific antibody comprises a VH region comprising an amino acid sequence of SEQ ID NO: 81 and a VL region comprising an amino acid sequence of SEQ ID NO: 82.

[0161] In some embodiments, the CD3-binding portion is an scFv. In some embodiments, the scFv comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In some embodiments, the scFv comprises an amino acid sequence of SEQ ID NO: 87.

[0162] In some embodiments, the bispecific antibody comprises a first scFv binding to B7- H3 and a second scFv binding to CD3. In some embodiments, the first scFv binding to B7-H3 comprises a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 56-69 and 92-105. In some embodiments, the second scFv binding to CD3 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti-B7H3 scFv comprising a sequence of any one of SEQ ID NOs: 56-69 and 92-105 and an anti-CD3 scFv comprising a sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 88-91 and 106-119. In some embodiments, the bispecific antibody comprises a sequence of any one of SEQ ID NOs: 88-91 and 106-119.

[0163] In some embodiments, the bispecific antibody comprises a first scFv binding to B7- H3 and a second scFv binding to CD3. In some embodiments, the first scFv binding to B7-H3 comprises a VH sequence that has at least 80% sequence identity to SEQ ID NO: 141 or 142 and a VL sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 143- 146. In some embodiments, the VH sequence comprises a sequence having at least 80%, 85%, or 90% identity to SEQ ID NO: 141 or 142, or comprises a sequence having at least 95%, 96%,97%, 98%, or 99% identity to SEQ ID NO: 141 or 142. In some embodiments, the VL sequence comprises a sequence having at least 80%, 85%, or 90% identity to any one of SEQ ID NOs: 143-146, or comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 143-146.

[0164] In some embodiments, the first scFv binding to B7-H3 comprises the VH sequence of SEQ ID NO: 141 or 142 and the VL sequence of any one of SEQ ID NOs: 143-146. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 143. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 142 and the VL sequence of SEQ ID NO: 144. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 142 and the VL sequence of SEQ ID NO: 145. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO: 142 and the VL sequence of SEQ ID NO: 146.

[0165] In some embodiments, the first scFv binding to B7-H3 comprises a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148. In some embodiments, the second scFv binding to CD3 comprises a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti-B7H3 scFv comprising the sequence of any one of SEQ ID NOs: 60, 61, 104, 147 and 148, and an anti-CD3 scFv comprising the sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti-B7H3 scFv comprising the sequence of SEQ ID NO: 60 and an anti-CD3 scFv comprising the sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti-B7H3 scFv comprising the sequence of SEQ ID NO: 61 and an anti-CD3 scFv comprising the sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti-B7H3 scFv comprising the sequence of SEQ ID NO: 104 and an anti-CD3 scFv comprising the sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti-B7H3 scFv comprising the sequence of SEQ ID NO: 147 and an anti-CD3 scFv comprising the sequence of SEQ ID NO: 87. In some embodiments, the bispecific antibody comprises an anti- B7H3 scFv comprising the sequence of SEQ ID NO: 148 and an anti-CD3 scFv comprising the sequence of SEQ ID NO: 87.Antibody Conjugates

[0166] As disclosed herein, the antibodies can be used to deliver desired payload, linked to the antibody, to the target cells (e.g., cancer cells), including without limitation therapeutic agents and / or detectable agents. In some embodiments, the anti-B7H3 antibody is attached to at least one payload. In some embodiments, one anti-B7H3 antibody can deliver one, two, three, four, five, six, seven, eight or more identical or different payloads into the cell. In some instances, one anti-B7H3 antibody delivers multiple copies of a payload molecule into the cell. In other instances, one anti-B7H3 antibody delivers multiple structurally different payloads into the cell. As used herein, a payload can be any desired molecule, complex, or other entity that can be attached to the anti-B7H3 antibody. The binding of one or more payloads to the anti-B7H3 antibody will not impact the binding affinity of the antibody to the target B7-H3.

[0167] Any useful and desired payload can be delivered using the method provided herein. Such flexibility allows the anti-B7H3 antibodies to be used in multiple applications, such as diagnostics, prognostics, or theranostics. The term “theranostics” refers to therapy-related diagnostics, including without limitation using diagnostic information to predict or monitor drug response.

[0168] In some embodiments, the antibody can be conjugated to a therapeutic agent. Examples of therapeutic agents that may be attached to the anti-H7B3 antibodies provided herein include, but are not limited to, antitumor agents, antineoplastic agents, prodrugs, lysosome destabilizing agents (e.g., chloroquine), alkylating agents, alkaloids, allosteric inhibitors, antifolics, anti-inflammatory agents, antibiotics, antibacterials, antifungals, antifibrotic agents, anti-infective agents, anti-parasitic agents, antiviral agents, antimycobacterial agents, antineoplastic agents, antiprotozoal agents, antiviral agents, drugs, bioactive peptides, steroid hormones, nucleic acids, photosensitizer substances, radio- pharmaceuticals, antiprion agents, and combinations thereof. In some embodiments, the therapeutic agent is an antitumor agent, a radioisotope, a drug agent, a nanoparticle, an immune toxin, or a combination thereof.

[0169] For example, the therapeutic agent may be an antitumor agent selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule active agent (e.g., a microtubule inhibitor); an alkylating agent; a retinoid, a carotenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; a mTOR inhibitor; an antimetabolite; a platincompound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparanase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; a PARP inhibitor, a Tyrosine kinase inhibitor, a PI3K inhibitor, an AKT inhibitor, an EGFR inhibitor, an antitumor antibiotic; a nitrosourea, a compound targeting / decreasing protein or lipid kinase activity, a compound targeting / decreasing protein or lipid phosphatase activity, any further anti-angiogenic compound, and combinations thereof.

[0170] Specific examples of antitumor agents include, but are not limited to, azacitidine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and combinations thereof. Additional examples of antitumor and other therapeutic agents are known in the art.

[0171] In some embodiments, the antitumor agent is a tubulin inhibitor. The terms “tubulin inhibitor”, “microtubule inhibitor” and “mitotic inhibitor” are used interchangeably herein to refer to a drug that inhibits mitosis, or cell division, and is used in treating cancer and other diseases. Specific examples of tubule inhibitors include, but are not limited to, monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), maytansine, maytansinoid, mertansine (emtansine, DM1), ravtansine (soravtansine, DM4), tubulysin, halichondrin (eribulin), cryptophycin, EG5 inhibitor, and any derivative thereof. MMAE and MMAF, both derived from Dolastatin 10, are antimitotic agents which inhibit cell division by blocking the polymerisation of tubulin. MMAE is more hydrophobic than MMAF. MMAF with a charged C-terminal phenylalanine that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE. Maytansine such as DM1 and DM4, blocks the polymerization of tubulin dimers by inhibiting the formation of mature microtubules. These tubulin inhibitors are the common payloads used in clinical ADC drugs. For example, MMAF is part of the approved drug belantamab mafodotin in multiple myeloma and some experimental anti-cancer antibody- drug conjugates (ADCs) such as vorsetuzumab mafodotin and SGN-CD19A. MMAE is another antimitotic auristatin that is often conjugated to a monoclonal antibody (MAb), such as Brentuximab (cAC10), Glembatumumab (CR011, CDX-011), AGS67E, Sofituzumab, Polatuzumab, Enfortumab, Pinatuzumab, Lifastuzumab, Brentuximab, Glembatumumab,Tisotumab, and Indusatumab. Any tubulin inhibitor and other ADC payload listed in Wang et al.

[0027] can be linked to an anti-B7H3 antibody as disclosed herein.

[0172] In some embodiments, the antitumor agent is a DNA inhibitor. DNA inhibitors act on the whole cell cycle by destroying DNA through double-strand breakage, alkylation, chimerism, crosslinking, causing cytotoxic effects, and having therapeutic effect on solid tumors. Specific examples of DNA inhibitors include, but are not limited to, alkylator, duocarmycin, duocarmycin DM, calicheamicin, pyrrolobenzodiazepine (PDB), enediyne, uncialamycin, topoisomerase inhibitor, topotecan, camptothecin (CPT), exatecan, and any derivative thereof.

[0173] In some embodiments, the antitumor agent is an RNA inhibitor. RNA inhibitors are small molecule agents that specifically target RNA to kill both dividing and dormant tumor cells. RNA inhibitors can be used as ADC payloads effective in both fast and slow- proliferating cells and against tumor drug resistance and tumor recurrence. Specific examples of RNA inhibitors include, but are not limited to, RNA splicing inhibitor, RNA polymerase II inhibitor, thailanstatin, amatoxin, and any derivative thereof.

[0174] In some embodiments, the antibody can be conjugated to a detection agent. In some embodiments, the detection agent is a diagnostic or imaging agent, or both. In some embodiments, the detection agent comprises at least one magnetic label, fluorescent moiety, enzyme, light emitting particle, chemiluminescent probe, metal particle, non-metal colloidal particle, polymeric dye particle, pigment molecule, electrochemically active species, semiconductor nanocrystal, nanoparticle, quantum dot, gold particles, fluorophore, or radioactive label.

[0175] In still other embodiments, the agent / payload may be used for both detection for diagnostic purposes and simultaneously for therapeutic purposes. As a non-limiting example, a radioactive label could be used to detect and / or kill target cells.

[0176] In some cases, the agent / payload may be attached to the Anti-B7H3 antibody covalently, including without limitation direct conjugation to the B7H3-binding antibody, or via a linker entity, or both. In other cases, the agent / payload may be attached to the anti-B7H3 antibody non-covalently. In a non-limiting example, the anti-B7H3 antibody may be conjugated to a biotin moiety, and the payload could be attached to a streptavidin. In this example, the biotin-streptavidin bond would provide the non-covalent attachment between the anti-B7H3 antibody and the payload. In another example of non-covalent binding between theanti-B7H3 antibody and the payload, the payload is conjugated to a second antibody, and the second antibody binds to the anti-B7H3 antibody. In yet other cases, e.g., in the case of multiple payloads linked to the anti-B7H3 antibodies, the payloads may be attached both covalently and non-covalently. Antibodies with Enhanced Affinities

[0177] In some embodiments, the present application provides an anti-B7H3 antibody with a high binding affinity. The term “high binding affinity”, as used herein refers to a high degree of tightness with which a particular ligand binds to its partner. Affinities can be measured by any available method, including those known in the art. In some embodiments, an affinity can be considered to be a high binding affinity if the Kdis about 500 pM or less (e.g., below about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, etc.) in binding assays.

[0178] In some embodiments, the present application provides an anti-B7H3 antibody with enhanced binding affinity. An antibody with “enhanced affinity” or “enhanced binding affinity” or “enhanced antigen-binding affinity” refers to an antibody, compared to a parent or reference antibody, that shows an improvement in the affinity for an antigen. In some embodiments, such improvement, or enhanced affinity, is caused by one or more amino acid substitutions in the sequence of the antibody. In some embodiments, one or more amino acid substitutions in the sequence of the antibody result in an improvement in the affinity of the antibody for an antigen compared to a parent or reference antibody which does not possess such substitutions. For example, an antibody with enhanced affinity for B7-H3 refers to an antibody that has greater affinity for B7-H3 than a reference antibody (e.g., ch8H9, ch8H9[3mut], hu[JBC]8H9-6m, or hu[1rd]8H9-6m), wherein the antibody and the reference antibody (e.g., ch8H9, ch8H9[3mut], hu[JBC]8H9-6m, or hu[1rd]8H9-6m) differ in at least one amino acid residue. In some instances, such amino acid substitutions occur in one or more complementarity determining regions (CDRs) of the antibody. In other instances, such substitutions occur in one or more framework regions (FRs) of the antibody. In yet other instances, such substitutions occur in both CDRs and FRs of the antibody.

[0179] In some embodiments, an antibody can be considered as having enhanced antigen- binding affinity over a reference antibody if the antibody shows a stronger affinity (e.g., the Kd is lower) for the antigen. In some embodiments, an anti-B7H3 antibody can be considered ashaving enhanced antigen-binding affinity than a reference antibody (e.g., ch8H9, ch8H9[3mut], hu[JBC]8H9-6m, or hu[1rd]8H9-6m) if the Kd of the anti-B7H3 antibody is about 90% or less (e.g., about 89%, about 88%, about 87%, about 86%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1% or less) of the Kdof the reference antibody. Similarly, an anti-B7H3 antibody can be considered as having enhanced antigen-binding affinity than a reference antibody (e.g., ch8H9, ch8H9[3mut], hu[JBC]8H9-6m, or hu[1rd]8H9-6m) if the “total stabilizing energy” as determined by PPCheck[7] of the anti-B7H3 antibody is about 0.5 kJ / mol or more (e.g., about 1 kJ / mol, about 2 kJ / mol, about 3 kJ / mol, about 4 kJ / mol, about 5 kJ / mol, about 6 kJ / mol, about 7 kJ / mol, about 8 kJ / mol, about 9 kJ / mol, about 10 kJ / mol, about 11 kJ / mol, about 12 kJ / mol, about 13 kJ / mol, about 14 kJ / mol, about 15 kJ / mol, about 16 kJ / mol, about 17 kJ / mol, about 18 kJ / mol, about 19 kJ / mol, about 20 kJ / mol, about 25 kJ / mol, about 30 kJ / mol about 35 kJ / mol, about 40 kJ / mol, about 45 kJ / mol, about 50 kJ / mol, about 55 kJ / mol, about 60 kJ / mol, about 65 kJ / mol, about 70 kJ / mol, about 75 kJ / mol, about 80 kJ / mol, about 85 kJ / mol, about 90 kJ / mol, about 95 kJ / mol, about 100 kJ / mol or more) lower than the reference antibody. Competing Antibodies

[0180] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that competes with the isolated antibody described herein for binding to the human B7-H3 (CD276) protein.

[0181] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising one or more complementarity determining regions (CDRs) as disclosed herein. In some embodiments, the antibody competes with an antibody comprising a HCDR1 sequence comprising the sequence of GYX1FTX2X3X4 (SEQ ID NO: 7), wherein X1 is T, D, N, S, or E; X2 is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3 is Y, E, F, or R; and X4is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6 is S or R; and / or a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein X7 is T, E, R, D, Y, W, N, Q, S, or H; X8is T or Y; X9is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10is T, Y, W, R, N, H, or F; and X11is W, Y, or F, and a LCDR1 sequence comprising the sequence of X14X15X16X17X18X19 (SEQ ID NO: 19), wherein X14 is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F,T, or N; X17 is S, D, E, N, M, G, or Y; X18 is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and / or a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein X20is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21 is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22 is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23 is F, W, or Y; X24 is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25is L, W, Y, F, R, K, E, N, H, or S.

[0182] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a HCDR1 sequence comprising the sequence of GYX1FTX2X3X4(SEQ ID NO: 7), wherein X1is T, D, N, S, or E; X2is N, L, W, Y, E, F, D, P, S, Q, T, H, V, R, M, or I; X3is Y, E, F, or R; and X4is D, W, R, Y, E, T, I, or H; a HCDR2 sequence comprising the sequence of IFPGDX5X6T (SEQ ID NO: 8), wherein X5 is G or D; and X6 is S or R; and a HCDR3 sequence comprising the sequence of ARQX7X8X9X10X11FAY (SEQ ID NO: 9), wherein X7is T, E, R, D, Y, W, N, Q, S, or H; X8is T or Y; X9is A, W, Y, F, Q, E, H, R, N, D, K, M, C, P, S, T, L, I, or V; X10 is T, Y, W, R, N, H, or F; and X11 is W, Y, or F, and a HFR1 sequence comprising the sequence of QVQLQQSGAELVKPGASVKLSCKX12S (SEQ ID NO: 15), wherein X12is A or T; a HFR2 sequence comprising the sequence of INWVRQRPX13QGLEWIGW (SEQ ID NO: 16), wherein X13 is E or G; a HFR3 sequence comprising the sequence of X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFC (SEQ ID NO: 17), where X31is Q or Y; and a HFR4 sequence comprising the sequence of WGQGTLVTVSA (SEQ ID NO: 18), and a LCDR1 sequence comprising the sequence of X14X15X16X17X18X19 (SEQ ID NO: 19), wherein X14 is Q, E, T, F, N, D, K, H, V, L, S, Y, or W; X15 is S, D, N, E, W, or Q; X16 is I, R, Y, K, E, H, D, Q, V, L, F, T, or N; X17is S, D, E, N, M, G, or Y; X18is D, N, S, or E; and X19 is Y, W, or R; a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNX20X21X22X23X24X25T (SEQ ID NO: 20), wherein X20is G, W, Y, H, F, R, E, Q, K, M, D, C, S, L, N, T, I, V, or P; X21is H, W, Y, R, D, N, Q, T, K, F, S, I, E, or V; X22is S, E, R, D, Y, H, K, Q, F, W, T, or N; X23is F, W, or Y; X24is P, Y, W, F, H, N, E, D, C, S, R, or T; and X25 is L, W, Y, F, R, K, E, N, H, or S, and a LFR1 sequence comprising the sequence of X26X27VMTQSPATLSVTPGDRVX28LSCRAS (SEQ ID NO: 23), wherein X26is D, Q, E, or N; X27is I, W, E, Y, F, T, N, R, K, or Q; and X28is S or T; a LFR2 sequence comprising the sequence of LX29WYQQKSHESPRLLIK (SEQ ID NO: 24), wherein X29is H, Y, N, or S; a LFR3 sequence comprising the sequence of QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC (SEQ ID NO: 25), wherein X30is S,D, E, A, N, Q, T, P, or V; and a LFR4 sequence comprising the sequence of FGAGTKLELK (SEQ ID NO: 26). In some embodiments, X2 is L, X4 is W, X5 is R, X6 is Y, X7 is E, X8 is Y, X9is W, X10is Y, X18is N or S, X19is W, X20is W, X21is W, X23is W, X24is Y, X25is W, and / or X27 is W.

[0183] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a HCDR1 sequence of GYTFTNYW (SEQ ID NO: 27). In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a HCDR2 sequence of IFPGDDRT (SEQ ID NO: 28). In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a HCDR3 sequence of ARQTTWTWFAY (SEQ ID NO: 29). In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a LFR1 sequence of DWVMTQSPATLSVTPGDRVSLSCRAS (SEQ ID NO: 30). In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a LCDR1 sequence of QSISNY (SEQ ID NO: 31).

[0184] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a HCDR1 sequence of GYTFTNYD (SEQ ID NO: 10); a HCDR2 sequence of IFPGDDST (SEQ ID NO: 12); a HCDR3 sequence of ARQTTGTWFAY (SEQ ID NO: 14); a LCDR1 sequence of QSISNY (SEQ ID NO: 31); a LCDR2 sequence of YAS; and a LCDR3 sequence of QNGHSFPLT (SEQ ID NO: 22).

[0185] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a VH region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 32-41, 141, and 142, and a VL region comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 42-54, and 143-146.

[0186] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an antibody comprising a HC comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 70-75, and 149, and a LC comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 76-80, and 150-152.

[0187] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with an anti-B7H3 scFv comprising a sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 56- 69, 92-105, 147, and 148. In some embodiments, the anti-B7H3 scFv comprises a sequence of any one of SEQ ID NOs: 56-69, 92-105, 147, and 148.

[0188] In some embodiments, the antibody is an anti-B7H3 antibody that competes for binding with a bispecific antibody comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 88- 91 and 106-119. B. Preparation of Antibodies

[0189] In another aspect, the present application provides a preparation method for the anti- B7H3 antibodies disclosed herein. The method may comprise culturing host cells under such a condition that the antibodies are expressed. For example, an appropriate medium, an appropriate temperature, a culture time and the like may be used, and these methods are understood by those of ordinary skills in the art.

[0190] Any method suitable for producing a monoclonal antibody may be used to produce the anti-B7H3 antibodies (for example, the anti-huB7H3 antibodies) of the present application. For example, animals may be immunized with linked or naturally occurring B7-H3 or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, and repeated booster immunizations, and one or more routes may be used.

[0191] Any suitable form of B7-H3 may be used as an immunogen (antigen) to produce a non-human antibody specific to B7-H3 and to screen the biological activity of the antibody. An eliciting immunogen may be a full-length mature human B7-H3, comprising natural homodimers, or peptides containing single / multiple epitopes. The immunogen may be used alone, or in combination with one or more immunogenicity enhancers known in the art. The immunogen may be purified from a natural source or produced in a genetically modified cell. A DNA encoding the immunogen may be genomic or non-genomic (for example, cDNA) in source. A suitable genetic vector may be used to express the DNA encoding the immunogen, and the vector comprises, but is not limited to, an adenovirus vector, an adeno-associated virus vector, a baculovirus vector, a material, and a non-viral vector.

[0192] In some embodiments, the antibody is a chimeric antibody. Methods for making chimeric antibodies are known in the art. For example, chimeric antibodies can be made in which the antigen binding region (heavy chain variable region and light chain variable region) from one species, such as a mouse, is fused to the effector region (constant domain) of another species, such as a human. As another example, “class switched” chimeric antibodies can be made in which the effector region of an antibody is substituted with an effector region of a different immunoglobulin class or subclass.

[0193] In some embodiments, the antibody is a humanized antibody. Generally, a non- human antibody is humanized in order to reduce its immunogenicity. Humanized antibodies typically comprise one or more variable regions (e.g., CDRs) or portions thereof that are non- human (e.g., derived from a mouse variable region sequence), and possibly some framework regions or portions thereof that are non-human, and further comprise one or more constant regions that are derived from human antibody sequences. Methods for humanizing non-human antibodies are known in the art. Transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies. Other methods of humanizing antibodies include, for example, variable domain resurfacing, CDR grafting, grafting specificity-determining residues (SDR), guided selection, and framework shuffling. A humanized antibody may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody can be an IgG antibody, and an IgG1 subtype is used. An essential constant domain sequence may be optimized by screening antibodies with the biological assays described in the Examples below, so as to produce the desired biological activity. Similarly, any type of light chain may be used in the compounds and methods herein. Specifically, κ and λ chains or their variants may be used in the compounds and methods of the present application.

[0194] As an alternative to humanization, fully human antibodies can be generated. As a non-limiting example, transgenic animals (e.g., mice) can be produced that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. As another example, humanantibodies can be produced by hybridoma-based methods, such as by using primary human B cells for generating cell lines producing human monoclonal antibodies.

[0195] Human antibodies can also be produced using phage display or yeast display technology. In phage display, repertoires of variable heavy chain and variable light chain genes are amplified and expressed in phage display vectors. In some embodiments, the antibody library is a natural repertoire amplified from a human source. In some embodiments, the antibody library is a synthetic library made by cloning heavy chain and light chain sequences and recombining to generate a large pool of antibodies with different antigenic specificity. Phage typically display antibody fragments (e.g., Fab fragments or scFv fragments), which are then screened for binding to an antigen of interest.

[0196] In some embodiments, antibody fragments (such as a Fab, a Fab’, a F(ab’)2, a scFv, a VH, or a VHH) are generated. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies. However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered from E. coli cells and chemically coupled to form F(ab’)2 fragments. According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0197] In some embodiments, an antibody or an antibody fragment is conjugated to another molecule, e.g., polyethylene glycol (PEGylation) or serum albumin, to provide an extended half-life in vivo.

[0198] In some embodiments, multispecific antibodies comprising an anti-B7H3 antibody (or antigen-binding fragment thereof) as described herein are provided, e.g., a bispecific antibody. Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) has a binding specificity for B7-H3 and has a binding specificity for at least one other antigen. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) binds to two different B7-H3 epitopes. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) is capable of inducing B7-H3 clustering at the cell surface. Methods of making multispecific antibodies (e.g., bispecific antibodies) include, but are not limited to, recombinant co- expression of two pairs of heavy chain and light chain in a host cell, “knobs-into-holes”engineering, intramolecular trimerization, and fusion of an antibody fragment to the N- terminus or C-terminus of another antibody, e.g., tandem variable domains.

[0199] The sequence of the DNA molecule of the antibodies or the fragment thereof in the present application may be obtained by conventional techniques, such as methods using PCR amplification or genomic library screening and the like. In addition, the coding sequences of the light and heavy chains may also be fused together to form a single-chain antibody.

[0200] Once relevant sequences are obtained, they may be obtained on a large scale by recombination. This is generally done by cloning them into vectors, then transferring them into cells, and then isolating the relevant sequences from the proliferated host cell by means of a conventional method.

[0201] In addition, the relevant sequences may also be synthesized by using an artificial synthesis method, in particular when a fragment is short. Generally, a fragment with a very long sequence may be obtained by first synthesizing multiple small fragments and then linking these small fragments. Then, the nucleic acid molecules may be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.

[0202] The present application also relates to vectors comprising the aforementioned appropriate nucleic acid molecules and appropriate promoters or control sequences. These vectors may be used for transforming appropriate host cells to enable them to express proteins. The host cells may be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. For example, the animal cells may comprise (but are not limited to): CHO-S, CHO-K1, and HEK-293 cells.

[0203] The step of transforming the host cells with recombinant DNAs in the present application may be performed using techniques well known in the art. An obtained transformant may be cultured by a conventional method, and it expresses the polypeptide encoded by the nucleic acid molecule(s) of the present application. According to the host cells used, they are cultured in a conventional medium under suitable conditions. Generally, the host cells are cultured and transformed under conditions suitable for the expression of the antibodies of the present application. Then, the antibodies of the present application are purified and obtained using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinitychromatography, and other conventional separation and purification means well known to those skilled in the art.

[0204] The resulting antibodies may be identified by a conventional means. For instance, the binding specificity of the antibodies may be determined by immunoprecipitation or in vitro binding assays, such as fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0205] Exemplary methods for producing and validating the anti-B7H3 antibodies of the present application are described in Example 7. C. Nucleic Acids, Vectors, and Host cells

[0206] In another aspect, the present application further provides an isolated nucleic acid molecule or isolated nucleic acid molecules. The nucleic acid molecule(s) may encode the antibodies of the present application. For example, each of the nucleic acid molecule(s) may encode a complete antibody, or a portion thereof such as an antigen-binding fragment thereof (e.g., one or more of HCDR1-3, LCDR1-3, VL, VH, LC, or HC).

[0207] The nucleic acid molecule(s) of the present application may be isolated. For example, the nucleic acid molecule(s) may be produced or synthesized by any one of the following methods: (i) in vitro amplification, for example by polymerase chain reaction (PCR) amplification; (ii) clonal recombination; (iii) purification, for example, by fractionation through restriction digestion and gel electrophoresis; or (iv) synthesis, for example, by chemical synthesis. In some embodiments, the isolated nucleic acid(s) is / are a nucleic acid molecule(s) prepared by recombinant DNA technology.

[0208] In the present application, the nucleic acid(s) encoding the antibody or the antigen- binding fragment thereof may be prepared by a variety of methods known in the art. These methods include, but are not limited to, the overlap extension PCR using restriction fragment operations or using synthetic oligonucleotides. For specific operations, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.

[0209] In another aspect, the present application provides a vector or vectors, each of which comprises the nucleic acid molecule(s) of the present application. Each vector may comprise one or more said nucleic acid molecule(s). In addition, the vector may also comprise othergenes, for example, a marker gene that is allowed to select this vector in a suitable host cell and under a suitable condition. In addition, the vector may also comprise an expression control element that allows a coding region to be expressed correctly in a suitable host. Such a control element is well known to those skilled in the art, which, for example, may include a promoter, a ribosome binding site, an enhancer, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatable element. A specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes a 5′ non-transcribed sequence and 5′ and 3′ non-translated sequences, for example, a TATA box, a capped sequence, a CAAT sequence, etc., which are involved in transcription and translation initiation, respectively. For example, the 5′ non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for functionally linked to the nucleic acid for transcriptional control. The expression control sequence may further comprise an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may comprise, for example, promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, CMV promoters, and their artificial hybrid promoters (such as CMV), wherein a portion of a promoter may be fused with a portion of a promoter of an additional cellular protein (such as human GAPDH and glyceraldehyde-3-phosphate dehydrogenase) gene, and the promoter may or may not contain additional introns. The nucleic acid molecule(s) of the present application may be operably linked to the expression control element. The vector may comprise, for example, a plasmid, a cosmid, a virus, a bacteriophage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0210] In another aspect, the present application provides a host cell, which may comprise the nucleic acid molecule(s) of the present application and / or the vector or vectors of the present application. In some embodiments, each type of or each host cell may comprise one or one type of nucleic acid molecule or vector of the present application. In some embodiments, each type of or each cell may comprise a plurality of (e.g., 2 or more) or a plurality of types of (e.g., 2 or more types of) vectors of the present application. For example, the vector of the present application may be introduced into the host cell, e.g., a eukaryotic cell, such as a plant- originated cell, a fungal cell, or a yeast cell, etc. The vector of the present application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.D. Compositions

[0211] In another aspect, the present application further provides a composition. In some cases, the composition may be a pharmaceutical composition, which comprises an antibody of the present application, or an ADC thereof, or corresponding CAR-T cells, and a pharmaceutically acceptable carrier. Generally, these substances may be formulated in a non- toxic, inert, and pharmaceutically acceptable aqueous carrier medium, of which the pH may generally be about 5-8, for example, about 6-8, and a pH value may vary with the nature of the formulated substances and the disorders to be treated. The formulated pharmaceutical composition may be administered by conventional routes, comprising (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0212] The antibody of the present application may also be expressed in a cell by means of a nucleotide sequence for cell therapy. For example, the antibody is used for the chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

[0213] The pharmaceutical composition of the present application may be directly used to bind a B7-H3 protein molecule, and thus may be used to prevent and treat cancers and other diseases. In addition, other therapeutic agents may also be used at the same time.

[0214] The pharmaceutical composition of the present application may comprise the antibody of the present application at a safe and effective amount (such as 0.001-99 wt %, 0.01- 90 wt %, or 0.1-80 wt %) and a pharmaceutically acceptable adjuvant (which may comprise a carrier or an excipient). Such carriers may comprise (but are not limited to): saline water, buffer, glucose, water, glycerol, ethanol, and combinations thereof. A pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present application may be prepared into an injection form, for example, by means of a conventional method using normal saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as an injection and a solution should be manufactured under an aseptic condition. The dosage of an active ingredient is a therapeutically effective amount, for example, about 1 mg / kg body weight to about 5 mg / kg body weight per day. In addition, the antibody of the present application may also be used together with other therapeutic agents.

[0215] The antibody or pharmaceutical composition described herein may be formulated, dosed, and administered in line with good medical practices. The considerations in this casecomprise the specific disorder being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of the disorder, the site of agent delivery, the method of administration, the schedule of administration, and other factors known to a medical practitioner. A therapeutic agent (e.g., an anti-B7H3 antibody) does not need to be but is optionally formulated and / or administered together with one or more agents that are currently used for preventing or treating the disorder in question. The effective amount of such other agents depends on the amount of the therapeutic agent (e.g., the anti-B7H3 antibody) existing in the preparation, the type of disorder or treatment, and other factors discussed above. Generally, these agents may be used at any dose that is empirically / clinically determined to be appropriate and via any route that is empirically / clinically determined to be appropriate.

[0216] The antibody or pharmaceutical composition described herein may be employed in combination therapies. The particular combination of therapies (e.g., therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. The pharmaceutical compositions of the present disclosure can be employed in combination therapies (e.g., combination antibody therapies), that is, the pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other therapeutic active ingredients. In some embodiments, other active ingredient(s) can be an anti-cancer agent, monoclonal antibody, polyclonal antibody, RNA polymerase inhibitors, protease inhibitors, helicase inhibitors, immunomodulators, antisense compounds, short interfering RNAs, short hairpin RNAs, micro RNAs, RNA aptamers, ribozymes, and combinations thereof. The combination therapies employed may achieve a desired effect for the same purpose, or they may achieve different effects (e.g., control of any adverse effects). In some embodiments, combination therapy may involve administrations of a plurality of antibody agents directed to a single epitope (e.g. a single conformational epitope) of B7-H3. In some embodiments, combination therapy can comprise a plurality of antibody agents that recognize distinct epitopes of B7-H3, for example to simultaneously interfere with multiple mechanisms in the tumorigenesis process. Compared with a single therapy, the dose of the antibody administered in a combination therapy may be reduced. The progress of such a therapy may be easily monitored by conventional techniques.E. Kits

[0217] In another aspect, the present disclosure provides a kit for treating a subject with a cancer. In some embodiments, the kit comprises an isolated anti-B7H3 antibody, a composition, and / or a pharmaceutical composition of the present disclosure described herein. The kits are useful for treating any cancer, some non-limiting examples of which include lung cancer, colorectal cancer, breast cancer, prostate cancer, gastric cancer, liver cancer, cervical cancer, brain cancer, ovarian cancer, pancreatic cancer, skin cancer, eye cancer, soft tissue cancer, renal cancer, bladder cancer, head and neck cancer, neuroblastoma, melanoma, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, a nasopharyngeal carcinoma, a lymphoepithelioma-like carcinoma, any other cancer described herein, and a combination thereof.

[0218] Materials and reagents to carry out the various methods of the present disclosure can be provided in kits to facilitate execution of the methods. As used herein, the term “kit” includes a combination of articles that facilitates a process, assay, analysis, or manipulation. In particular, kits of the present disclosure find utility in a wide range of applications including, for example, diagnostics, prognostics, therapy, and the like.

[0219] Kits can contain chemical reagents as well as other components. In addition, the kits of the present disclosure can include, without limitation, instructions to the kit user, apparatus and reagents for sample collection and / or purification, apparatus and reagents for product collection and / or purification, apparatus and reagents for administering antibodies or other composition(s) of the present disclosure, apparatus and reagents for determining the level(s) of biomarker(s) and / or the activity and / or number of immune cells, apparatus and reagents for detecting B7-H3, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and / or control samples. Kits of the present disclosure can also be packaged for convenient storage and safe shipping, for example, in a box having a lid. For instance, the kits may be stored and shipped at room temperature, on wet ice or with cold packs, or frozen in the vapor phase of liquid nitrogen or in dry ice.

[0220] In some embodiments, the kits also contain negative and positive control samples for detection of B7-H3, immune cell activity and / or number, and / or the presence or level of biomarkers. In some embodiments, the negative control samples are non-cancer cells, tissue, or biofluid obtained from the subject who is to be treated or is already undergoing treatment.In other embodiments, the negative control samples are obtained from individuals or groups of individuals who do not have cancer. In other embodiments, the positive control samples are obtained from the subject, or other individuals or groups of individuals, who have cancer. In some embodiments, the kits contain samples for the preparation of a titrated curve of one or more biomarkers in a sample, to assist in the evaluation of quantified levels of the activity and / or number of one or more immune cells and / or biomarkers in a biological sample. In some embodiments, the kit further comprises instructions for the use of the isolated anti-B7H3 antibodies, compositions, and / or pharmaceutical compositions of the present disclosure described herein. F. Therapeutic Methods Using the Antibodies

[0221] In another aspect, the present application further provides uses of the anti-B7H3 antibodies, nucleic acid molecule(s), vector(s), host cell(s), and / or pharmaceutical composition(s) in the preparation of a medicament. In some instances, the medicament can be used for preventing, alleviating and / or treating a cancer, or for modulating the immune system in a subject. In other instances, the medicament can be used for prognosis and / or diagnosis of a cancer / disease, such as detecting B7-H3 expression in a biological sample. Methods for Treating Cancers

[0222] The present disclosure provides a method of treating a B7H3-related disease in a subject. For example, the B7H3-related disease may be a tumor, a cancer, an immune disease, or an infectious disease. In some embodiments, the cancer or tumor has abnormal B7-H3 expression (e.g., upregulated B7-H3 expression). In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the anti-B7H3 antibody or the pharmaceutical composition comprising the antibody described herein. In some embodiments, the method can inhibit cancer proliferation and / or tumor growth in the subject. In some embodiments, the cancer comprises B7H3-positive cancer cells. In some embodiments, the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, an chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.In some embodiments, the cancer is a neuroblastoma. In some embodiments, the cancer is a cervical cancer.

[0223] In some embodiments, treating the subject comprises inhibiting cancer cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell invasion, ameliorating or eliminating the symptoms of cancer, reducing the size (e.g., volume) of a cancer tumor, reducing the number of cancer tumors, reducing the number of cancer cells, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death, or enhancing the therapeutic effects of a composition or pharmaceutical composition. In some embodiments, treating the subject results in an increased survival time. In some instances, overall survival is increased. In other instances, disease-free survival is increased. In some instances, progression-free survival is increased. In particular embodiments, treating the subject results in a reduction in tumor volume and / or increased survival time.

[0224] In particular embodiments, treating the subject enhances the therapeutic effects of an anti-cancer therapy such as a chemotherapeutic agent, an immunotherapeutic agent, radiotherapy, hormone therapy, a differentiating agent, and / or a small-molecule drug. Methods for Modulating the Immune System

[0225] The present disclosure also provides a method of modulating the immune system in a subject by administering to the subject a therapeutically effective amount of the anti-B7H3 antibody or the pharmaceutical composition comprising the antibody described herein. In some embodiments, the method enhances T-cell mediated cytotoxicity in the subject. In some embodiments, the method suppresses the inhibitory effect of B7-H3 on T cell proliferation and / or function in the subject. In some embodiments, the method enhances NK cell mediated functions in the subject. In some embodiments, the method suppresses the inhibitory effect of B7-H3 on NK cell activity and / or function in the subject.

[0226] Whether the administration of the antibody or the pharmaceutical composition modulates the immune system (e.g., enhances T-cell mediated cytotoxicity, enhances NK cell activity, suppresses the inhibitory effect of B7-H3 on T cells or NK cells) can be determined by known methodologies, including those described herein. In some embodiments, the results of the immune cell activity and / or number measurement, and / or biomarker presence or level determinations, are recorded in a tangible medium. For example, the results of assays (e.g., the activity level and / or number of immune cells, the presence or level (e.g., expression) of one ormore biomarkers and / or a prognosis or diagnosis (e.g., of whether or not there is the presence of cancer, the prediction of whether the subject will respond to an antibody treatment, or whether the subject is responding to an antibody treatment) can be recorded, e.g., on paper or on electronic media (e.g., audio tape, a computer disk, a CD, a flash drive, etc.). Methods for Detecting B7-H3 Expression

[0227] The present application further provides a method for detecting B7-H3 expression in a biological sample as described below. In some cases, the method comprises contacting a biological sample with the anti-B7H3 antibody of the present application under such a condition that the anti-B7H3 antibody is allowed to bind to B7-H3, and detecting whether a complex is formed between the anti-B7H3 antibody and the antigen B7-H3. For example, the biological sample can be a tumor sample (e.g., biopsy) with elevated B7-H3 expression as compared to a non-tumor sample. Such a method may be an in vitro or in vivo method. The anti-B7H3 antibody of the present application may be used in, for example, immunoassays, which include, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blotting), flow cytometry (for example, FAGS), and enzyme-linked immunosorbent assay (ELISA). In some cases, for example, when the B7-H3 is used as a biomarker for selection of patients, the anti-B7H3 antibody is used to select subjects suitable for treatment with the anti-B7H3 antibody of the present application.

[0228] The application further provides the use of the anti-B7H3 antibody in a method for diagnosing a subject suffering from a disorder (e.g., a cancer or an immune dysfunction). The method comprises: contacting a sample from the subject with the anti-B7H3 antibody of the present application; and detecting the presence of B7-H3 or the expression level of B7-H3 in the sample, thereby determining the presence of the disorder. In some embodiments, the disorder is a cancer. In some embodiments, the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, an chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.

[0229] In some embodiments, a sample is obtained from the subject. In other embodiments, a sample is obtained from a different subject or a population of subjects. Samples obtainedfrom a different subject and / or a population of subjects can be used, for example, to establish reference ranges to facilitate comparisons that are part of the methods of the present disclosure. Samples can be obtained at any time, including before and / or after administration of the antibody of the present application, pharmaceutical composition(s), and / or other composition(s) of the present disclosure. In some embodiments, the sample comprises whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, buccal cells, tumor tissue, urine, fluid obtained from a pleural effusion, hair, skin, or a combination thereof. In general, the sample can comprise any biofluid. In some instances, the sample comprises circulating tumor cells (CTCs). The sample can also be made up of a combination of normal and cancer cells. In particular embodiments, the sample comprises circulating tumor cells (CTCs). The sample can be obtained, for example, from a biopsy, from a surgical resection, and / or as a fine needle aspirate (FNA). Samples can be used to determine, measure, or detect B7-H3, immune cell activity and / or number, and / or biomarker(s), as described herein.

[0230] In other embodiments, the methods further comprise the step of providing the results of assays, prognosis, and / or diagnosis to the patient (i.e., the subject) and / or the results of treatment. IV. Examples

[0231] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1: Generation and Verification of the ch8H9[3mut]-huB7H3 model in3cl6

[0232] Ahmed et al. and Nai-Kong et al. described anti-human B7-H3 (CD276) antibodies derived from ch8H9 (PDB ID: 5CMA), and a 3-dimensional (3D) model of ch8H9 bound to human B7-H3 (huB7H3). Some of the ch8H9 derivatives contain affinity-enhancing mutations as identified by yeast display[1-2]. The 3D model of ch8H9-huB7H3 can serve as a template for generating and / or verifying amino acid changes that can improve the ch8H9 antibody’s affinity for huB7H3.

[0233] Since the atomic coordinates of the ch8H9-huB7H3 model have not been deposited in the public domain, we aimed at reconstructing them using publicly available tools. Belowsteps outline a procedure yielding the ch8H9[3mut]-huB7H3 structural (3D) model “in3cl6” which is similar to Ahmed’s ch8H9-huB7H3 model but with three affinity-enhancing mutations located in the complementary determining regions (CDRs, as defined in Kabat definition) identified by Ahmed et al. using yeast display [1]. For those skilled in the art, it is imperative that similar models can also be obtained using alternative methods. Step 1: In silico generation of the huB7H3 structure

[0234] An X-ray crystal structure of murine B7-H3 is available from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) under ID 4I0K. To date, no 3D structure of human B7-H3 is available in the PDB. However, a 3D model of huB7H3 predicted by FoldSeek / AlphaFold has been published (https: / / search.foldseek.com / search?accession=Q5ZPR3&source=AlphaFoldDB). Figure 1 shows the murine B7-H3 protein sequence as indicated in Uniprot Q8VE98 with the sequence in PDB ID 4I0K underlined, and the human B7-H3 sequence as shown in Uniprot Q5ZPR3 and NCBI NP_001019907.1 https: / / www.ncbi.nlm.nih.gov / protein / NP_001019907.1). Human B7-H3 occurs in at least two versions. One version 4Ig-B7-H3 contains two variable (V)-like plus constant (C)-like immunoglobulin (Ig) domains, as shown in Figure 1. The other version 2Ig-B7-H3 contains only one such V-like + C-like Ig domain [1, 3]. A sequence alignment of the outer and inner Ig domains of 4Ig-B7-H3 demonstrates a high degree of similarity between the two domains (Figure 2). High degrees of similarity are also shown between murine 2Ig- B7-H3 and the outer Ig domain of human B7-H3 (Figure 3), and between murine 2Ig-B7-H3 and the inner Ig domain of human B7-H3 (Figure 4).

[0235] To obtain a model of huB7H3, the SWISS-MODEL modeling tool (https: / / swissmodel.expasy.org / ) [28, 29] can be utilized. Ahmed et al. emphasized amino acids 123-130 of human B7-H3 as particularly relevant for ch8H9 antibody binding[1]. Since amino acids 123-130 are part of the outer V-like + C-like Ig domain and since the murine B7- H3 sequence in PDB ID 4I0K only represents one such domain, we can choose an amino acid sequence only covering the outer V-like + C-like Ig domain, such as amino acids 1-251 of huB7H3 (Figure 1) as the SWISS-MODEL input sequence. We also chose PDB ID: 4I0K as the SWISS-MODEL template structure. Prior to using PDB ID: 4I0K as the template, we fixed its geometry using PDBFixer[4] and added hydrogen atoms using MolProbity[5] (http: / / molprobity.biochem.duke.edu / ). MolProbity can also be used to add hydrogen atoms to the SWISS-MODEL output file. With MolProbity, the ATOM serial numbers of the newly added hydrogen atoms are set to 0 but can be updated manually in, e.g., Microsoft Excel, or byemploying another software tool. This strategy yielded a structure of huB7H3 with the amino acids 35-240 of the Uniprot Q5ZPR3 sequence, as the SWISS-MODEL output sequence (Figure 1). A sequence alignment demonstrates a high degree of similarity between murine 2Ig-B7-H3 and the SWISS-MODEL output sequence of human B7-H3 (Figure 5). Step 2: In silico introduction of affinity-enhancing mutations in anti-huB7H3 Fab antibody ch8H9, yielding ch8H9[3mut] Fab antibody

[0236] Ahmed et al. identified three affinity-enhancing amino acid substitutions of the antibody ch8H9, located within the Kabat definition-based complementary determining regions (CDRs): H34Y in light chain (LC) CDR1 region, and G56D and A102G in heavy chain (HC) CDR2 and CDR3 region, respectively [1], with "34", "56", and "102" referring to consecutively numbered amino acids starting with the first amino acid of the variable chains as 1. To virtually introduce these three mutations into the antibody ch8H9, the SWISS-MODEL modeling tool can be utilized with the heavy and light chain sequences from the fragment antigen-binding (Fab) antibody in PDB ID: 5CMA modified to contain the three mutations as the target sequence and PDB ID: 5CMA as the template structure. Thereafter, MolProbity can be utilized to add hydrogen atoms. Following this procedure, the resulting Fab antibody is herein referred as ch8H9[3mut] with the “3mut” referring to the three CDR substitutions identified by Ahmed et al. Step 3: Docking of ch8H9[3mut] Fab antibody to huB7H3 using AbAdapt

[0237] To obtain a model similar to Ahmed’s ch8H9-huB7H3 model [1], corresponding epitope restrains of antibody-antigen interaction sites should be specified. AbAdapt (https: / / sysimm.org / abadapt / )[6] can be utilized to dock the antibody ch8H9[3mut] structure from Step 2 to the antigen huB7H3 structure from Step 1, with the following variables accessible from the “Structure” tab: ^ Antibody PDB: the antibody structure from Step 2 as a .pdb file ^ Antibody chains: the chain IDs of the heavy and light chains of the structure from Step 2 ^ Antigen PDB: the huB7H3 structure from Step 1 as a .pdb file ^ Antigen chains: the chain IDs of the huB7H3 chain of the structure from Step 1 ^ Optional Inputs: can be left blank ^ Epitope Restraints: upload as text file with the following content (“C” reflects the chain ID of huB7H3): 123 C F 1 124 C V 1 125 C S 1126 127 128 129 130 ^ Radius to mask non-restraints: 10 (default) ^ Piper sampling frequency: 0.9 (default)

[0238] After docking, the output structure can be hydrogen-supplemented using MolProbity. To obtain an energetically more favorable structure, the hydrogen-adjusted, docked structure can be subjected to energy minimization using obabel / minimize (Open Babel) in, e.g., 1500 steps and a general amber force field using a steepest decent method (https: / / openbabel.org / docs / Command-line_tools / babel.html#forcefield-energy-and- minimization). With this approach, some hydrogen atoms might get duplicated with alternative X, Y, Z coordinates. This conflict can be resolved by manually deleting one set of the extra hydrogens in, e.g., Microsoft Excel, or by employing another software tool. In silico verification of the in3cl6 model

[0239] The ch8H9[3mut]-huB7H3 model obtained via Steps 1-3 is referred as “in3cl6”. Views of the in3cl6 model, in a zoomed-out (Figure 6A) and zoomed-in (Figure 6B) view, show that the amino acid R127 of the antigen huB7H3 interacts with the variable light (VL) region of the antibody ch8H9[3mut], and the amino acid D128 of the antigen interacts with the variable heavy (VH) chain of the antibody. Figures 7 and 8 show non-covalent interactions between ch8H9[3mut] and huB7H3 in model in3cl6, as demonstrated in 5 hydrogen bond pairs (Figure 7A), 2 pi-cation pairs (Figure 7B), 1 salt bridge / ionic interaction pair (Figure 7C), 1 pi-stacking pair (Figure 7D), and 42 residue pairs in contacts (Figure 8). This data indicates that the in3cl6 model shares similar features of the Ahmed’s model [1], such as sharing the same orientation of the antibody versus the antigen and predicting the same epitope region (FVSIRDFG, 123-130 of huB7H3).

[0240] To further verify the in3cl6 model, we used two methods: PPCheck [7],

[0022] (http: / / caps.ncbs.res.in / ppcheck / ) and Protein-Ligand Interaction Profiler (PLIP) [8] (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index), to determine potential hotspots in the epitope FVSIRDFG (123-130) of huB7H3. All residues except Val-124 and Ile-126 were predicted to be critical in the interaction with the ch8H9[3mut] antibody as assessed by hotspot analysis using PPCheck and binding type determination using PLIP (Table 5).Table 5. Prediction of residues in the FVSIRDFG epitope interacting with the ch8H9[3mut] antibody in the in3cl6 model.Example 2: In silico mutagenesis based on the in3cl6 model

[0241] To screen antibody variants with potentially enhanced antigen-binding affinities, in silico saturation mutagenesis can be conducted using, for instance, mCSM-AB2[9] (https: / / biosig.lab.uq.edu.au / mcsm_ab2 / ). Using such a tool, each residue of the antibody in the antibody-antigen interface can be virtually replaced with one of the remaining 19 natural amino acids, then binding strengths for each substitute can be estimated. Residue replacements resulting in higher binding strengths compared to the wild-type residue indicate such substitutes can improve the antibody’s binding affinity. Saturation mutagenesis with the in3cl6 model

[0242] For the in silico saturation mutagenesis the method (webtool) mCSM-AB2 was employed. As input, a PDB file with a structure in which the heavy and light chains had the same chain ID was used. We do not know if using unique chain IDs for each antibody chain would have yielded vastly different results. However, using an alternative method to predict the effects of a subset of the mutations identified by mCSM-AB2, a strong concordance between the results from mCSM-AB2 and the alternative method was noticed (see “Confirmation of the affinity-enhancing single mutations” below). Using the method mCSM-AB2 for the saturation mutagenesis as just described, 217 mutations in the antibody portion of the in3cl6 model were identified with enhanced antigen binding affinities (Table 6). All 217 mutations occurred in 32 antibody residues (Table 7). Table 7 lists L-31 twice since the predicted ^^G energy was identical with two mutations (D31N and D31S). For 25 of these 32 residues, the mutation with the highest predicted affinity was selected for further analysis. Table 7 shows mutations identified by in silico saturation mutagenesis using mCSM-AB2 which were predicted to result in higher binding affinities. Forboth Tables 6 and 7, “Chain” refers to the light (L) or heavy (H) chain of the ch8H9[3mut] antibody in the in3cl6 model, “Position” refers to the relative amino acid positions within either the L or H chain, “WT” refers to the wild-type amino acids at given positions, “Mut.” refers to the alternative amino acid at these positions, “Distance (Angstrom)” refers to the closest distances in Angstrom (Å) between the mutation sites and the antigen (huB7H3). “Predicted (ΔΔG_affinity)” refers to the predicted changes in affinity / binding strength in kcal / mol (increase in affinity predicted if value > 0), and “in silico confirmed” indicates whether the modeling using SWISS-MODEL and the binding strength assessment of the modeled structures using PPCheck confirmed the predicted increase in affinity proposed by mCSM-AB2 (saturation mutagenesis). The individual binding strengths (total stabilizing energies) of the modeled structures are visualized in Figures 9 and 10A. Table 6.217 mutations identified in the in3cl6 model with predicted increased antigen binding affinities by in silico saturation mutagenesis using mCSM-AB2.Table 7. 33 mutations in 32 residues identified in the in3cl6 model with predicted increased antigen binding affinities by in silico saturation mutagenesis using mCSM-AB2 (subset of mutations from Table 6 showing for each residue only the mutation with the highest predicted increase in affinity, except for position 31 on the light (L) chain for which two mutations share the highest predicted increase in affinity).26 of the 33 mutations were modeled using SWISS- MODEL then the “total stabilization energy” assessed using PPCheck (Figures 9 and 10). “Yes” under “in silico confirmed” indicates that an increased affinity was also predicted for the modeled mutation.Visual inspection of the in3cl6 model to identify mutations potentially suitable for affinity-enhancing mutagenesis

[0243] The in3cl6 model was visually inspected using iCn3D[10-11] and assessed for regions / residues potentially suitable for mutagenesis to increase affinity. The heavy chain mutations VH-S57R and VH-Q59Y appeared to be particularly suitable. To assess their potential effects on affinity, the mCSM-AB2[9] (https: / / biosig.lab.uq.edu.au / mcsm_ab2 / ), mCSM-PPI2

[0012] (https: / / biosig.lab.uq.edu.au / mcsm_ppi2 / ), and SSIPe

[0013] (https: / / zhanggroup.org / SSIPe / ) tools were employed. Table 8 shows that at least two of these three methods predicted increased affinity for both mutations. To ensure ^^G energies were calculated between the antibody and huB7H3 (and not between the antibody’s heavy and light chains) a PDB file with a structure in which the heavy and light chains had the same chain ID was used as input for the mCSM-PPI2 and SSIPe tools. Table 8. Predicted effect of mutations identified by visual inspection of the in3cl6 model.*sum of VH-S57R and VH-Q59Y ^^G values

[0244] To verify these results, both mutations were modeled using SWISS-MODEL and energy-minimized using obabel / minimize (Open Babel) in 1500 steps and a general amber force field using a steepest decent method.

[0245] PPCheck was utilized to estimate the binding strengths, through investigating total stabilizing energies (TSEs), between the ch8H9[3mut] antibody and the huB7H3 antigen in the in3cl6 model. A lower TSE indicates a higher binding strength or binding affinity. Table 9 demonstrates that these two mutations, VH-S57R and VH-Q59Y, can enhance antigen-binding affinities as they exhibit higher absolute TSEs in comparison to the two references: ch8h9[3mut] and VH-G8A antibodies. It has been previously predicted that a VH-G8A mutation of the ch8H9[3mut] antibody does not affect the huB7H3 binding. Table 9. PPCheck-predicted total stabilizing energies (TSEs) for mutant ch8H9[3mut] antibodies in the in3cl6 model.Confirmation of the affinity-enhancing single mutations

[0246] To confirm the saturation mutagenesis results, 26 mutations in 25 residues shown in Table 7 and the VH-S57R and VH-Q59Y mutations were each modeled using SWISS- MODEL and energy-minimized using obabel / minimize (Open Babel) in 250 steps and a general amber force field using a steepest decent method.

[0247] PPCheck was utilized to estimate the binding strengths via TSEs between the reference and mutant ch8H9[3mut] antibodies and huB7H3 in the in3cl6 model. Figures 9 and 10A demonstrate that the mutations confirmed in silico (Table 7) are also predicted to have enhanced binding affinity because their estimated absolute total stabilizing energies (TSEs) are higher than the TSEs of the two references: ch8h9[3mut] and VH-G8A antibodies.

[0248] Next, we used iCn3D [10-11] (https: / / www.ncbi.nlm.nih.gov / Structure / icn3d / full.html) to visualize the 6 mutations with the lowest predicted TSEs (i.e., highest predicted affinities): VH G102W, VL D31N, VH D33W, VL D31S, VH T101Y, and VL I2W. For most mutations, we observed more non-covalent bonds between the mutated amino acid and huB7H3 than the ones between the reference antibodies and the antigen (Figures 10B-10G).

[0249] Figures 11A and 11B show the amino acid substitutions in the heavy chain and light chain sequences of ch8H9[3mut] which were predicted to result in enhanced binding affinity for huB7H3. Confirmation of the affinity-enhancing mutations with multiple amino acid substitutions

[0250] Combinations of mutations might have an additive or synergistic effect on antigen- binding affinity. Five of the previously studied mutations (referred to as “top-5 mutations”, Table 13): VL-I2W, VL-D31N, VH-D33W, VH-S57R, and VH-G102W, were selected and modeled in combinations of 2, 3, 4, and 5 mutations. The mutations were selected based on their predicted affinity increase indicated by mCSM-AB2 (Figures 9 and 10A). If neighboring residues both had high increases, or, as in the case of VL-D31, one residue had two substitutions with high predicted affinity increases (VL-D31N, VL-D31S), only the mutation with the higher ΔΔG value was selected. Introducing both mutations was either not possible(VL-D31) or might lead to a local structure distortion negatively affecting affinity. Table 10 shows the combinations selected. Table 10. Top-5 mutations introduced individually or in combinations into the ch8H9[3mut] Fab antibody in the in3cl6 model.

[0251] PPCheck was utilized to estimate the binding strengths via total stabilizing energies (TSEs) between the antigen huB7H3 and the references (ch8H9[3mut] wild-type and VH-G8A Fabs), single, dual, triple, quadruple, or quintuple mutations in the in3cl6 model. Figures 12 and 13 demonstrate that generally, higher numbers of mutations of the antibody correlate with stronger predicted antigen-binding affinities (lower TSEs). Example 3: Alternative models to verify the mutagenesis results

[0252] Antibody-antigen modeling, especially if it entails docking, involves complex processes which are not guaranteed to yield the best results. The in3cl6 model is one of multiple models obtained using different methods and input parameters. It replicates several features ofAhmed et al.’s model[1] for the reasons outlined above. To further confirm that we virtually generated a series of antibody variants with higher antigen-binding affinities compared to the ch8H9[3mut] Fab antibody, we also modeled and predicted binding strengths of several mutations also virtually inserted into the ch8H9[3mut] Fab sequence but modeled using alternative structural models (in3cl0 and in4cl0) as templates. Model in3cl0

[0253] Model in3cl0 was generated with the same methods including input files and software parameters as model in3cl6, outlined above: ^ Step 1: Generation of huB7H3 model. ^ Step 2: In silico introduction of affinity-enhancing mutations in anti-huB7H3 Fab antibody ch8H9, yielding ch8H9[3mut]. ^ Step 3: Docking of ch8H9[3mut] Fab antibody to huB7H3 using AbAdapt[6].

[0254] Whereas model in3cl6 is the model with the seventh best score (“cluster-6”), model in3cl0 is the model with the best score (“cluster 0”) as determined by the AbAdapt tool.

[0255] Model in3cl0 was also subjected to in silico saturation mutagenesis using mCSM- AB2[9]. From the 18 mutations identified in the in3cl6 model (Figure 10A), four mutations were identified having enhanced binding affinities in the in3cl0 model (Table 11). Table 11. Mutations predicted to have enhanced binding affinities in both the in3cl6 and in3cl0 models.

[0256] To verify the saturation mutagenesis results, each of the 4 mutations (Table 11) was modeled using SWISS-MODEL and energy-minimized using obabel / minimize (Open Babel) in 250 steps and a general amber force field using a steepest decent method.

[0257] PPCheck[7] was utilized to estimate the binding strengths via TSEs between the mutant ch8H9[3mut] antibodies and huB7H3 in the in3cl0 model. Figure 14 demonstrates that the four mutations (Table 11) are indeed predicted to enhance the bindings as their estimated absolute TSEs are higher than the ones of the two references. Model in4cl0

[0258] Model in4cl0 was generated like models in3cl6 and in3cl0 outlined above, except that for the docking step with AbAdapt the “Radius to mask non-restraints” was set to 5 [Angstrom] instead of 10. Model in4cl0 was the model with the best score (“cluster 0”) as determined by the AbAdapt tool.

[0259] Model in4cl0 was also subjected to in silico saturation mutagenesis using mCSM- AB2[9]. From the 18 mutations identified in the in3cl6 model (Figure 10A), five mutations were identified having enhanced binding affinities in the in4cl0 model (Table 12). Table 12. Mutations predicted to have enhanced binding affinities in both the in3cl6 and in4cl0 models.

[0260] To verify the saturation mutagenesis results, each of the 5 mutations (Table 12) were modeled using SWISS-MODEL and energy-minimized using obabel / minimize (Open Babel) in 250 steps and a general amber force field using a steepest decent method.

[0261] PPCheck[7] was utilized to estimate the binding strengths via TSEs between the mutant ch8H9[3mut] antibodies and huB7H3 in the in4cl0 model. Figure 15 demonstrates that, with the exception of VH-Q59Y, the four mutations indicated in Table 12 are indeed predicted to enhance the bindings as their estimated absolute TSEs are higher than the ones of the two references. Example 4: In silico humanization of ch8H9[3mut] to generate hu8H9[3mut] (hu[1rd]8H9, hu[2rd]8H9, hu[3rd]8H9)

[0262] To minimize immunogenicity towards the antibody, murine sequences in framework regions can be replaced with human sequences. The design of such humanized antibodies can be done using software tools such as BioPhi (https: / / biophi.dichlab.org / )

[0014] .

[0263] BioPhi was employed for in silico humanization of the variable sequences of ch8H9[3mut], yielding hu8H9[3mut] VH and VL sequences. BioPhi yielded one hu8H9[3mut] VH sequence and three hu8H9[3mut] VL sequences over 1-5 iterations / rounds of humanization with prevalence stringency settings of >10% or >=90%, as shown by multiple sequence alignment using Clustal Omega

[0015] . Individual humanized fragment variable (Fv) sequences consisting of one humanized VL sequence and the humanized VH sequence are referred to ashu8H9[3mut_Xrd], whereby “X” refers to the number of iterations / rounds of humanization employed and “3mut” to the 3 CDR mutations of the “6m” mutations described by Ahmed et al. Similarly, humanized Fv sequences carrying all 6 of the “6m” mutations are referred to as hu[Xrd]8H9-6m, whereby “X” refers to the number of iterations / rounds of humanization performed. Figure 16 indicates that only one hu8H9[3mut] VH sequence was obtained irrespective of the number of iteration rounds or the prevalence stringency settings. Figure 17 indicates three hu8H9[3mut] VL sequences were obtained after 5 rounds of humanization. The non-human fraction was increased from 38% to 42% from 1 to 2 or more iterations, suggesting that the first iteration yielded the sequence with the lowest potential immunogenicity. No additional sequence changes occurred after 3 rounds of humanization (Figure 17). Example 5: Introduction of the top-5 mutations into the hu8H9[3mut] Fv antibodies

[0264] To assess whether the top-5 mutations: VH-D33W, VH-S57R, VH-G102W, VL- I2W, and VL-D31N, in the hu8H9[3mut] fragment variable (Fv) antibodies also improve the binding affinities for the antigen, virtual structures for each mutation were generated using SWISS-MODEL in the in3cl6 model and energy-minimized using obabel / minimize (Open Babel) with 250 steps with a general amber force field using a steepest decent method.

[0265] PPCheck[7] was utilized to estimate the binding strengths via TSEs between the mutant hu8H9[3mut] Fv antibodies and huB7H3. Figures 18A-18C demonstrate that all the top-5 mutations (Table 13) are indeed predicted to enhance the bindings as their estimated absolute TSEs are higher than the ones of the two references. The VL-D31N mutation, along with the framework obtained after 2 rounds of iteration, shows the highest antigen-binding affinity (the lowest TSE value) across all the mutations and all the rounds of humanization (Figure 18B). Table 13. Top-5 mutations identified in the ch8H9[3mut] Fab with enhanced antigen-binding e in3cl6 model.Example 6: Binding affinity comparisons to ch8H9-6m and hu8H9-6m antibodies

[0266] To assess whether the antibodies we designed have higher binding affinities than other ch8H9 / hu8H9-based antibodies developed by others, such as ch8H9-6m and hu8H9-6m by Ahmed et al., we further modeled the following constructs.

[0267] We modeled ch8H9-6m Fab constructs with the “6m” mutations identified by Ahmed et al. [1] using the in3cl6 structural model with the ch8H9[3mut] Fab as the modeling template and heavy and light chain sequence variants as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. Figure 19 shows the TSE values of such antibody variants for both the heavy and light chains combined (sum of H TSE and L TSE). The data shows that VH-D33W, VH-G102W, VL-D31N, all top-5 mutations in combination, and potentially VL-I2W, yield lower TSEs in comparison to the “ch8H9-6m” and other reference antibodies, suggesting our antibodies have higher binding affinities for huB7H3 than the antibodies developed by others.

[0268] We modeled hu8H9-6m fragment variable (Fv) (i.e., VL + VH domains only) constructs with the “6m” mutations identified by Ahmed et al. [1] using the in3cl6 structural model with the ch8H9[3mut] Fab as the modeling template and heavy and light chain variable sequence variants as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. Figure 20 shows the TSE values of such antibody variants for both the variable heavy (VH) and variable light (VL) chains combined (sum of VH TSE and VL TSE). The data suggests that the top-5 mutations we identified, either alone or in combination, yield higher binding affinities for huB7H3 than the humanized antibodies hu[JBC]8H9 and hu[JBC]8H9-6m described and referred to by Ahmed et al. [1] as hu8H9 and hu8H9-6m, respectively. The inclusion of the “6m” mutations in hu[JBC]8H9 did in our system not increase the predicted affinity, potentially due to the modeling template already including 3 of the 6 mutations which might have biased the wild-type antibody towards a configuration with an artificially increased predicted binding affinity.

[0269] We further modeled ch8H9-6m Fab constructs with the “6m” mutations identified by Ahmed et al. [1] using the in3cl6 structural model with the ch8H9 Fab as the modeling template and heavy and light chain sequence variants as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. Figure 21 shows the TSE values of such antibody variants for both the heavy and light chains combined (sum of H TSE and L TSE). Using the ch8H9 Fab as part of the template, the data shows that from thetop-5 mutations (Table 13), VH-D33W, VH-S57R, VH-G102W, and VL-D31N alone, and all the 5 mutations in combination, yield lower TSEs in comparison to the “ch8H9-6m” and other reference Fab antibodies, suggesting our antibodies have higher binding affinities for huB7H3 than the antibodies developed by others.

[0270] We also modeled hu8H9-6m fragment variable (Fv) (i.e., VL + VH domains only) constructs with the “6m” mutations identified by Ahmed et al. [1] using the in3cl6 structural model with the ch8H9 Fab as the modeling template and heavy and light chain variable sequence variants as target sequences. The modeled constructs were energy-minimized using Open Babel (obabel, minimize) with 250 steps. Figure 22 shows the TSE values of such antibody variants for both the variable heavy (VH) and variable light (VL) chains combined (sum of VH TSE and VL TSE). Using the ch8H9 Fab as part of the template, the data suggests that the top-5 mutations we identified, either alone or in combination, yield higher binding affinities for huB7H3 than the humanized antibodies hu[JBC]8H9 and hu[JBC]8H9-6m described by Ahmed et al. [1], especially for the antibodies in silico humanized with one round / iteration (Figure 22). Example 7: Wet-lab validation Prioritizing constructs for wet lab validation

[0271] With budgetary constraints in mind, mutations predicted to enhance ch8H9[3mut] or hu8H9 affinity for huB7H3 may be wet lab-validated in several iterations. First, individual mutations could be tested for huB7H3 binding affinities. Those confirmed with enhanced binding affinities could then be validated in combinations. However, adding construct(s) with the highest expected affinity early on might be beneficial also as it may lead to a “quick win”.

[0272] To further prioritize among the combinations, predicted interaction scores could be calculated to identify the combinations with a cooperative effect, i.e., those combinations with a higher predicted affinity than any of the mutations individually[16-17]. For combinations consisting of three or more mutations, testing for cooperative effects could also be conducted relative to sub-combinations, e.g., dual mutation combinations for triple mutation combinations. Figure 23 demonstrates that all but two combinations contain components acting cooperatively to enhance the affinity. The two combinations with antagonistic components are the following:^ VL-I2W_VH-D33W_VH-S57R whereby VH-D33W_VH-S57R was predicted to act antagonistically. ^ VH-D33W_VH-S57R_VH-G102W whereby VH-D33W_VH-S57R was predicted to act antagonistically.

[0273] In both cases, the dual mutation VH-D33W_VH-S57R yielded a higher predicted affinity (lower TSE) than the triple combinations, suggesting that this dual mutation would provide more favorable benefits than the triple combinations. Below indicates the suggestion of the first set of constructs planned for wet-lab validation. “_1rd” refers to the in silico humanization conducted with 1 round / iteration of humanization using BioPhi. ^ Reference Constructs 1. ch8H9 2. ch8H9-6m 3. hu8H9 by Ahmed et al. (2015) [1] 4. hu8H9-6m by Ahmed et al. (2015) [1] ^ Test Constructs 1. ch8H9[3mut]_VH-D33W 2. ch8H9[3mut]_Top5 Combination (all 5 mutations) 3. hu8H9[3mut_1rd]_VH-D33W 4. hu8H9[3mut_1rd]_Top5 Combination (all 5 mutations) 5. hu[1rd]8H9-6m_VH-D33W 6. hu[1rd]8H9-6m_VH-S57R 7. hu[1rd]8H9-6m_VH-G102W 8. hu[1rd]8H9-6m_VL-I2W 9. hu[1rd]8H9-6m_VL-D31N 10. hu[1rd]8H9-6m_Top5 Combination (all 5 “top-5” mutations) Methods for synthesis and purification of constructs

[0274] For each construct of interest, a protein sequence and one or more corresponding DNA sequences are derived. Depending on the expression system (e.g., mouse, hamster, human) and other factors (e.g., RNA stability), a specific logic to select the DNA codons is applied.

[0275] Whereas several methods can be used to obtain the DNA molecules, often, they are synthetically made then cloned into an expression vector such as a plasmid. The expression vector is then mixed with the expression system, such as hamster cells [especially Chinese Hamster Ovary (CHO) cells or derivatives therefrom] or cells from human origin. Plasmid vectors are generally inserted into the expression system using a transfection reagent or by electroporation. The choice of expression system depends on various factors, such as expected / desired expression levels or considerations around species- or cell type-specific post- translational modifications (such as glycosylation)

[0018] .

[0276] To purify the constructs of interest from the host cells or their supernatant, a variety of methods can be applied. If the construct of interest is a single chain fragment variable (scFv) or a tandem-scFv (taFv) (“BiTE” format), genetically encoded affinity tags can be added to a region not involved in antigen-binding. The C-terminus of the construct can be a suitable location for such a tag. A commonly used affinity tag is the hexa-histidine (6xHis, HHHHHH, SEQ ID NO: 85) tag (“His tag”), allowing purification by metal-chelate affinity chromatography.

[0277] For those skilled in the art, it is obvious that additional and alternative methods could be applied to synthesize and purify the binding agent of interest. Methods for measuring affinity for antigen

[0278] Once purified, it might be desirable to assess the affinity of the binding agent for the antigen. A commonly used strategy is based on surface plasmon resonance (SPR) using a Biacore™ system. In essence, one of the binding partners (i.e., theoretically, either the binding agent (“antibody”) or the antigen) is immobilized on the sensor chip surface then a series of concentrations of the other binding partner is injected. Changes in the index of refraction at the interaction surface are used to determine the affinity (https: / / en.wikipedia.org / wiki / Biacore). Another approach to measure the affinity between the binding agent and the antigen is the enzyme-linked immunosorbent assay (ELISA). For both SPR and ELISA with ch / hu8H9[3mut]-based constructs, suitable antigens include 2Ig-B7-H3 and 4Ig-B7-H3 (Figure 1) [1, 3]. Methods for measuring bioactivity

[0279] It is further desirable to determine the potential of the hu8H9[3mut] binding unit to induce target cell lysis. This can be accomplished by generating constructs with said bindingunit connected to an effector domain such as an anti-CD3 domain to activate T cells for target cell-directed lysis or a fragment constant (Fc) domain to promote natural killer cell-mediated target cell destruction.

[0280] The following is an example in which the hu8H9[3mut] binding unit is a single-chain fragment variable (scFv) and the effector domain an anti-CD3 domain, yielding a tandem scFv (taFv) construct of the bispecific T cell engager (BiTE) class.

[0281] Sequence options of the taFv binding agent are described below. Each construct carries a hexa-histidine tag at the C-terminus. After expression in CHO cells and purification by metal-chelate affinity chromatography, the binding agent and CD3-sorted human T cells are added to target cells such as prostate cancer cells in a 96-well plate. After 4-6 hours, target cell viability is assessed using, e.g., a lactate dehydrogenase (LDH) release assay. To obtain half- maximal effective concentrations (EC50), multiple concentrations of the binding agents are added to the target cells. To identify the optimal ratio of T cell effector (E) to target (T) cells, corresponding E / T optimization experiments are conducted prior to the dose-response experiments yielding EC50 values. Design of anti-huB7H3 antibodies at the sequence level

[0282] The ch8H9[3mut] and hu8H9[3mut] antibodies, with or without additional mutations, can be expressed as a variety of formats, such as a Fab, an scFv, a taFv of the BiTE class, or a full-length IgG antibody with or without a “warhead” (e.g., a chemotherapeutic agent).

[0283] To validate an in silico predicted affinity increase by a mutation, simple formats with inexpensive manufacturing processes are preferred. Particularly suitable are scFv and taFv formats as they can be expressed from a single coding sequence.

[0284] It is possible that the orientation (VL-VH versus VH-VL) can affect the binding characteristics of scFv-based binding agents[19-21]. Therefore, constructs with both orientations might be desirable.

[0285] The present application also provides exemplary sequences relevant to this disclosure in the section “Informal Sequence Listing”. It is imperative that the disclosure also includes additional sequences related to ch8H9 or hu8H9, e.g., with combinations of mutations not shown.Example 8: Affinity and Specificity Analysis of an Exemplary VL-D31N scFv Antibody for Human B7-H3

[0286] This example illustrates the evaluation of an exemplary VL-D31N scFv antibody for its affinity to and specificity for the antigen B7-H3 using Surface Plasmon Resonance (SPR) assays.

[0287] To assess the binding strengths between the antigen human B7-H3 and its antibodies, an SPR assay was performed using Series S Sensor Chip Protein A (Cytiva). The antigen (Fc- tagged B7-H3) was injected onto the sensor chip as the capture molecule. The antibodies (scFvs), including ch8H9-6m (SEQ ID NO: 93), hu[JBC]8H9 (SEQ ID NO: 94), hu[JBC]8H9- 6m (SEQ ID NO: 95), hu[1rd]8H9-6m (SEQ ID NO: 120), and hu[1rd]8H9-6m_VL-D31N (SEQ ID NO: 104), were diluted and injected over the surface of flow cell 1 and 2 during the association phase, followed by the injection of running buffer during the dissociation phase. As shown in Figure 25, all of the antibodies exhibited measurable binding affinities to the antigen human B7-H3. The sensor-grams data (Figure 25) were analyzed and converted into binding measurement data, which were quantified in Table 14 and Figure 26. These data demonstrate that the antibody hu[1rd]8H9-6m_VL-D31N shows the lowest KD [nM] value, indicating the highest binding affinity to the antigen human B7-H3. Table 14. Affinity of VL-D31N scFv for Human B7-H3 by Surface Plasmon Resonance (SPR) in first SPR experiment.

[0288] We further investigated the specificity of the antibody hu[1rd]8H9-6m_VL-D31N for human B7-H3 using an SPR assay. Four Fc-tagged B7 proteins, including B7-H3, B7-H2 (ICOS ligand), B7-H4 (VTCN1), and B7-H1 (PD-L1), were injected onto Series S Sensor Chip Protein A (Cytiva) as distinct capture molecules in the assay. The antibodies (scFvs) hu[1rd]8H9-6m (SEQ ID NO: 120), hu[1rd]8H9-6m_VL-D31N (SEQ ID NO: 104), andhu[JBC]8H9-6m (SEQ ID NO: 95) were diluted and injected over the surface of flow cell 1 and 2 during the association phase, followed by the injection of running buffer during the dissociation phase. As shown in Figure 27, binding was only observed with the antigen B7- H3 but not with other antigens, i.e., B7-H2 (ICOS ligand), B7-H4 (VTCN1), and B7-H1 (PD- L1). The sensor-grams data (Figure 27) were analyzed and converted into binding measurement data, which were quantified in Table 15 and Figure 28. These data demonstrate that the antibody hu[1rd]8H9-6m_VL-D31N specifically binds to the antigen B7-H3 without cross-reactivity to the other antigens, indicating its specificity for human B7-H3. Table 15. Specificity of VL-D31N scFv for Human B7-H3 but not B7-H1, B7-H2, or B7-H4 by Surface Plasmon Resonance in second SPR experiment. NA, not applicable (no obvious binding)

[0289] We further evaluated the predicted stabilizing energy of the VL-D31N, VL-F94W, VH-N31L, and VH-G102D mutations, alone or in combination, in hu[1rd]8H9-6m Fv constructs. Shown in Figure 29 are total stabilizing energy (TSE) values from PPCheck (https: / / caps.ncbs.res.in / ppcheck / ) for both the variable heavy (VH) and light (VL) chains combined (sum of VH TSE and VL TSE). PPCheck input structures were modeled using SWISS-MODEL with the ch8H9[3mut] Fab structure as template and then energy-minimizedusing Open Babel (obabel, minimize) with 250 steps. Using a cutoff at the lowest reference TSE value (among VH-G8A and WT), each of the four mutations tested (VL-D31N, VL- F94W, VH-N31L, VH-G102D), alone or in combination, yielded a TSE value smaller than the cutoff given by the references.

[0290] Figure 30 shows binding sensor-grams of ligand to analyte for scFv binding to human B7-H3 for hu[JBC]8H9-6m, hu[1rd]8H9-6m, and hu[1rd]8H9-6m_VL-D31N scFvs as assessed by SPR in a third SPR experiment. Two sets of scFvs were tested, generated in different production runs (Prod. Round B, Prod. Round C). The sensor-grams data (Figure 30) were analyzed and converted into binding measurement data, which were quantified in Table 16 and Figure 31. Table 16. Specificity of VL-D31N scFv for Human B7-H3 by Surface Plasmon Resonance in third SPR experiment.

[0291] We further assessed the affinity increase by hu[1rd]8H9-6m_VL-D31N relative to hu[JBC]8H9-6m for scFv constructs. Figure 32 depicts a statistical assessment of the affinity increase by hu[1rd]8H9-6m_VL-D31N relative to hu[JBC]8H9-6m. Three SPR experiments (#1-3) were conducted with scFvs generated in 2-3 production rounds (referred to as production rounds A, B, or C). Figure 32A shows KD values in nanomoles (nM). Figure 32B shows the purity of the scFvs as assessed by SDS-PAGE and SEC-HPLC. Figure 32C shows arithmetic means and standard error of the mean (SEM) values of the KD values from the three experiments. For hu[1rd]8H9-6m and hu[1rd]8H9-6m_VL-D31N in SPR #3, the average KD values for production rounds B and C were used as the SPR #3 contribution to the means. For hu[JBC]8H9-6m, the mean was directly calculated with the 3 values shown. For hu[1rd]8H9- 6m_VL-D31N, compared to hu[JBC]8H9-6m, there is a trend for higher affinity for human B7-H3 (lower KD values; p = 0.081; one-tailed t-test). Figure 32D shows arithmetic means and SEM values of the KD values from the three SPR experiments after normalizing (dividing)each value with the hu[JBC]8H9-6m KD value from the same experiment. This approach corrects for potential systemic effects equally affecting all scFvs within an experiment. For SPR #3, the KD values prior to normalization were handled as for Figure 32C. The affinity for human B7-H3 is significantly higher (lower KD value) for hu[1rd]8H9-6m_VL-D31N than for hu[JBC]8H9-6m (p = 0.020; one-sample, one-tailed t-test).

[0292] Since low purities may negatively affect SPR results, comparing affinity levels obtained with scFvs of suboptimal purity may not yield an accurate ranking of the scFvs based on their true affinities. Figures 33-40 summarize the purities obtained by SDS-PAGE and SEC-HPLC for the scFvs with the sequences hu[JBC]8H9-6m, hu[1rd]8H9-6m, and hu[1rd]8H9-6m_VL-D31N for the different production rounds.

[0293] Figure 33 depicts purity data of the hu[JBC]8H9-6m scFv generated in production round A. Figure 34 depicts purity data of the hu[JBC]8H9-6m scFv generated in production round B. Figure 35 depicts purity data of the hu[1rd]8H9-6m scFv generated in production round A. Figure 36 depicts purity data of the hu[1rd]8H9-6m scFv generated in production round B. Figure 37 depicts purity data of the hu[1rd]8H9-6m scFv generated in production round C. Figure 38 depicts purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round A. Figure 39 depicts purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round B. Figure 40 depicts SEC-HPLC purity data of the hu[1rd]8H9- 6m_VL-D31N scFv generated in production round B undiluted (Figure 40A) and diluted to 0.5 mg / ml (Figure 40B). Figure 41 depicts purity data of the hu[1rd]8H9-6m_VL-D31N scFv generated in production round C. References 1. Ahmed, M.; Cheng, M.; Zhao, Q.; Goldgur, Y.; Cheal, S. M.; Guo, H. 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[0294] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. V. Informal Sequence Listing SEQ ID NO: 1 Murine B7-H3 (Uniprot Q8VE98) MLRGWGGPSVGVCVRTALGVLCLCLTGAVEVQVSEDPVVALVDTDATLRCS FSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYSNRTALFPDLLVQGNASLRL QRVRVTDEGSYTCFVSIQDFDSAAVSLQVAAPYSKPSMTLEPNKDLRPGNMVTITCS SYQGYPEAEVFWKDGQGVPLTGNVTTSQMANERGLFDVHSVLRVVLGANGTYSCL VRNPVLQQDAHGSVTITGQPLTFPPEALWVTVGLSVCLVVLLVALAFVCWRKIKQSC EEENAGAEDQDGDGEGSKTALRPLKPSENKEDDGQEIA SEQ ID NO: 2 Human B7-H3 (Uniprot Q5ZPR3, NCBI NP_001019907.1) MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCC SFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASL RLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCL VRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLA QLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADE GSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAE VFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQD AHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAED QDGEGEGSKTALQPLKHSDSKEDDGQEIA SEQ ID NO: 3 human B7-H3 Repeats of postulated core binding motif of ch8H9 antibody FVSIRDFG SEQ ID NO: 4 hB7-H3 Transmembrane region LWVTVGLSVCLIALLVALAFV SEQ ID NO: 5 SWISS-MODEL input sequence MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCC SFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASL RLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITC SSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCL VRNPVLQQDAHSSVTITPQRSPTGAVEVQV SEQ ID NO: 6 SWISS-MODEL output sequence EDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGS AYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSK PSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQG LFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQ SEQ ID NO: 7 HCDR1 w / Xs GYX1FTX2X3X4SEQ ID NO: 8 HCDR2 w / Xs IFPGDX5X6T SEQ ID NO: 9 HCDR3 w / Xs ARQX7X8X9X10X11FAY SEQ ID NO: 10 HCDR1 GYTFTNYD SEQ ID NO: 11 HCDR2 (G) IFPGDGST SEQ ID NO: 12 HCDR2 (D) IFPGDDST SEQ ID NO: 13 HCDR3 (A) ARQTTATWFAY SEQ ID NO: 14 HCDR3 (G) ARQTTGTWFAY SEQ ID NO: 15 HFR1 w / Xs QVQLQQSGAELVKPGASVKLSCKX12S SEQ ID NO: 16 HFR2 w / Xs INWVRQRPX13QGLEWIGW SEQ ID NO: 17 HFR3 w / Xs X31YNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCSEQ ID NO: 18 HFR4 WGQGTLVTVSA SEQ ID NO: 19 LCDR1 w / Xs X14X15X16X17X18X19SEQ ID NO: 20 LCDR3 w / Xs QNX20X21X22X23X24X25T SEQ ID NO: 21 CDRL1 QSISDY SEQ ID NO: 22 CDRL3 QNGHSFPLT SEQ ID NO: 23 LFR1 w / Xs X26X27VMTQSPATLSVTPGDRVX28LSCRAS SEQ ID NO: 24 LFR2 w / Xs LX29WYQQKSHESPRLLIK SEQ ID NO: 25 LFR3 w / Xs QSIX30GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC SEQ ID NO: 26 LFR4 FGAGTKLELK SEQ ID NO: 27 VH-D33W GYTFTNYW SEQ ID NO: 28 VH-S57R IFPGDDRT SEQ ID NO: 29 VH-G102W ARQTTWTWFAY SEQ ID NO: 30 VL-I2W DWVMTQSPATLSVTPGDRVSLSCRAS SEQ ID NO: 31 VL-D31N QSISNY SEQ ID NO: 32 Anti-huB7H3 VH of ch8H9[3mut] QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSA Bold, underscored: CDR mutations shown to increase affinity by yeast display [1] SEQ ID NO: 33 Anti-huB7H3 VH of ch8H9[3mut] with VH-D33W mutation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAY WGQGTLVTVSA SEQ ID NO: 34 Anti-huB7H3 VH of ch8H9[3mut] with VH-S57R mutation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSA SEQ ID NO: 35 Anti-huB7H3 VH of ch8H9[3mut] with VH-G102W mutation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAYW GQGTLVTVSASEQ ID NO: 36 Anti-huB7H3 VH of ch8H9[3mut] with VH-D33W, VH-S57R, VH- G102W mutations QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAY WGQGTLVTVSA SEQ ID NO: 37 VH of hu8H9[3mut] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSS Bold, underscored: CDR mutations shown to increase affinity by yeast display[1]. SEQ ID NO: 38 VH of hu8H9[3mut] with A24T mutation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSS Bold, underscored: CDR mutations shown to increase affinity by yeast display[1]. Bold, underscored, italics: A24T framework mutation shown to increase affinity by yeast display[1]. SEQ ID NO: 39 VH of hu8H9[3mut] with D33W mutation QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFA YWGQGTLVTVSS Bold, underscored: CDR mutations shown to increase affinity by yeast display[1]. Bold, underscored, italics: VH-D33W mutation. SEQ ID NO: 40 VH of hu8H9[3mut] with S57R mutation QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSS Bold, underscored: CDR mutations shown to increase affinity by yeast display[1]. Bold, underscored, italics: VH-S57R mutation. SEQ ID NO: 41 VH of hu8H9[3mut] with G102W mutation QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWFAY WGQGTLVTVSS Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VH-G102W mutation. SEQ ID NO: 42 Anti-huB7H3 VL of ch8H9[3mut] DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK Bold, underscored: CDR mutation shown to increase affinity by yeast display [1] SEQ ID NO: 43 Anti-huB7H3 VL of ch8H9[3mut] with VL-I2W mutation DWVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYAS QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKSEQ ID NO: 44 Anti-huB7H3 VL of ch8H9[3mut] with VL-D31N mutation DIVMTQSPATLSVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK SEQ ID NO: 45 Anti-huB7H3 VL of ch8H9[3mut] with VL-I2W, VL-D31N mutations DWVMTQSPATLSVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYAS QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK SEQ ID NO: 46 VL of hu8H9[3mut] after 1 iteration of humanization (hu8H9[3mut_1rd]) DIVMTQSPATLSVSPGDRVTLSCRASQSISDYLYWYQQKPGQSPRLLIKYASQSISGIP SRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. SEQ ID NO: 47 VL of hu8H9[3mut] after 2 iterations of humanization (hu8H9[3mut_2rd]) DIVMTQSPATLSVSPGDRVTLSCRASQSISDYLYWYQQKPGQSPRLLIYYASQSISGIP SRFSGSGSGTDFTLTINSLEPEDFAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. SEQ ID NO: 48 VL of hu8H9[3mut] after 3-5 iterations of humanization (hu8H9[3mut_3rd]) DIVMTQSPATLSVSPGDRVTLSCRASQSISDYLYWYQQKPGQAPRLLIYYASQSISGIP SRFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. SEQ ID NO: 49 VL of hu8H9[3mut_1rd] with I2W mutation DWVMTQSPATLSVSPGDRVTLSCRASQSISDYLYWYQQKPGQSPRLLIKYASQSISGI PSRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VL-I2W mutation. SEQ ID NO: 50 VL of hu8H9[3mut_1rd] with D31N mutation DIVMTQSPATLSVSPGDRVTLSCRASQSISNYLYWYQQKPGQSPRLLIKYASQSISGIP SRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VL-D31N mutation. SEQ ID NO: 51 VL of hu8H9[3mut_2rd] with I2W mutation DWVMTQSPATLSVSPGDRVTLSCRASQSISDYLYWYQQKPGQSPRLLIYYASQSISGI PSRFSGSGSGTDFTLTINSLEPEDFAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VL-I2W mutation. SEQ ID NO: 52 VL of hu8H9[3mut_2rd] with D31N mutation DIVMTQSPATLSVSPGDRVTLSCRASQSISNYLYWYQQKPGQSPRLLIYYASQSISGIP SRFSGSGSGTDFTLTINSLEPEDFAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VL-D31N mutation.SEQ ID NO: 53 VL of hu8H9[3mut_3rd] with I2W mutation DWVMTQSPATLSVSPGDRVTLSCRASQSISDYLYWYQQKPGQAPRLLIYYASQSISGI PSRFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VL-I2W mutation. SEQ ID NO: 54 VL of hu8H9[3mut_3rd] with D31N mutation DIVMTQSPATLSVSPGDRVTLSCRASQSISNYLYWYQQKPGQAPRLLIYYASQSISGIP SRFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTFGQGTKLEIK Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. Bold, underscored, italics: VL-D31N mutation. SEQ ID NO: 55 Intra-scFv linker GGGGSGGGGSGGGGS SEQ ID NO: 56 Anti-huB7H3 scFv of ch8H9[3mut], VL-VH orientation DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGS GGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLE WIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGT WFAYWGQGTLVTVSA Bold, underscored: CDR mutations shown to increase affinity by yeast display [1] SEQ ID NO: 57 Anti-huB7H3 scFv of ch8H9[3mut], VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDY LYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG HSFPLTFGAGTKLELK Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]1 SEQ ID NO: 58 Anti-huB7H3 scFv of ch8H9[3mut] with VL-I2W mutation, VL-VH orientation DWVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYAS QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGG SGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGL EWIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTG TWFAYWGQGTLVTVSA Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. Bold, underscored, italics: VL-I2W mutation. SEQ ID NO: 59 Anti-huB7H3 scFv of ch8H9[3mut] with VL-I2W mutation, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDWVMTQSPATLSVTPGDRVSLSCRASQSISD YLYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQN GHSFPLTFGAGTKLELK Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. Bold, underscored, italics: VL-I2W mutation.SEQ ID NO: 60 Anti-huB7H3 scFv of ch8H9[3mut] with VL-D31N mutation, VL-VH orientation DIVMTQSPATLSVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGS GGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLE WIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGT WFAYWGQGTLVTVSA Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. Bold, underscored, italics: VL-D31N mutation. SEQ ID NO: 61 Anti-huB7H3 scFv of ch8H9[3mut] with VL-D31N mutation, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISNY LYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG HSFPLTFGAGTKLELK Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. Bold, underscored, italics: VL-D31N mutation. SEQ ID NO: 62 Anti-huB7H3 scFv of ch8H9[3mut] with VH-D33W mutation, VL-VH orientation DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGS GGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGL EWIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTG TWFAYWGQGTLVTVSA Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. Bold, underscored, italics: VH-D33W mutation. SEQ ID NO: 63 Anti-huB7H3 scFv of ch8H9[3mut] with VH-D33W mutation, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAY WGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISD YLYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQN GHSFPLTFGAGTKLELK SEQ ID NO: 64 Anti-huB7H3 scFv of ch8H9[3mut] with VH-S57R mutation, VL-VH orientation DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGS GGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLE WIGWIFPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGT WFAYWGQGTLVTVSA SEQ ID NO: 65 Anti-huB7H3 scFv of ch8H9[3mut] with VH-S57R mutation, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDY LYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG HSFPLTFGAGTKLELK SEQ ID NO: 66 Anti-huB7H3 scFv of ch8H9[3mut] with VH-G102W mutation, VL-VH orientation DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGS GGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLE WIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWT WFAYWGQGTLVTVSA SEQ ID NO: 67 Anti-huB7H3 scFv of ch8H9[3mut] with VH-G102W mutation, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDY LYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG HSFPLTFGAGTKLELK SEQ ID NO: 68 Anti-huB7H3 scFv of ch8H9[3mut] with VL-I2W, VL-D31N, VH- D33W, VH-S57R, VH-G102W mutations, VL-VH orientation DWVMTQSPATLSVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYAS QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGG SGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQG LEWIGWIFPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTT WTWFAYWGQGTLVTVSA SEQ ID NO: 69 Anti-huB7H3 scFv of ch8H9[3mut] with VL-I2W, VL-D31N, VH- D33W, VH-S57R, VH-G102W mutations, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAY WGQGTLVTVSAGGGGSGGGGSGGGGSDWVMTQSPATLSVTPGDRVSLSCRASQSIS NYLYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQ NGHSFPLTFGAGTKLELK SEQ ID NO: 70 Heavy chain (VH-CH1) of ch8H9 QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 71 Heavy chain (VH-CH1) of ch8H9[3mut] QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV Bold, underscored: CDR mutations shown to increase affinity by yeast display[1]. SEQ ID NO: 72 Heavy chain (VH-CH1) of ch8H9[3mut] with VH-D33W mutationQVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 73 Heavy chain (VH-CH1) of ch8H9[3mut] with VH-S57R mutation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 74 Heavy chain (VH-CH1) of ch8H9[3mut] with VH-G102W mutation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAYW GQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 75 Heavy chain (VH-CH1) of ch8H9[3mut]] with VH-D33W, VH-S57R, VH-G102W mutations QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 76 Light chain (VL-CL; kappa) of ch8H9 DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE SEQ ID NO: 77 Light chain (VL-CL; kappa) of ch8H9[3mut] DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE Bold, underscored: CDR mutation shown to increase affinity by yeast display[1]. SEQ ID NO: 78 Light chain (VL-CL; kappa) of ch8H9[3mut] with VL-I2W mutation DWVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYAS QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE SEQ ID NO: 79 Light chain (VL-CL; kappa) of ch8H9[3mut] with VL-D31N mutation DIVMTQSPATLSVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESEQ ID NO: 80 Light chain (VL-CL; kappa) of ch8H9[3mut] with VL-I2W, VL-D31N mutations DWVMTQSPATLSVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYAS QSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE SEQ ID NO: 81 Anti-CD3 VH DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYI NPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTTLTVSS SEQ ID NO: 82 Anti-CD3 VL DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSK VASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK SEQ ID NO: 83 Intra-scFv linker VEGGSGGSGGSGGSGGVD SEQ ID NO: 84 Signal peptide MGWSCIILFLVATATGVHS SEQ ID NO: 85 Hexa histidine tag HHHHHH SEQ ID NO: 86 Inter-scFv linker GGGGS SEQ ID NO: 87 Anti-CD3 scFv in VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYI NPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASS SVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAAT YYCQQWSSNPLTFGAGTKLELK SEQ ID NO: 88 huB7H3 x CD3 taFv (“BiTE”) in VL-VH (anti-huB7H3; ch8H9[3mut]) x VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 MGWSCIILFLVATATGVHSDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLY WYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHS FPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGY TFTNYDINWVRQRPEQGLEWIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLS RLTSEDSAVYFCARQTTGTWFAYWGQGTLVTVSAGGGGSDIKLQQSGAELARPGAS VKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTT DKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSG GSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWI YDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLE LKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. SEQ ID NO: 89 huB7H3 x CD3 taFv (“BiTE”) in VH-VL (anti-huB7H3; ch8H9[3mut]) x VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 MGWSCIILFLVATATGVHSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYD INWVRQRPEQGLEWIGWIFPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLS VTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFT LSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGSDIKLQQSGAELARPGAS VKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTT DKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSG GSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWI YDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLE LKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. SEQ ID NO: 90 huB7H3 x CD3 taFv (“BiTE”) in VL-VH (anti-huB7H3; ch8H9[3mut]) x VH-VL orientation, with VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations MGWSCIILFLVATATGVHSDWVMTQSPATLSVTPGDRVSLSCRASQSISNYLY WYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHS FPLTFGAGTKLELKGGGGSGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASGY TFTNYWINWVRQRPEQGLEWIGWIFPGDDRTQYNEKFKGKATLTTDTSSSTAYMQL SRLTSEDSAVYFCARQTTWTWFAYWGQGTLVTVSAGGGGSDIKLQQSGAELARPGA SVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLT TDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGS GGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRW IYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLE LKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. SEQ ID NO: 91 huB7H3 x CD3 taFv (“BiTE”) in VH-VL (anti-huB7H3; ch8H9[3mut]) x VH-VL orientation, with VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations MGWSCIILFLVATATGVHSQVQLQQSGAELVKPGASVKLSCKASGYTFTNYW INWVRQRPEQGLEWIGWIFPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSED SAVYFCARQTTWTWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDWVMTQSPATL SVTPGDRVSLSCRASQSISNYLYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDF TLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGSDIKLQQSGAELARPGA SVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLT TDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGS GGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRW IYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLE LKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. SEQ ID NO: 92 Anti-huB7H3 scFv of ch8H9, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWG QGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLH WYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHS FPLTFGAGTKLELKHHHHHH His tag: HHHHHH SEQ ID NO: 93 Anti-huB7H3 scFv of ch8H9-6m, VH-VL orientationQVQLQQSGAELVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYWG QGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVTLSCRASQSISDYL YWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGH SFPLTFGAGTKLELKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display [1]. His tag: HHHHHH SEQ ID NO: 94 Anti-huB7H3 scFv of hu[JBC]8H9 (hu8H9 VH and VL from Table 2 by Ahmed et al. (2015) [1] with added His tag, HHHHHH), VH-VL orientation QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWI FPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVSLSCRASQSISD YLHWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQN GHSFPLTFGQGTKLELKRHHHHHH His tag: HHHHHH SEQ ID NO: 95 Anti-huB7H3 scFv of hu[JBC]8H9-6m, VH-VL orientation QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTGTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISD YLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQN GHSFPLTFGQGTKLELKRHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. His tag: HHHHHH SEQ ID NO: 96 Anti-huB7H3 scFv of ch8H9[3mut] with VH-D33W mutation (SEQ ID NO: 13) with His Tag, HHHHHH, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDYL YWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGH SFPLTFGAGTKLELKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-D33W mutation. His tag: HHHHHH SEQ ID NO: 97 Anti-huB7H3 scFv of ch8H9[3mut] with VL-I2W, VL-D31N, VH- D33W, VH-S57R, VH-G102W mutations (SEQ ID NO: 19) with His Tag, HHHHHH, VH-VL orientation QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDWVMTQSPATLSVTPGDRVSLSCRASQSISNY LYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG HSFPLTFGAGTKLELKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. His tag: HHHHHH SEQ ID NO: 98 Anti-huB7H3 scFv of hu[1rd]8H9 with VH-D33W mutation, VH-VL orientationQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFA YWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSIS DYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQ NGHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR mutation shown to increase affinity by yeast display. Bold, underscored, italics: VH-D33W mutation. His tag: HHHHHH SEQ ID NO: 99 Anti-huB7H3 scFv of hu[1rd]8H9 with VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations (SEQ ID NO: 19) with His Tag, HHHHHH, VH-VL orientation QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWF AYWGQGTLVTVSSGGGGSGGGGSGGGGSDWVMTQSPATLSVSPGDRVTLSCRASQ SISNYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYY CQNGHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display [1]. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. His tag: HHHHHH SEQ ID NO: 100 Anti-huB7H3 scFv of hu[1rd]8H9-6m with VH-D33W mutation, VH- VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFA YWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSIS DYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQ NGHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-D33W mutation. His tag: HHHHHH SEQ ID NO: 101 Anti-huB7H3 scFv of hu[1rd]8H9-6m with VH-S57R mutation, VH-VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISD YLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQN GHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-S57R mutation. His tag: HHHHHH SEQ ID NO: 102 Anti-huB7H3 scFv of hu[1rd]8H9-6m with VH-G102W mutation, VH- VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISD YLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQN GHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-G102W mutation. His tag: HHHHHHSEQ ID NO: 103 Anti-huB7H3 scFv of hu[1rd]8H9-6m with VL-I2W mutation, VH-VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSDWVMTQSPATLSVSPGDRVTLSCRASQSIS DYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQ NGHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W mutation. His tag: HHHHHH SEQ ID NO: 104 Anti-huB7H3 scFv of hu[1rd]8H9-6m with VL-D31N mutation, VH- VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISN YLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQN GHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-D31N mutation. His tag: HHHHHH SEQ ID NO: 105 Anti-huB7H3 scFv of hu[1rd]8H9-6m with VL-I2W, VL-D31N, VH- D33W, VH-S57R, VH-G102W mutations, VH-VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWF AYWGQGTLVTVSSGGGGSGGGGSGGGGSDWVMTQSPATLSVSPGDRVTLSCRASQ SISNYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYY CQNGHSFPLTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. His tag: HHHHHH Bispecifics (CD3) SEQ ID NO: 106 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of ch8H9, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWG QGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLH WYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHS FPLTFGAGTKLELKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHH Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag.SEQ ID NO: 107 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of ch8H9-6m, VH- VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLQQSGAELVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIF PGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYWG QGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVTLSCRASQSISDYL YWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGH SFPLTFGAGTKLELKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQ RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYD DHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCR ASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 108 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[JBC]8H9 (hu8H9 VH and VL from Table 2 by Ahmed et al. (2015) [1], VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIF PGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWG QGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVSLSCRASQSISDYLH WYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHS FPLTFGQGTKLELKRGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQ RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYD DHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCR ASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHH Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 109 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[JBC]8H9-6m, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTGTWFAYW GQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISDYL YWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGH SFPLTFGQGTKLELKRGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVK QRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTC RASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 110 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of ch8H9[3mut] with VH-D33W mutation (SEQ ID NO: 13), VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTGTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPATLSVTPGDRVSLSCRASQSISDYL YWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGH SFPLTFGAGTKLELKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYD DHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCR ASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-D33W mutation. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 111 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of ch8H9[3mut] with VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations (SEQ ID NO: 19), VH- VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWVRQRPEQGLEWIGWI FPGDDRTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTWTWFAYW GQGTLVTVSAGGGGSGGGGSGGGGSDWVMTQSPATLSVTPGDRVSLSCRASQSISNY LYWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNG HSFPLTFGAGTKLELKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVK QRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTC RASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 112 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9 with VH-D33W mutation, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISD YLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQN GHSFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWV KQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARY YDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMT CRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAAT YYCQQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR mutation shown to increase affinity by yeast display. Bold, underscored, italics: VH-D33W mutation. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 113 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9 with VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations (SEQ ID NO: 19), VH- VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWFA YWGQGTLVTVSSGGGGSGGGGSGGGGSDWVMTQSPATLSVSPGDRVTLSCRASQSIS NYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQ NGHSFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHW VKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAA TYYCQQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 114 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9-6m with VH-D33W mutation, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAY WGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISD YLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQN GHSFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWV KQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARY YDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMT CRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAAT YYCQQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-D33W mutation. Italics: [Inter-scFv linker]-[anti- CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 115 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9-6m with VH-S57R mutation, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAYW GQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISDYL YWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGH SFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-S57R mutation. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 116 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9-6m with VH-G102W mutation, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWFAYW GQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISDYL YWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGH SFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHHBold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VH-G102W mutation. Italics: [Inter-scFv linker]-[anti- CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 117 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9-6m with VL-I2W mutation, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAYW GQGTLVTVSSGGGGSGGGGSGGGGSDWVMTQSPATLSVSPGDRVTLSCRASQSISDY LYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQNG HSFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVK QRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTC RASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYY CQQWSSNPLTFGAGTKLELKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W mutation. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 118 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9-6m with VL-D31N mutation, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGW IFPGDDSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAYW GQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISNYL YWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGH SFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD HYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRA SSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLTFGAGTKLELKHHHHHH Italics: linker-[anti-CD3 (VH-VL) scFv]-His tag Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-D31N mutation. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 119 huB7H3 x CD3 taFv (“BiTE”), anti-huB7H3 scFv of hu[1rd]8H9-6m with VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations, VH-VL orientation x anti-CD3, VH-VL orientation, as in TCT001 of PCT / US2014 / 033068 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYWINWVRQAPGQGLEWMG WIFPGDDRTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTWTWFA YWGQGTLVTVSSGGGGSGGGGSGGGGSDWVMTQSPATLSVSPGDRVTLSCRASQSI SNYLYWYQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQ NGHSFPLTFGQGTKLEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHW VKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTM TCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAA TYYCQQWSSNPLTFGAGTKLELKHHHHHHBold, underscored: CDR and framework mutations shown to increase affinity by yeast display. Bold, underscored, italics: VL-I2W, VL-D31N, VH-D33W, VH-S57R, VH-G102W mutations. Italics: [Inter-scFv linker]-[anti-CD3 (VH-VL) scFv]-His tag. SEQ ID NO: 120 Anti-huB7H3 scFv of hu[1rd]8H9-6m, VH-VL orientation QVQLVQSGAEVKKPGASVKVSCKTSGYTFTNYDINWVRQAPGQGLEWMGWIFPGD DSTQYNEKFKGRVTLTTDTSTSTAYMELSRLTSEDTAVYFCARQTTGTWFAYWGQG TLVTVSSGGGGSGGGGSGGGGSDIVMTQSPATLSVSPGDRVTLSCRASQSISDYLYW YQQKPGQSPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSLEPEDVAVYYCQNGHSFP LTFGQGTKLEIKHHHHHH Bold, underscored: CDR and framework mutations shown to increase affinity by yeast display. His tag: HHHHHH

Claims

WHAT IS CLAIMED IS:

1. An isolated antibody that specifically binds to a human B7-H3 (CD276) protein, wherein the antibody comprises: a heavy chain complementarity determining region 1 (HCDR1) sequence comprising the sequence of GYTFTNYD (SEQ ID NO: 10); a HCDR2 sequence comprising the sequence of IFPGDDST (SEQ ID NO: 12); a HCDR3 sequence comprising the sequence of ARQTTGTWFAY (SEQ ID NO: 14); a light chain complementarity determining region 1 (LCDR1) sequence comprising the sequence of QSISNY (SEQ ID NO: 31); a LCDR2 sequence comprising the sequence of YAS; and a LCDR3 sequence comprising the sequence of QNGHSFPLT (SEQ ID NO: 22).

2. The isolated antibody of claim 1, wherein the antibody comprises: a heavy chain framework region 1 (HFR1) sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121 or 122; a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125 or 126; and / or a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127 or 128.

3. The isolated antibody of claim 2, wherein the antibody comprises: a HFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 121 or 122;a HFR2 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 123 or 124; a HFR3 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 125 or 126; and a HFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 127 or 128.

4. The isolated antibody of any one of claims 1-3, wherein the antibody comprises: a light chain framework region 1 (LFR1) sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 135-138; and / or a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139 or 140.

5. The isolated antibody of claim 4, wherein the antibody comprises: a LFR1 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 129 or 130; a LFR2 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 131-134; a LFR3 sequence comprising a sequence having at least 80% identity to any one of SEQ ID NOs: 135-138; and a LFR4 sequence comprising a sequence having at least 80% identity to SEQ ID NO: 139 or 140.

6. The isolated antibody of any one of claims 1-5, wherein the antibody comprises a VH sequence that has at least 85% sequence identity to SEQ ID NO: 141 or 142.

7. The isolated antibody of any one of claims 1-6, wherein the antibody comprises a VL sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 143-146.

8. The isolated antibody of any one of claims 1-7, wherein the antibody comprises the VH sequence of SEQ ID NO: 141 or 142 and the VL sequence of any one of SEQ ID NOs: 143-146.

9. The isolated antibody of any one of claims 1-8, wherein the antibody is a full-length monoclonal antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a chimeric antibody, a camelized single-domain antibody, a Fab, a F(ab’)2, a Fab′, a F(ab)2, a Fv fragment, a scFv, a di-scFv, a BiTE, a dAb, or a bivalent scFv.

10. The isolated antibody of claim 9, wherein the antibody is a scFv comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148.

11. The isolated antibody of any one of claims 1-9, wherein the antibody comprises a heavy chain (HC) comprising a sequence that has at least 85% sequence identity to SEQ ID NO:

149.

12. The isolated antibody of claim 11, wherein the antibody comprises a light chain (LC) comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 150-152.

13. The isolated antibody of claim 11 or 12, wherein the antibody comprises a HC comprising the sequence of SEQ ID NO: 149 and a LC comprising the sequence of any one of SEQ ID NOs: 150-152.

14. The isolated antibody of any one of claims 1-13, wherein the antibody is a monoclonal antibody.

15. The isolated antibody of any one of claims 1-13, wherein the antibody is a chimeric antibody.

16. The isolated antibody of any one of claims 1-13, wherein the antibody is a humanized antibody.

17. The isolated antibody of any one of claims 1-13, wherein the antibody is a fully human antibody.

18. The isolated antibody of any one of claims 1-13, wherein the antibody is a bispecific antibody.

19. The isolated antibody of claim 18, wherein the bispecific antibody binds to both B7-H3 and CD3.

20. The isolated antibody of claim 19, wherein the CD3-binding portion of the bispecific antibody comprises a VH sequence that has at least 85% sequence identity to SEQ ID NO: 81 and a VL sequence that has at least 85% sequence identity to SEQ ID NO:

82.

21. The isolated antibody of claim 19 or 20, wherein the CD3-binding portion is an scFv.

22. The isolated antibody of claim 21, wherein the scFv comprises the sequence of SEQ ID NO:

87.

23. The isolated antibody of any one of claims 19-22, wherein the B7-H3- binding portion of the bispecific antibody comprises a VH sequence that has at least 85% sequence identity to SEQ ID NO: 141 or 142 and a VL sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 143-146.

24. The isolated antibody of claim 23, wherein the B7-H3-binding portion is an scFv comprising a sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 60, 61, 104, 147 and 148.

25. The isolated antibody of any one of claims 1-24, wherein the antibody is conjugated to a therapeutic agent or detection agent.

26. The isolated antibody of claim 25, wherein the therapeutic agent is an antitumor agent, a radioisotope, a drug agent, a nanoparticle, an immune toxin, or a combination thereof.

27. The isolated antibody of claim 25, wherein the detection agent is a diagnostic or imaging agent, or both.

28. A pharmaceutical composition comprising the isolated antibody any one of claims 1-27, and a pharmaceutically acceptable carrier.

29. A kit comprising: the isolated antibody of any one of claims 1-27 or the pharmaceutical composition of claim 28, and instructions for use thereof.

30. A method of treating a cancer in a subject, comprising administering to the subject the isolated antibody of any one of claims 1-27 or the pharmaceutical composition of claim 28.

31. The method of claim 30, wherein the cancer comprises B7-H3-positive tumor cells.

32. The method of claim 30 or 31, wherein the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, a chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.

33. A method of modulating the immune system of a subject, comprising administering to the subject the isolated antibody of any one of claims 1-27 or the pharmaceutical composition of claim 28.

34. A method of enhancing T-cell mediated cytotoxicity in a subject, comprising administering to the subject the isolated antibody of any one of claims 1-27 or the pharmaceutical composition of claim 28.

35. A method of detecting B7-H3 in a biological sample, comprising contacting a biological sample with the isolated antibody of any one of claims 1-27 or the pharmaceutical composition of claim 28.

36. A method of diagnosing a subject suffering from a cancer, comprising contacting a biological sample from the subject with the isolated antibody of any one of claims 1-27 or the pharmaceutical composition of claim 28, wherein detecting the presence of B7-H3 or the expression level of B7-H3 in the sample determines the presence of the disorder.

37. The method of claim 36, wherein the disorder is a cancer.

38. The method of claim 37, wherein the cancer is selected from the group consisting of a lung cancer, a colorectal cancer, a breast cancer, a prostate cancer, a gastric cancer, a liver cancer, a cervical cancer, a brain cancer, a ovarian cancer, a pancreatic cancer, a skin cancer, an eye cancer, a soft tissue cancer, a renal cancer, a bladder cancer, a head and neck cancer, a neuroblastoma, a melanoma, a mesothelioma, an acute leukemia, a chronic leukemia, a medulloblastoma, a multiple myeloma, a sarcoma, a nasopharyngeal carcinoma, and a lymphoepithelioma-like carcinoma.

39. An antibody or antigen-binding fragment thereof that competes with the isolated antibody of any one of claims 1-27 for binding to the human B7-H3 protein.

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