Anti-B7H3 antibodies and methods of use

Abstract

The present disclosure provides antibodies, or antigen-binding fragments thereof, that bind to human 4Ig-B7H3, multispecific antibodies, or antigen-binding fragments thereof, that recognize human 4Ig-B7H3 as one antigen and at least one other antigen, anti-human 4Ig-B7H3 antibodies, or antigen-binding fragments thereof, further conjugated to cytotoxins, pharmaceutical compositions comprising the antibodies, or antigen-binding fragments thereof, and methods of preparing the same. And the use of such antibodies, multispecific antibodies or such compositions for the treatment of diseases such as cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to application No. PCT / CN2022 / 143248, filed on December 29, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] Disclosed herein are antibodies that specifically bind to human 4Ig-B7H3, as well as isolated nucleic acids, vectors, and host cells. These antibodies can be used to construct multispecific antibodies, construct antibody-drug conjugates (ADCs), or form fusion proteins with other modalities (e.g., a second tumor-associated antigen (TAA), immune checkpoints, or immunostimulatory factors). Finally, the anti-human 4Ig-B7H3 antibodies disclosed herein can be used to treat a variety of cancers. Background Art

[0003] B7H3 or B7-H3 (CD276 or B7RP-2) is a type I transmembrane protein identified from a dendritic cell (DC) cDNA library in 2001 (Chapoval et al., (2001) Nat. Immunol. 2(3):269-274). As part of the B7 immunoregulatory family, B7H3 has two isoforms in humans: 4Ig-B7H3 and 2Ig-B7H3. 4Ig-B7H3 is the predominant isoform expressed in malignant cells (Steinberger et al., (2004) J. Immunol. 172(4):2352-2359). The extracellular domain of mouse B7H3 consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, while human B7H3 contains a single or identical pair of these domains due to exon duplication (Sun et al., (2002) J. Immunol. 168(12):6294-6297; Steinberger et al., (2004) J. Immunol. 172(4):2352-2359). The intracellular domain of B7H3 is shorter and has no known signaling motifs (Picarda et al., (2016) Clin. Cancer. Res. 22(14):3425-3431). In addition to the transmembrane form, B7H3 can also exist in a soluble form that is cleaved from membrane B7H3 by proteases or by alternative splicing (Zhang et al. (2008) Immunology. 123(4):538-546).

[0004] B7H3 belongs to the B7 protein family and shares 20%-27% amino acid identity with other B7 family ligands, but the receptor remains to be elucidated (Chapoval et al., (2001) Nat. Immunol. [Natural - Immunology] 2(3): 269-274). To date, the molecular mechanism by which B7H3 participates in immune evasion is unclear, and the function of B7H3 in T cell-mediated adaptive immunity remains controversial. Although initially identified as a T cell co-stimulatory molecule (Chapoval et al., (2001) Nat. Immunol. [Natural - Immunology] 2(3): 269-274; Zhang et al., (2004) Acta Biochim. Biophys. Sin. 36(6):430-436), but later studies showed that it mainly plays an immunosuppressive role (Suh et al. (2003) Nat. Immunol. 4(9):899-906; Prasad et al. (2004) J. Immunol. 173(4):2500-2506; Fukushima et al. (2007) Immunol. Lett. 113(1):52-57; Veenstra et al. (2015) Blood. 125(21):3335-3346). At the same time, the crystal structure of mouse B7H3 shows that the FG loop is very important for mB7H3-mediated inhibition of T cell proliferation (Vigdorovich et al., (2013) Structure. [Structure] 21 (5): 707-717). Notably, NSCLC with high B7H3 expression is associated with a lower number of tumor-infiltrating lymphocytes and is refractory to anti-PD1 therapy, suggesting that B7H3 plays a role in immune evasion. Therefore, B7H3-targeted therapy combined with anti-PD-1 / PD-L1 antibody therapy is a promising approach for B7H3-expressing NSCLC (Altan et al., (2017) Clin. Cancer. Res. [Clinical Cancer Research] 23 (17): 5202-5209).

[0005] In addition to immune regulation, B7H3 also has intrinsic tumor-promoting functions. TCGA analysis has shown that B7H3 expression is associated with the EGFR / PI3K / AKT pathway, the MAPK pathway, and the EMT process in various cancer types. Aberrant B7H3 expression promotes tumor metastasis, angiogenesis, glycolytic metabolism, and drug resistance (Tekle et al. (2012) Int. J. Cancer. 130(10):2282-2290; Liu et al. (2015) Mol. Med. Rep. 12(4):5455-5460; Lee et al. (2017) Cell Res. 27(8):1034-1045; Flem-Karlsen et al. (2018) Trends Cancer. 4(6):401-404; Liu et al. (2019) Oncogene. 38(1):88-102; Lai et al. (2020) Immunol. Res. 68(3):177). Thus, in many cancer types, B7H3 overexpression is associated with poor prognosis and poor clinical outcome (Crispen et al. (2008) Clin. Cancer Res. 14(16):5150-5157; Bachawal et al. (2015) Cancer Res. 75(12):2501-2509; Fan et al. (2016) Pak. J. Med. Sci. 32(6):1568-1573; Song et al. (2016) Onco. Targets Ther. 9:6257-6263; Wu et al. (2016) Oncotarget. 7(49):81750-81756; Benzon et al. (2017) Prostate Cancer Prostatic Dis.[Prostate Cancer and Prostate Diseases] 20(1):28-3).

[0006] Many studies have reported that B7H3 is highly overexpressed in a variety of solid tumors and tumor vasculature, but has limited expression in normal tissues (Loo et al., (2012) Clin. Cancer Res. [Clinical Cancer Research] 18(14):3834-3845; Seaman et al., (2017) Cancer Cell. [Cancer Cell] 31(4):501-515e508; Du et al., (2019) Cancer Cell. [Cancer Cell] 35(2):221-237e228; Yamato et al., (2022) Mol. Cancer Ther. [Molecular Cancer Ther.] 21(4):635-646), making it an attractive target for cancer therapy. Most B7H3-targeted products are in the early stages of development. Y-Mabs has developed omburtamab with radionuclides (including I131 or Lu177), and its main product is I131-omburtamab targeting brain tumors. Enoblituzumab is an Fc-enhanced B7H3 Ab in Phase I / II and has shown limited anti-tumor efficacy in anti-PD1 / PD-L1 refractory patients when combined with an anti-PD1 antibody. B7H3 ADCs have shown preliminary anti-tumor activity in Phase I studies and have a well-tolerated safety profile, including MacroGenics' MGC018 and Daiichi Sankyo's DS-7300.

[0007] There are no approved therapeutic antibodies against B7H3, and there remains an unmet medical need for therapeutics targeting B7H3. Summary of the Invention

[0008] The present disclosure includes specific antibodies and antibody fragments thereof for human 4Ig-B7H3. In addition, the antibodies and antibody fragments disclosed herein can be used to construct multispecific antibodies, or to construct antibody-drug conjugates (ADCs), or to form fusion proteins with other forms (e.g., a second tumor-associated antigen (TAA), an immune checkpoint, or an immunostimulatory factor). 4Ig-B7H3 antibodies, alone or in combination with other therapeutic agents, can potentially be used to treat or prevent cancer.

[0009] The present disclosure relates to antibodies and antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3.

[0010] This disclosure encompasses the following embodiments.

[0011] An antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, the antibody or antigen-binding fragment thereof comprising: (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising: (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 20, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; or (viii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 28; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

[0012] The antibody or antigen-binding fragment thereof, wherein: (i) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (ii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8; (iii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (iv) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (v) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (vi) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21; (vii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24; or (viii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24.

[0013] The antibody or antigen-binding fragment thereof, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids have been inserted, deleted or substituted in SEQ ID NOs: 26 and 24, SEQ ID NOs: 7 and 8, SEQ ID NOs: 12 and 8, SEQ ID NOs: 15 and 8, SEQ ID NOs: 18 and 8, SEQ ID NOs: 7 and 21, SEQ ID NOs: 7 and 24, and SEQ ID NOs: 29 and 24.

[0014] The antibody or antigen-binding fragment thereof, wherein: (i) the heavy chain variable region comprises SEQ ID NO: 26, and the light chain variable region comprises SEQ ID NO: 24; (ii) the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 8; (iii) the heavy chain variable region comprises SEQ ID NO: 12, and the light chain variable region comprises SEQ ID NO: 8; (iv) the heavy chain variable region comprises SEQ ID NO: 15, and the light chain variable region comprises SEQ ID NO: 8; (v) the heavy chain variable region comprises SEQ ID NO: 18, and the light chain variable region comprises SEQ ID NO: 8; (vi) the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 21; (vii) the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 24; or (viii) the heavy chain variable region comprises SEQ ID NO: 29, and the light chain variable region comprises SEQ ID NO: 24.

[0015] The antibody or antigen-binding fragment thereof is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment or a F(ab')2 fragment.

[0016] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a scFv comprising a VH having amino acids of SEQ ID NO: 26 and a VL having amino acids of SEQ ID NO: 24, optionally the VH and VL are connected via an amino acid linker, optionally the amino acid linker is any sequence of SEQ ID NO: 35 to SEQ ID NO: 77.

[0017] The antibody or antigen-binding fragment thereof, wherein the amino acid linker is SEQ ID NO: 77.

[0018] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a scFv having the amino acid sequence of SEQ ID NO: 32.

[0019] A multispecific antibody or antigen-binding fragment thereof, comprising at least a first antigen-binding domain that specifically binds to human 4Ig-B7H3, wherein the first antigen-binding domain is an antibody or antigen-binding fragment thereof described herein; and at least a second antigen-binding domain that specifically binds to a second human tumor-associated antigen (TAA).

[0020] The multispecific antibody or antigen-binding fragment thereof, wherein the multispecific antibody is a bispecific antibody.

[0021] The multispecific antibody or antigen-binding fragment thereof further comprises an amino acid linker, wherein the amino acid linker is any sequence of SEQ ID NO: 35 to SEQ ID NO: 77.

[0022] The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass and / or a light chain constant region of the κ or λ type.

[0023] The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1 subclass and a light chain constant region of the κ type.

[0024] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0025] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or is hypofucosylated.

[0026] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an added bisecting GlcNac structure.

[0027] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0028] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin via a cytotoxin linker.

[0029] The antibody or antigen-binding fragment thereof, wherein: (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 80); and / or (2) The antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0030] In some embodiments, (3) the antibody or antigen-binding fragment thereof does not bind to an epitope comprising or consisting of amino acid residues 145-238 of human 4Ig-B7H3 (SEQ ID NO: 80); and / or (4) the antibody or antigen-binding fragment thereof does not bind to an epitope comprising or consisting of amino acid residues 363-456 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0031] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0032] A method for treating cancer, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment thereof or pharmaceutical composition described herein.

[0033] The method, wherein the cancer is 4Ig-B7H3 positive.

[0034] The method, wherein the cancer is colorectal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, lung cancer or esophageal cancer.

[0035] The method, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

[0036] The method, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.

[0037] The method, wherein the esophageal cancer is esophageal squamous cell carcinoma.

[0038] The method, wherein the antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

[0039] The method, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide or 5-azacytidine.

[0040] The method, wherein the therapeutic agent is an immune checkpoint inhibitor.

[0041] The method, wherein the therapeutic agent is an anti-PD-1 antibody.

[0042] The method, wherein the anti-PD1 antibody is Tislelizumab.

[0043] An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof described herein.

[0044] A vector comprising a nucleic acid described herein.

[0045] A host cell comprising a nucleic acid or a vector described herein.

[0046] A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell described herein and recovering the antibody or antibody fragment from the culture.

[0047] An antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein: (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 80); and / or (2) The antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0048] In some embodiments, (3) the antibody or antigen-binding fragment thereof does not bind to an epitope comprising or consisting of amino acid residues 145-238 of human 4Ig-B7H3 (SEQ ID NO: 80); and / or (4) the antibody or antigen-binding fragment thereof does not bind to an epitope comprising or consisting of amino acid residues 363-456 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0049] In some embodiments, the present disclosure provides anti-human 4Ig-B7H3 antibodies or antigen-binding fragments thereof that exhibit specific binding to human 4Ig-B7H3 and have high affinity for it.

[0050] In some embodiments, the present disclosure provides anti-human 4Ig-B7H3 antibodies or antigen-binding fragments thereof that exhibit increased binding affinity and / or increased internalization (eg, increased internalization rate). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1The affinity determination of the purified B7H3 mAb BGA-3726 (mouse chimeric) for the human B7H3 antigen by SPR is shown. In particular, the sensorgram shows the change of the combination as a signal derived from the glass chip surface. The glass chip is a plasma resonance surface that is bound to BGA-3726. Anti-BGA-3726 antibodies are captured and immobilized on the chip by anti-mouse IgG Fc antibodies. After fixation, serial dilutions of human or cyno B7H3 outer domains are passed through the chip. Since B7H3 is the target of BGA-3726, the degree of binding is visualized as a response unit (RU) (Y-axis) and is proportional to the binding time (X-axis) in seconds. Therefore, the light gray line is a serial dilution of human B7H3 protein, and the dark gray line is a fitting curve corresponding to different human B7H3 concentrations. The dotted line is the highest concentration of B7H3 protein. The results are used to calculate the association rate (Kon) and dissociation rate (Koff) of BGA-3295.

[0052] Figure 2 A- Figure 2 B shows the affinity of purified anti-4Ig-B7H3 mAb BGA-3726 to human 4Ig-B7H3 ( Figure 2 A) and cyno B7H3( Figure 2 B) Binding affinity of overexpressing cells.

[0053] Figure 3 A- Figure 3 B shows the comparison of binding affinity of engineered antibodies derived from BGA-3726 to human 4Ig-B7H3 overexpressing cells as assayed by FACS. Figure 3 A shows the dose-dependent binding curve of BGA-3726 to human 4Ig-B7H3 overexpressing cells. Figure 3 B shows the dose-dependent binding curves of BGA-6938 and BGA-5488 to human 4Ig-B7H3 overexpressing cells.

[0054] Figure 4 Shown is a comparison of the binding affinity of BGA-6938 and MABX-9001a to B7H3-positive tumor cells H358 cells as determined by FACS. A hIgG isotype Ab was used as a non-binding control.

[0055] Figure 5 Comparison of the internalization activity of BGA-6938 and MABX-9001a into B7H3-positive tumor cells H358 cells at the indicated time points by FACS assay is shown. hIgG isotype Ab was used as a non-binding control.

[0056] Figure 6The cell killing activities of BGA-6938-GGFG-DXd(DAR8), MABX-9001a-GGFG-DXd(DAR8) and MABX-9001a-GGFG-DXd(DAR4) in H1650, H441 and H1048 cell models are shown. Iso-GGFG-DXd(DAR8) was used as a non-targeting control.

[0057] Figure 7 The binding affinity of the scFv of BGA-6938 to H358 cells is shown.

[0058] Figure 8 A-8B shows epitope mapping of BGB-6938. Figure 8 A shows the construct design of truncated human B7H3. Figure 8 B shows epitope identification of BGB-6938 by domain truncation and comparison with the epitope of DS-7300. DETAILED DESCRIPTION

[0059] The present disclosure provides anti-human 4Ig-B7H3 antibodies and antigen-binding fragments thereof. In addition, the present disclosure provides antibodies with desired binding affinity, desired internalization (e.g., increased internalization rate), and other desired properties. Anti-human 4Ig-B7H3 antibodies can be used to construct multispecific antibodies with other forms (e.g., a second tumor-associated antigen (TAA), immune checkpoints, or immunostimulatory factors), or to construct antibody-drug conjugates (ADCs), or to fuse with other domains to form fusion proteins. In addition, anti-human 4Ig-B7H3 antibodies and their constructs can be used to treat cancer and related disorders. I. Anti-4Ig-B7H3 Antibody

[0060] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof generated as described below.

[0061] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having an amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 29 (Table 7). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment comprises a HCDR having an amino acid sequence of any one of the HCDRs listed in Table 7. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibody comprises one, two, three, or more HCDRs having an amino acid sequence of any one of the HCDRs listed in Table 7.

[0062] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VL domain having an amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 8, or SEQ ID NO: 21 (Table 7). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment comprises a LCDR having an amino acid sequence of any one of the LCDRs listed in Table 7. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibody comprises one, two, three, or more LCDRs having an amino acid sequence of any one of the LCDRs listed in Table 7.

[0063] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 23.

[0064] In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (a) a heavy chain variable region (HCDR) comprising one or more complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, and SEQ ID NO: 28, and / or (b) a light chain variable region (LCDR) comprising one or more complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 20, and SEQ ID NO: 23.

[0065] In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (a) a heavy chain variable region (HCDR) comprising three complementarity determining regions, wherein the HCDRs are HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 11; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 28, or SEQ ID NO: 17; and / or (b) a light chain variable region (LCDR) comprising three complementarity determining regions, wherein the LCDRs are LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 20, or SEQ ID NO: 23; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0066] In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (a) a heavy chain variable region (HCDR) comprising three complementarity determining regions, wherein the HCDRs are: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 14; a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; a HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or (b) a light chain variable region (LCDR) comprising three complementarity determining regions, wherein the LCDRs are LCDR1 comprising the amino acid sequence of SEQ ID NO:4, LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and LCDR3 comprising the amino acid sequence of SEQ ID NO:6; LCDR1 comprising the amino acid sequence of SEQ ID NO:20, LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and LCDR3 comprising the amino acid sequence of SEQ ID NO:6; or LCDR1 comprising the amino acid sequence of SEQ ID NO:23, LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0067] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (i) HCDR1 (heavy chain complementarity determining region 1), HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: 26; (ii) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: 7; (iii) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: 12; (iv) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: 15; (v) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: 18; or (vi) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: 29; and / or (i) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region (VH) shown in SEQ ID NO: (i) LCDR1, LCDR2 and LCDR3 from the light chain variable region (VL) shown in SEQ ID NO:21; (ii) LCDR1, LCDR2 and LCDR3 from the light chain variable region (VL) shown in SEQ ID NO:8.

[0068] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising: (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 20, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; or (viii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 28; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6, according to the Kabat definition.

[0069] In one embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure comprise: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 29, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 29, and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 8, or SEQ ID NO: 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 24, SEQ ID NO: 8, or SEQ ID NO: 21. Any of NO:21 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

[0070] In one embodiment, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (ii) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8; (iii) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (iv) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (v) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (vi) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21; (vii) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24; or (viii) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24.

[0071] In one embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids have been inserted, deleted or substituted in SEQ ID NOs: 26 and 24, SEQ ID NOs: 7 and 8, SEQ ID NOs: 12 and 8, SEQ ID NOs: 15 and 8, SEQ ID NOs: 18 and 8, SEQ ID NOs: 7 and 21, SEQ ID NOs: 7 and 24, SEQ ID NOs: 29 and 24.

[0072] In one embodiment, the present disclosure provides the antibody or antigen-binding fragment thereof, comprising: (i) a heavy chain variable region comprising SEQ ID NO: 26, and a light chain variable region comprising SEQ ID NO: 24; (ii) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 8; (iii) a heavy chain variable region comprising SEQ ID NO: 12, and a light chain variable region comprising SEQ ID NO: 8; (iv) a heavy chain variable region comprising SEQ ID NO: 15, and a light chain variable region comprising SEQ ID NO: 8; (v) a heavy chain variable region comprising SEQ ID NO: 18, and a light chain variable region comprising SEQ ID NO: 8; (vi) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 21; (vii) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 24; or (viii) a heavy chain variable region comprising SEQ ID NO: 29, and a light chain variable region comprising SEQ ID NO: 24.

[0073] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acids that have been altered but have a percent identity of at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% in the CDR regions compared to the CDR regions disclosed in Table 7. In some aspects, they include amino acid alterations (insertions, deletions or substitutions, optionally conservative amino acid substitutions) wherein no more than 1, 2, 3, 4 or 5 amino acids are altered in the CDR regions when compared to the CDR regions depicted in the sequences described in Table 7 while maintaining binding specificity and affinity.

[0074] Other antibodies of the disclosure include those in which amino acids in the variable region (e.g., the framework region of the variable region) or nucleic acids encoding amino acids have been altered; but have at least 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of the variable region described in Table 7 while retaining binding specificity / affinity, optionally without altering the corresponding sequence of the CDRs. In some aspects, it includes changes in the amino acid sequence, wherein no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in the variable region (e.g., the framework region of the variable region) are changed when compared to the variable region depicted in the sequence described in Table 7, while retaining binding specificity / affinity, optionally without changing the corresponding sequence of the CDR.

[0075] In another embodiment, the present disclosure provides antibodies or antigen-binding fragments thereof that are expressed in 1 x 10 -6 M to 1x 10 -11 The binding affinity (K D ) specifically binds to human 4Ig-B7H3. In another embodiment, the anti-4Ig-B7H3 antibody or antigen-binding fragment thereof is present at a concentration of about 1 x 10 -6 M, about 1x 10 -7 M, about 1x 10-8 M, about 1x 10 - 9 M, about 1x 10 -10 M or about 1x 10 -11 The binding affinity (K D ) binds to human 4Ig-B7H3.

[0076] The present disclosure also provides nucleic acid sequences encoding VH or VL of antibodies that specifically bind to human 4Ig-B7H3. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0077] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-4Ig-B7H3 antibodies described in Table 7. Thus, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with (e.g., competitively inhibit their binding in a statistically significant manner) the antibodies described in Table 7 in a binding assay. The ability of a test antibody to inhibit the binding of an antibody or antigen-binding fragment thereof disclosed herein to 4Ig-B7H3 demonstrates that the test antibody can compete with the antibody or antigen-binding fragment thereof for binding to 4Ig-B7H3. Without being bound by any one theory, such an antibody may bind to the same or related (e.g., structurally similar or spatially adjacent) epitope on 4Ig-B7H3 as the antibody or antigen-binding fragment thereof that it competes with. In certain aspects, an antibody that binds to the same epitope on 4Ig-B7H3 as an antibody or antigen-binding fragment thereof disclosed herein is a human or humanized monoclonal antibody. Such a human or humanized monoclonal antibody can be prepared and isolated as described herein.

[0078] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0079] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0080] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof specifically binds to the IgV1 domain of human 4Ig-B7H3. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof specifically binds to the IgV2 domain of human 4Ig-B7H3. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof specifically binds to the IgV1 and IgV2 domains of human 4Ig-B7H3.

[0081] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 145-238 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0082] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 363-456 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0083] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof does not bind to the IgC1 domain of human 4Ig-B7H3. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof does not bind to the IgC2 domain of human 4Ig-B7H3. In some embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof does not bind to the IgC1 and IgC2 domains of human 4Ig-B7H3.

[0084] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope that does not overlap with the epitope of the reference antibody DS-7300.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0086] In one embodiment, the antibody or antigen-binding fragment thereof is in the form of an scFv comprising a VH-VL in an N-terminal to C-terminal direction or a VL-VH in an N-terminal to C-terminal direction. In some embodiments, the VH and VL are connected via an amino acid linker as described herein. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 29. In some embodiments, the VL comprises the amino acid sequence of any one of SEQ ID NO: 24, SEQ ID NO: 8, or SEQ ID NO: 21. In some embodiments, the VH is any one of the VHs described in Table 7. In some embodiments, the VL is any one of the VLs described in Table 7. In some embodiments, the amino acid linker has an amino acid sequence comprising any one of SEQ ID NOs: 35-77. In some embodiments, the amino acid linker is any sequence of SEQ ID NOs: 35-77. In one embodiment, the amino acid linker is SEQ ID NO: 77.

[0087] In one embodiment, the antibody or antigen-binding fragment thereof comprises a scFv having the amino acid sequence of SEQ ID NO: 32. In another embodiment, the antibody or antigen-binding fragment thereof comprises a scFv of SEQ ID NO: 32. II. Anti-4Ig-B7H3 Multispecific Antibody

[0088] In one embodiment, the anti-4Ig-B7H3 antibodies disclosed herein can be used to construct multispecific antibodies together with other modalities (e.g., a second human tumor-associated antigen (TAA), immune checkpoint or immune stimulatory factor), wherein 4Ig-B7H3 serves as the first TAA.

[0089] In one embodiment, the anti-4Ig-B7H3 antibodies disclosed herein can be incorporated into anti-4Ig-B7H3xTAA multispecific antibodies, wherein TAA is an antibody or fragment thereof directed against any human tumor-associated antigen. The antibody molecule is a multispecific antibody molecule, for example, comprising multiple antigen-binding domains, wherein at least one antigen-binding domain sequence specifically binds to 4Ig-B7H3 as a first epitope, and a second antigen-binding domain sequence specifically binds to a second TAA as a second epitope. In one embodiment, the multispecific antibody comprises a third, fourth, or fifth antigen-binding domain. In one embodiment, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0090] In one embodiment, the multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds only to two antigens. The bispecific antibody comprises a first antigen-binding domain that specifically binds to human 4Ig-B7H3 and a second antigen-binding domain that specifically binds to a second TAA. This includes a bispecific antibody that comprises a second heavy chain variable domain and a second light chain variable domain that specifically bind to a second TAA and a first heavy chain variable domain and a first light chain variable domain that specifically bind to human 4Ig-B7H3. In some embodiments, the bispecific antibody comprises an antigen-binding fragment, wherein the antigen-binding fragment can be Fab, F(ab')2, Fv, single-chain Fv (scFv) or a single-domain antibody.

[0091] In one embodiment, the multispecific antibodies of the present disclosure are expressed in 1 x 10 -6 M to 1x 10 -10 M or even 1x 10 -11 The binding affinity (K D ) binds to a second human TAA and / or human 4Ig-B7H3. In another embodiment, the multispecific antibodies disclosed herein are present at about 1 x 10 -6 M, about 1x 10 -7 M, about 1x 10 -8 M, about 1x 10 -9 M, about 1x 10 -10 M or about 1x 10 -11 The binding affinity (K D ) binds to a second human TAA and / or human 4Ig-B7H3.

[0092] In one embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein the first antigen-binding domain that specifically binds to human 4Ig-B7H3 comprises: (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising: (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 20, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; or (viii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 28; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; and The second antigen binding domain specifically binds to a second human TAA.

[0093] In another embodiment, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein the first antigen-binding domain that specifically binds to human 4Ig-B7H3 comprises: (i) a heavy chain variable region comprising SEQ ID NO: 26, and a light chain variable region comprising SEQ ID NO: 24; (ii) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 8; (iii) a heavy chain variable region comprising SEQ ID NO: 12, and a light chain variable region comprising SEQ ID NO: 8; (iv) a heavy chain variable region comprising SEQ ID NO: 15, and a light chain variable region comprising SEQ ID NO: 8; (v) a heavy chain variable region comprising SEQ ID NO: 18, and a light chain variable region comprising SEQ ID NO: 8; (vi) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 21; (vii) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 24; or (viii) a heavy chain variable region comprising SEQ ID NO: 29, and a light chain variable region comprising SEQ ID NO: 24, and The second antigen binding domain specifically binds to a second human TAA.

[0094] Previous experiments (Coloma and Morrison Nature Biotech. [Nature Biotechnology] 15: 159-163 (1997)) described tetravalent bispecific antibodies engineered by fusing DNA encoding a single-chain anti-dansyl antibody Fv (scFv) to the C-terminus (CH3-scFv) or to the hinge (hinge-scFv) of an IgG3 anti-dansyl antibody. The present disclosure provides multivalent antibodies (e.g., tetravalent antibodies) having at least two antigen-binding domains that can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibodies herein comprise three to eight, but preferably four, antigen-binding domains that specifically bind to at least two antigens.

[0095] In one embodiment, the multispecific antibody is a bispecific antibody.In another embodiment, the multispecific antibody further comprises an amino acid linker as described herein, such as any one of SEQ ID NO: 35 to SEQ ID NO: 77. III. Anti-human 4Ig-B7H3 antibody conjugated to cytotoxin

[0096] Anti-human 4Ig-B7H3 antibodies can be used to construct antibody drug conjugates (ADCs). In one embodiment, the antibody or its antigen-binding fragment is conjugated to a cytotoxin. In another embodiment, the antibody or its antigen-binding fragment is conjugated to a cytotoxin via a cytotoxin linker. Cytotoxins

[0097] Cytotoxins or cytotoxic agents include any agents that are detrimental to cell growth, viability, or proliferation, including, but not limited to, microtubule-interacting agents and DNA-damaging agents. Examples of suitable cytotoxic and chemotherapeutic agents that can be conjugated to the antibodies of the present disclosure include, for example, 1-(2-chloroethyl)-1,2-dimethylsulfonyl hydrazide, 1,8-dihydroxy-bicyclo[7.3.1]13-4,9-diene-2,6-diyn-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitin, aminopterin, anguidine, anthracycline, anthramycin, daptom ... , AMC), auristatins, bleomycin, busulfan, butyric acid, calicheamicins (e.g., calicheamicin gamma 1), camptothecins, carminomycins, carmustine, cemadotine, cisplatin, colchicine, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxyanthraquinone, disorazoles, Aplysia caudalis ( For example, Aplysia 10), doxorubicin, duocarmycin, echinomycins, eleutherobins, emetine, epothilone, esperamicin, estramustine, ethidium bromide, etoposide, fluorouracil, geldanamycin, gramicidin D, glucocorticoids, irinotecan, kinesin spindle (KSP) inhibitors, leptomycin, lonoxane (l eurosines), lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercaptopurines, methopterins, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxin, procaine, propranolol, pteridines, puromycin, pyrrolobenzodiazepines pyrrolobenzodiazepines (PBD), rhizoxin, streptozotocin, tallysomycins, paclitaxel, teniposide, tetracaine, thioepa chlorambucil, tomaymycins, topotecan, tubulysin, vinblastine, vincristine, vindesine, vinorelbine, and any derivatives thereof. Cytotoxic linkers

[0098] A cytotoxin linker or ADC linker is any group or moiety that links, connects, or bonds an antibody or antigen-binding protein described herein to a therapeutic moiety (e.g., a cytotoxic agent). Suitable linkers can be found, for example, in Antibody-Drug Conjugates and Immunotoxins; Phillips, GL, ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L, ed.; Humana Press, 2013; Antibody-Drug Conjugates; Wang, J., Shen, W.-C, and Zaro, JL, eds.; Springer International Publishing, 2015, the contents of each of which are incorporated herein by reference in their entirety.

[0099] Suitable adhesives or cytotoxic joints for antibody conjugates as described herein are sufficiently stable to utilize the circulating half-life of the antibody, and simultaneously capable of releasing its payload after antigen-mediated conjugate internalization. Joints can be cleavable or non-cleavable. Cleavable joints include joints that are cut by intracellular metabolism after internalization, such as by hydrolysis, reduction, or enzymatic reaction cutting. Non-cleavable joints include joints that release the attached payload by lysosomal degradation of the antibody after internalization. Suitable joints include, but are not limited to, acid-labile joints, hydrolytically unstable joints, enzymatically cleavable joints, reduction-unstable joints, self-incinerating joints, and non-cleavable joints. Suitable joints also include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, maleimido-hexanoyl (mal-caproyl) units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.

[0100] Any cytotoxic linker molecule or linker technology known in the art can be used to create or construct the ADC of the present disclosure. In certain embodiments, the cytotoxic linker is a cleavable linker. According to other embodiments, the linker is a non-cleavable linker. Exemplary linkers that can be used in the context of the present disclosure include, but are not limited to, linkers comprising or consisting of, for example, GGFG, MC (6-maleimidocaproyl), MP (maleimidopropionyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide site in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide site in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate), SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate) and variants and combinations thereof. Examples of additional linkers that can be used in the context of the present disclosure are provided, for example, in US 7,754,681 and Ducry, Bioconjugate Chem., 2010, 27: 5-13 and references cited therein, the contents of which are incorporated herein by reference in their entirety.

[0101] In certain embodiments, the cytotoxic linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, for example, capable of releasing at least a portion of the payload in the presence of an enzyme or at a specific pH range or value. In some embodiments, the linker comprises an enzyme-cleavable portion. Illustrative enzyme-cleavable portions include, but are not limited to, peptide bonds, ester bonds, hydrazone bonds, and disulfide bonds. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0102] In some embodiments, the cytotoxic linker comprises a non-cleavable portion.

[0103] Suitable cytotoxic linkers also include, but are not limited to, those that chemically bond to two cysteine ​​residues of a single binder (e.g., an antibody). Such linkers can be used to mimic the disulfide bonds of an antibody that are disrupted during the conjugation process.

[0104] In certain embodiments, the cytotoxin linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic and L- or D-amino acids. In certain embodiments, the cytotoxin linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or citrulline, its derivatives or combinations thereof. In certain embodiments, one or more side chains of amino acids are connected to the side chain group, as described below. In certain embodiments, the linker comprises valine and citrulline. In certain embodiments, the cytotoxin linker comprises lysine, valine and citrulline. In certain embodiments, the linker comprises lysine, valine and alanine. In certain embodiments, the linker comprises valine and alanine. IV. Fusion Protein Targeting Human 4Ig-B7H3

[0105] Anti-human 4Ig-B7H3 antibodies can be used to fuse with other proteins or other functional domains to form fusion proteins or chimeric proteins.

[0106] In some embodiments, an anti-human 4Ig-B7H3 antibody is fused to an immune checkpoint, an immune stimulatory factor, a cytokine, or a second TAA directly or indirectly via an amino acid linker described herein.

[0107] In one embodiment, anti-human 4Ig-B7H3 antibodies are fused to functional domains or receptor subunits that can be used to transduce signals from scFv and confer antibody specificity to immune cells, such as T cells, NK cells, and other effector cells.

[0108] In some embodiments, anti-human 4Ig-B7H3 antibodies are used to construct chimeric antigen receptors (CARs). Detailed information about CAR constructs can be found in Guedan et al., (2019) Mol. Ther. Methods Clin. Dev. [Molecular Therapeutic Approaches and Clinical Development] 12: 145-156.

[0109] Functional domains or receptor subunits include transmembrane domains, hinge regions, intracellular signaling domains, costimulatory domains, etc. V. Other modifications Constant region and Fc region

[0110] The heavy chain constant region can be a wild-type sequence of a heavy chain constant region from the IgG1, IgG2, IgG3, or IgG4 subclass. The light chain constant region can be a wild-type sequence of a light chain from a kappa or lambda type. In one embodiment, the heavy chain constant region is a wild-type sequence of a constant region from an IgG1. The light chain constant region is a wild-type sequence of a light chain from a kappa chain. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 20, and the light chain constant region has the amino acid sequence of SEQ ID NO: 21. In another embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 1, which includes the mutations E116P, L117A, L118A, G119del, and P212A. These amino acid mutations correspond to E233P, L234A, L235A, G236del, and P329A in EU numbering.

[0111] In one embodiment, the Fc region may be a wild-type Fc region of the IgG1, IgG2, IgG3 or IgG4 subclass. In one embodiment, the antibody or antigen-binding fragment thereof comprises an Fc domain of IgG1 or IgG4 with reduced effector function.

[0112] In one embodiment, the antibody or its antigen-binding fragment comprises an Fc domain with an extended half-life. In another embodiment, the antibody or its antigen-binding fragment comprises an Fc domain of IgG1, wherein a YTE mutation (M252Y / S254T / T256E, EU numbering, as described in US7658921) is introduced into the CH2 of the IgG Fc region.

[0113] In another embodiment, the antibodies of the present disclosure have potent Fc-mediated effector functions, and the antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells.

[0114] In other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue so that the antibody has an altered affinity for an effector ligand, but retains the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0115] In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0116] On the other hand, one or more amino acid residues are changed so as to change the ability of the antibody to fix complement. This method is described in, for example, the disclosure WO 94 / 29351 of Bodmer et al. In a specific aspect, one or more amino acids of the antibody or its antigen-binding fragment of the present disclosure are replaced by one or more allotypic amino acid residues of IgG1 subclass and κ isotype. Allotypic amino acid residues also include but are not limited to the heavy chain constant region of IgG1, IgG2 and IgG3 subclasses and the light chain constant region of κ isotype, as described in Jefferis et al., (2009) MAbs.[monoclonal antibodies] 1:332-338.

[0117] In another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in publication WO 00 / 42072 by Presta. In addition, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., (2001) J. Biol. Chem. 276: 6591-6604).

[0118] On the other hand, the glycosylation of the antibody is modified. For example, aglycosylated antibodies can be prepared (i.e., the antibody lacks or has reduced glycosylation). For example, glycosylation can be changed to increase the affinity of the antibody to the antigen. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at the site. Such aglycosylation can increase the affinity of the antibody to the antigen. This method is described in, for example, U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0119] Additionally or alternatively, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells in which recombinant antibodies are expressed to produce antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lec13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al., (1999) Nat. Biotech. 17:176-180).

[0120] On the other hand, if reduction of ADCC is desired, many previous reports have shown that human antibody subclass IgG4 has only moderate ADCC and almost no CDC effector function (Moore GL et al. (2010) Mabs. [monoclonal antibodies] 2: 181-189). However, it was found that native IgG4 is less stable under stress conditions (such as in acidic buffer or at elevated temperatures) (Angal, S. (1993) Mol Immunol. [molecular immunology] 30: 105-108; Dall'Acqua, W. et al. (1998) Biochemistry. [biochemistry] 37: 9266-9273; Aalberse et al. (2002) Immunol. [immunology] 105: 9-19). Reducing ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with a combination of alterations that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable inherent properties of IgG4 is that its two heavy chains dynamically separate in solution to form half antibodies, which leads to the generation of bispecific antibodies in vivo through a process called "Fab arm exchange" (Vander Neut Kolfschoten M et al., (2007) Science. [Science] 317: 1554-157). Mutation of serine at position 228 (EU numbering system) to proline inhibits the separation of IgG4 heavy chains (Angal, S. (1993) Mol Immunol. [Molecular Immunology] 30: 105-108; Aalberse et al., (2002) Immunol. [Immunology] 105: 9-19). It has been reported that some amino acid residues in the hinge region and the gamma Fc region have an influence on the interaction of antibodies with Fc gamma receptors (Chappel SM et al., (1991) Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., (1995) FASEB J. [Journal of the Federation of American Societies for Experimental Biology] 9:115-119; Armour, KL et al., (1999) Eur. J. Immunol. 29:2613-2624; Clynes, RA et al., (2000) Nature Medicine. 6:443-446; Arnold JN, (2007) Annu. Rev. Immunol. 25:21-50).In addition, some rare IgG4 isotypes in the human population can also cause different physicochemical properties (Brusco, A. et al., (1998) Eur J Immunogenet. [European Journal of Immunogenetics] 25: 349-55; Aalberse et al., (2002) Immunol. [Immunology] 105: 9-19). In order to generate multispecific antibodies with low ADCC and CDC but good stability, the hinge and Fc regions of human IgG4 can be modified and many changes can be introduced. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of U.S. Patent No. 8,735,553 to Li et al.

[0121] In another embodiment, an antibody of the disclosure comprises an Fc domain of human IgG4 having an S228P and / or R409K substitution (according to the EU numbering system). Amino acid linker

[0122] It will be understood that the presence or absence of an amino acid linker has minimal effect on the activity of the antibodies or proteins of the disclosure.

[0123] It should also be understood that the domains and / or regions of the polypeptide chains of antibodies or proteins can be separated by linker regions of various lengths. In some embodiments, the antigen-binding domain is separated from the CL, CH1, hinge, CH2, CH3 or the entire Fc region by a linker region. For example, VL1-CL-(linker) VH2-CH1. Such a linker region can comprise randomly sorted amino acids, or a group of restricted amino acids. Such linker regions can be flexible or rigid (see US2009 / 0155275).

[0124] Multispecific antibodies have been constructed by genetically fusing two single-chain Fv (scFv) or Fab fragments with or without a flexible linker (Mallender et al., (1994) J. Biol. Chem. 269:199-206; Mack et al., (1995) Proc. Natl. Acad. Sci. USA. 92:7021-5; Zapata et al., (1995) Protein Eng. 8:1057-62), via a dimerization apparatus such as a leucine zipper (Kostelny et al., (1992) J. Immunol. 148:1547-53; de Kruif et al. (1996) J. Biol. Chem. 271:7630-4) and an Ig C / CH1 domain (Muller et al., (1998) FEBS Lett. 422:259-64); through diabodies (Holliger et al., (1993) Proc. Nat. Acad. Sci. USA. 1998 90:6444-8; Zhu et al., (1996) Bio / Technology (NY). 14:192-6); Fab-scFv fusions (Schoonjans et al., (2000) J. Immunol. 165:7050-7); and miniantibody formats (Pack et al., (1992) Biochemistry 31:1579-84; Pack et al., (1993) Bio / Technology. 11:1271-7).

[0125] The antibodies or proteins disclosed herein comprise an amino acid linker region of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more amino acid residues between one or more antigen binding domains, CL domains, CH1 domains, hinge regions, CH2 domains, CH3 domains or Fc regions. In some embodiments, the amino acids glycine and serine are contained within the linker region. In another embodiment, the linker can be GS (SEQ ID NO:35), GGS (SEQ ID NO:36), GSG (SEQ ID NO:37), SGG (SEQ ID NO:38), GGG (SEQ ID NO:39), GGGS (SEQ ID NO:40), SGGG (SEQ ID NO:41), GGGGS (SEQ ID NO:42), GGGGSGS (SEQ ID NO:42) NO:43), GGGGSGS (SEQ ID NO:44), GGGGSGGS (SEQ ID NO:45), GGGGSGGGGS (SEQ ID NO:46), GGGGSGGGGSGGGGS (SEQ ID NO:47), AKTTPKLEEGEFSEAR (SEQ ID NO:48), AKTTPKLEEGEFSEARV (SEQ ID NO:49), AKTTPKLGG (SEQ ID NO:50), SAKTTPKLGG (SEQ ID NO:51), AKTTPKLEEGEFSEARV (SEQ ID NO:52), SAKTTP (SEQ ID NO:53), SAKTTPKLGG (SEQ ID NO:54), RADAAP (SEQ ID NO:55), RADAAPTVS (SEQ ID NO:56), RADAAAAGGPGS (SEQ ID NO:57), RADAAAA(G4S)4 (SEQ ID NO:58), SAKTTP (SEQ ID NO:59), SAKTTPKLGG (SEQ ID NO:60), SAKTTPKLEEGEFSEARV (SEQ ID NO:61), ADAAP (SEQ ID NO:62), ADAAPTVSIFPP (SEQ ID NO:63), TVAAP (SEQ ID NO:64), TVAAPSVFIFPP (SEQ ID NO:65), QPKAAP (SEQ ID NO:66), QPKAAPSVTLFPP (SEQ ID NO:67), AKTTPP (SEQ IDNO: 68), AKTTPPSVTPLAP (SEQ ID NO: 69), AKTTAP (SEQ ID NO: 70), AKTTAPSVYPLAP (SEQ ID NO: 71), ASTKGP (SEQ ID NO: 72), ASTKGPSVFPLAP (SEQ ID NO: 73), GENKVEYAPALMALS (SEQ ID NO: 74), GPAKELTPLKEAKVS (SEQ ID NO: 75), and GHEAAAVMQVQYPAS (SEQ ID NO: 76), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 77), or any combination thereof (see WO 2007 / 024715). Dimerization-specific amino acids

[0126] In one embodiment, the multivalent antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change causes a "protrusion into the hole" interaction and increases the assembly of the correct multivalent antibody. The dimerization-specific amino acid can be in the CH1 domain or the CL domain or a combination thereof. The dimerization-specific amino acids for pairing the CH1 domain with other CH1 domains (CH1-CH1) and the CL domain with other CL domains (CL-CL) can be found at least in the disclosures of WO2014082179, WO 2015181805 family, and WO 2017059551. The dimerization-specific amino acid can also be in the Fc domain and can be combined with the dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair. Antibody production

[0127] Antibodies and antigen-binding fragments thereof can be produced by any method known in the art, including but not limited to recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any appropriate host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0128] The present disclosure further provides polynucleotides encoding antibodies or proteins described herein, such as polynucleotides encoding heavy chain variable regions or light chain variable regions comprising complementary determining regions described herein. In some aspects, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 27 or SEQ ID NO: 30. In some aspects, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from SEQ ID NO: 10, SEQ ID NO: 22 or SEQ ID NO: 25.

[0129] The present disclosure also provides polynucleotides encoding the scFv described herein, such as a polynucleotide encoding the amino acid sequence of SEQ ID NO:32.

[0130] The polynucleotides disclosed herein can encode the variable region sequences of the multispecific antibodies described herein. They can also encode the variable and constant regions of the multispecific antibodies. In another embodiment, the polynucleotides disclosed herein can encode the amino acid sequences of the fusion proteins described herein.

[0131] In some embodiments, the polynucleotides described herein can be codon-optimized for expression in a host cell, eg, a eukaryotic cell, more particularly, a mammalian cell (eg, a CHO cell).

[0132] The present disclosure also provides expression vectors and host cells for producing antibodies herein. The selection of expression vectors depends on the expected host cell to be expressed in the vector. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the antibody chain or Fab. In some aspects, in addition to being under the control of inducing conditions, an inducible promoter is used to prevent the expression of the inserted sequence. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The culture of the organism transformed can be amplified under non-inducing conditions without causing the colony to be biased towards the coding sequence that its expression product is better tolerated by the host cell. In addition to the promoter, other regulatory elements may also be required or desired for effective expression of the antibody or Fab. These elements typically include an ATG start codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency can be increased by including an enhancer appropriate for the cell system being used (see, for example, Scharf et al., (1994) Results Probl. Cell Differ. 20:125; Bittner et al., (1987) Meth. Enzymol. 153:516). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[0133] The host cell for carrying and expressing the antibody chain can be a prokaryotic cell or a eukaryotic cell. Escherichia coli is a prokaryotic host that can be used for cloning and expressing the polynucleotides of the present disclosure. Other applicable microbial hosts include bacillus, such as Bacillus subtilis (Bacillus subtilis), and other enterobacteriaceae (enterobacteriaceae), such as Salmonella (Salmonella), Serratia (Serratia) and various Pseudomonas (Pseudomonas) species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (for example, replication origin) compatible with the host cell. In addition, there will be any number of well-known promoters, such as lactose promoter system, tryptophan (trp) promoter system, beta-lactamase promoter system or promoter system from bacteriophage lambda. Promoter typically optionally controls expression with an operator sequence, and has a ribosome binding site sequence, etc., for starting and completing transcription and translation. Other microorganisms such as yeast can also be used for expressing antibodies. The combination of insect cells and baculovirus vectors can also be used. In other aspects, mammalian host cells are used to express and produce the antibodies of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These include any normal death or normal or abnormal immortalized animal or human cells. For example, several suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B cells and hybridomas. Using mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, New York, New York, 1987. Expression vectors for mammalian host cells may include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., (1986) Immunol. Rev. [Immunol. Rev.] 89: 49-68), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or regulatable or adjustable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRPpolIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art. Detection and diagnostic methods

[0134] The antibodies or antigen-binding fragments disclosed herein can be used in a variety of applications, including but not limited to methods for detecting 4Ig-B7H3. In one aspect, the antibodies or antigen-binding fragments can be used to detect the presence of 4Ig-B7H3 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain aspects, the biological sample includes cells or tissues. In other aspects, such tissues include normal and / or cancerous tissues that express 4Ig-B7H3 at higher levels relative to other tissues.

[0135] In one aspect, the present disclosure provides methods for detecting the presence of 4Ig-B7H3 in a biological sample. In certain aspects, the method comprises contacting the biological sample with an anti-4Ig-B7H3 antibody under conditions permissive for binding of the antibody to the antigen, and detecting whether a complex forms between the antibody and the antigen. The biological sample may include, but is not limited to, urine, tissue, sputum, or blood samples.

[0136] Also included are methods for diagnosing disorders associated with 4Ig-B7H3 expression. In certain aspects, the method comprises contacting a test cell with an anti-4Ig-B7H3 antibody; determining the expression level (quantitatively or qualitatively) of 4Ig-B7H3 expressed by the test cell by detecting binding of the anti-4Ig-B7H3 antibody to a 4Ig-B7H3 polypeptide; and comparing the expression level of the test cell with the expression level of 4Ig-B7H3 in a control cell (e.g., a normal cell of the same tissue origin as the test cell or a cell that does not express 4Ig-B7H3), wherein a higher level of 4Ig-B7H3 expression in the test cell compared to the control cell indicates the presence of a disorder associated with 4Ig-B7H3 expression. Treatment

[0137] The antibodies or antigen-binding fragments disclosed herein can be used in a variety of applications, including but not limited to methods for treating 4Ig-B7H3-related disorders or diseases. In one aspect, the 4Ig-B7H3-related disorder or disease is cancer. In some embodiments, the cancer is 4Ig-B7H3-positive.

[0138] In one aspect, the present disclosure provides methods for treating cancer. In certain aspects, the methods comprise administering an effective amount of an anti-4Ig-B7H3 antibody or antigen-binding fragment, or a multispecific antibody comprising a 4Ig-B7H3 antibody to a patient in need thereof. In another aspect, the present disclosure provides anti-4Ig-B7H3 antibodies or antigen-binding fragments or multispecific antibodies, or pharmaceutical compositions, for use in treating cancer. In another aspect, the present disclosure provides uses of anti-4Ig-B7H3 antibodies or antigen-binding fragments, multispecific antibodies or antigen-binding fragments thereof, or pharmaceutical compositions in the manufacture of a medicament for treating cancer.

[0139] Cancer can include but is not limited to colorectal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, lung cancer or esophageal cancer. In one embodiment, lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In another embodiment, non-small cell lung cancer is squamous non-small cell lung cancer. In another embodiment, esophageal cancer is esophageal squamous cell carcinoma.

[0140] Antibodies or antigen-binding fragments disclosed herein can be administered in any suitable manner, including parenteral, intrapulmonary and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at different time points, push injection administration and pulse infusion.

[0141] The antibodies or antigen-binding fragments of the present disclosure can be formulated, dosed, and applied in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the administration regimen, and other factors known to medical practitioners. The antibody need not but is optionally formulated with one or more agents currently used to prevent or treat the disorder being studied. The effective amount of such other agents depends on the amount of the antibody present in the formulation, the type of disorder or treatment, and other factors discussed above.

[0142] For the prevention or treatment of disease, the appropriate dosage of the antibodies or antigen-binding fragments of the disclosure will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. Combination therapy

[0143] In one aspect, the anti-4Ig-B7H3 antibodies or multispecific antibodies containing anti-4Ig-B7H3 antibodies of the present disclosure can be used in combination with other therapeutic agents. Other therapeutic agents that can be used with the 4Ig-B7H3 antibodies of the present disclosure include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents; (e.g., ), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib)), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20-targeted agents (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52-targeted agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, levofloxacin Lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19 targeting agents (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), EGEN-001, Polo-like kinase 1 inhibitors (e.g., BI 672)).

[0144] The anti-4Ig-B7H3 antibodies disclosed herein can be used in combination with other therapeutic agents (e.g., other immune checkpoint antibodies). Such immune checkpoint antibodies may include anti-PD1 antibodies. Anti-PD1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab, or nivolumab. Tislelizumab (SEQ ID No: 22 and 23 in Table 7) is disclosed in US 8,735,553. Pembrolizumab (formerly known as MK-3475) is disclosed in US 8,354,509 and US 8,900,587 and is a humanized lgG4-K immunoglobulin that targets PD1 receptors and inhibits the binding of PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is in clinical studies for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human IgG4-κ monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. US 8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0145] In one embodiment, the present disclosure provides a use of a combination of an anti-4Ig-B7H3 antibody or a multispecific antibody comprising an anti-4Ig-B7H3 antibody and an anti-PD-1 antibody (e.g., tislelizumab or other anti-PD-1 antibodies described above) in the manufacture of a medicament for treating cancer (e.g., the cancers described above). In another embodiment, the present disclosure provides a combination of an anti-4Ig-B7H3 antibody or a multispecific antibody comprising an anti-4Ig-B7H3 antibody and an anti-PD-1 antibody (e.g., tislelizumab or other anti-PD-1 antibodies described above) for use in treating cancer (e.g., the cancers described above).

[0146] Combination therapy is intended to mean and does mean and include any of the following: administering such a combination therapy simultaneously to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components to said patient substantially simultaneously, administering such a combination to a patient in need of treatment at substantially the same time, where such components are formulated separately from one another into separate dosage forms to be taken by said patient at substantially the same time, whereby said components are released to said patient at substantially the same time, administering such combination therapy sequentially to a patient in need thereof, where such components are formulated separately from one another into separate dosage forms to be taken by said patient at consecutive times with substantial time intervals between each administration, whereby said components are released to said patient at substantially different times; and Such a combination is administered sequentially to a patient in need of treatment, when such components are formulated together in a single dosage form that releases said components in a controlled manner, whereby they are released to said patient simultaneously, successively and / or overlappingly at the same and / or different times, wherein each part can be administered by the same or different routes. Pharmaceutical composition

[0147] Also provided are compositions comprising anti-4Ig-B7H3 antibodies or antigen-binding fragments thereof (including multispecific antibodies) or polynucleotides containing sequences encoding antibodies or antigen-binding fragments, including pharmaceutical formulations. In certain embodiments, the compositions comprise one or more anti-4Ig-B7H3 antibodies or antigen-binding fragments, or one or more polynucleotides containing sequences encoding one or more anti-4Ig-B7H3 antibodies or antigen-binding fragments. These compositions may further comprise a suitable carrier, such as a pharmaceutically acceptable excipient well known in the art, including a buffer.

[0148] Pharmaceutical formulations of the anti-4Ig-B7H3 antibodies or antigen-binding fragments described herein are prepared by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc. Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0149] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0150] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0151] Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes. definition

[0152] Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0153] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0154] Unless the context clearly indicates otherwise, the term "or" means and is used interchangeably with the term "and / or."

[0155] As used herein, the term "anti-cancer agent" refers to any agent useful in treating a cell proliferative disorder such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0156] The term "human 4Ig-B7H3" refers to the 4Ig isoform of the type I transmembrane protein B7H3 in humans. In some embodiments, the amino acid sequence of human 4Ig-B7H3 comprises SEQ ID NO:80.

[0157] As used herein, the terms "administration / administering" and "treating / treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid when applied to an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. The treatment of cells encompasses the contact of an agent with a cell and the contact of an agent with a fluid, wherein the fluid is in contact with the cell. The terms "administration" and "treatment / treatment" also refer to in vitro and ex vivo treatments, for example, the treatment of a cell by a reagent, a diagnostic agent, a binding compound or by another cell. The terms "subject" herein include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human. On the one hand, treating any disease or disorder refers to improving the disease or disorder (i.e., slowing down or preventing or reducing the development of the disease or at least one clinical symptom thereof). On the other hand, "treat / treating / treatment" refers to alleviating or improving at least one physical parameter, including those that the patient may not be able to discern. In yet another aspect, "treat," "treating," or "treatment" refers to modulating a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both. In yet another aspect, "treat," "treating," or "treatment" refers to preventing or delaying the onset, development, or progression of a disease or disorder.

[0158] In the context of the present disclosure, the term "subject" is a mammal, eg, a primate, preferably a higher primate, such as a human (eg, a patient having or at risk of having a disorder described herein).

[0159] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. Within an antigen, the variable region of an antibody interacts with the antigen at many sites through non-covalent forces. Generally, the more interactions there are, the stronger the affinity.

[0160] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0161] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies and anti-idiotypic (anti-Id) antibodies, human engineered antibodies, single-chain antibodies (scFv), single domain antibodies, Fab fragments, Fab' fragments or F(ab')2 fragments. Antibodies can belong to any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). In addition, antibodies include derivatives thereof, such as fusion proteins, multispecific antibodies or antibody-drug conjugates (ADC). In addition, antibodies include derivatives thereof, by being directly or indirectly connected to or forming a complex with another agent (e.g., other drugs or antibodies).

[0162] The term "chimeric antibody" refers to a molecule composed of domains from different species, i.e., the variable domains of an antibody from one host species (e.g., mouse, rabbit, llama, etc.) are fused to the constant domains of an antibody from a different species (e.g., human).

[0163] In some embodiments, the anti-4Ig-B7H3 antibodies comprise at least one antigen-binding site and at least one variable region. In some embodiments, the anti-4Ig-B7H3 antibodies comprise antigen-binding fragments of the 4Ig-B7H3 antibodies described herein. In some embodiments, the anti-4Ig-B7H3 antibodies are isolated or recombinant. In some embodiments, the anti-4Ig-B7H3 antibodies also encompass multispecific antibodies that target 4Ig-B7H3 as at least one arm and one or more other antigens as additional arms.

[0164] The term "monoclonal antibody" or "mAb" or "Mab" herein refers to a colony of substantially homogeneous antibodies, that is, the antibody molecules included in the colony are identical in amino acid sequence except for the possible naturally occurring mutations that may be present in a small amount. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies with different amino acid sequences in their variable domains, particularly their complementary determining regions (CDRs), which are typically specific for different epi-positions. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody colony and should not be construed as requiring antibody production by any ad hoc method. Monoclonal antibodies (mAb) can be obtained by methods well known to those skilled in the art. See, e.g., Kohler et al., (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al., (1992) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY; Harlow et al., (1988) ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory; and Colligan et al., (1993) CURRENT PROTOCOLS IN IMMUNOLOGY. The antibodies disclosed herein can belong to any immunoglobulin class (including IgG, IgM, IgD, IgE, IgA), and any subclass thereof (e.g., IgG1, IgG2, IgG3, IgG4). Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be obtained in vivo by injecting cells from a single hybridoma intraperitoneally into mice, such as primary primed Balb / c mice, to produce ascites containing high concentrations of the desired antibody. Monoclonal antibodies of the isotype IgM or IgG can be purified from such ascites, or from culture supernatants, using column chromatography methods well known to those skilled in the art.

[0165] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer comprises two pairs of identical polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain can be defined as a constant region that is primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as alpha, delta, epsilon, gamma, or mu, and define the isotype of the antibody as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

[0166] The variable regions of each light chain / heavy chain (VL / VH) pair form an antibody binding site. Thus, in general, a complete antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically identical in primary sequence.

[0167] Typically, the variable domains of the heavy and light chains contain three hypervariable regions, also referred to as "complementarity determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions, enabling binding to specific epitopes. In general, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The positions of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (see, e.g., Johnson et al., (2001) Nucleic Acids Res. 29:205-206; Chothia and Lesk, (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature. 342:877-883; Chothia et al., (1992) J. Mol. Biol. 227:799-817; Al-Lazikani et al. (1997) J. Mol. Biol. 273:927-748; ImMunoGenTics (IMGT)) numbering (Lefranc, M.-P., (1999) The Immunologist. 7, 132-136; Lefranc, M.-P. et al., (2003) Dev. Comp. Immunol. 27, 55-77 ("IMGT" numbering scheme).The definition of antigen combining sites is also described in the following literature: Ruiz et al., (2000) Nucleic Acids Res. [Nucleic Acids Research] 28:219-221; and Lefranc, MP, (2001) Nucleic Acids Res. [Nucleic Acids Research] 29:207-209; MacCallum et al., (1996) J. Mol. Biol. [Journal of Molecular Biology] 262:732-745; and Martin et al., (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 86:9268-9272; Martin et al., (1991) Methods Enzymol [Enzymology Methods]. 203:121-153; and Rees et al., in Sternberg MJE (ed.), Protein Structure Prediction [Protein Structure Prediction], Oxford University Press. Press [Oxford University Press], Oxford, 141-172 (1996). For example, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to Chothia, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to IMGT, the CDR amino acid residues in VH are numbered as approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered as approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0168] The term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region includes amino acid residues from "CDR" (e.g., LCDR1, LCDR2, and LCDR3 in the light chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain). See, Kabat et al., (1991) Sequences of Proteins of Immunological Interest [immunology-related protein sequences], 5th Edition Public Health Service [Public Health Service], National Institutes of Health [National Institutes of Health], Bethesda, Maryland (defining the CDR regions of antibodies by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. [Molecular Biology Journal] 196: 901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0169] Unless otherwise indicated, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen to which the full-length antibody binds, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies; and multispecific antibodies formed from antibody fragments.

[0170] As used herein, an antibody "specifically binds" a target protein, meaning that the antibody exhibits preferential binding to the target protein compared to other proteins, but such specificity does not require absolute binding specificity. "Specific binding" or "selective binding" of an antibody, as used in the context of describing an interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological agents (e.g., in a biological sample, blood, serum, plasma, or tissue sample). Thus, under certain specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a particular antigen at least twice the background level and does not specifically bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a particular antigen at least ten (10) times the background binding level and does not specifically bind to other antigens present in the sample in significant amounts.

[0171] As used herein, an "antigen binding domain" comprises at least six CDRs and specifically binds to an epitope (or three CDRs for single domain antibodies). The "antigen binding domain" of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen binding domain that specifically binds to a first epitope and a second antigen binding domain that specifically binds to a second epitope. A multispecific antibody can be a bispecific antibody, a trispecific antibody, a tetraspecific antibody, etc., having an antigen binding domain for each specific epitope. A multispecific antibody can be a multivalent antibody (e.g., a bispecific tetravalent antibody) comprising multiple antigen binding domains, such as 2, 3, 4 or more antigen binding domains that specifically bind to a first epitope and 2, 3, 4 or more antigen binding domains that specifically bind to a second epitope.

[0172] The term "human antibody" as used herein means an antibody that comprises only human immunoglobulin protein sequences. If produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, a human antibody may contain murine carbohydrate chains. Similarly, a "mouse antibody" or a "rat antibody" means an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.

[0173] The term "humanization" or "humanized antibody" means an antibody form containing sequences from non-human (e.g., mouse, rabbit, llama, etc.) antibodies and human antibodies. Such antibodies contain the minimum sequence derived from non-human immunoglobulin. Typically, a humanized antibody will comprise substantially all of at least one and typically two variable domains, all or substantially all of its hypervariable loops corresponding to those of non-human immunoglobulins, and all or substantially all of FRs are those of human immunoglobulin sequences. Humanized antibodies will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. When it is necessary to distinguish humanized antibodies from parent rodent antibodies, prefixes "hum," "hu," "Hu," or "h" are added to the antibody clone name. The humanized form of rodent / camelid antibodies typically comprises the identical CDR sequence of the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0174] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has the highest determined amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. A corresponding human germline sequence can also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be only the framework region, only the complementarity determining regions, the framework and complementarity determining regions, the variable segment (as defined above), or other combinations of sequences or subsequences comprising a variable region. Sequence identity can be determined using the methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. A corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference variable region nucleic acid or amino acid sequence. Furthermore, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., human germline sequences or mutated forms of human germline sequences, or antibodies containing consensus framework sequences derived from analysis of human framework sequences, e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86.

[0175] The term "equilibrium dissociation constant (K D , M)" refers to the dissociation rate constant (kd, time -1 ) divided by the association rate constant (ka, time -1 , M -l The equilibrium dissociation constant can be measured using any method known in the art. The antibodies disclosed herein will typically have a dissociation constant of less than about 10 -7 or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, in some aspects, is less than about 10 -11 M, 10 -12 M or 10 -13 The equilibrium dissociation constant of M.

[0176] The term "cancer" or "tumor" herein has the broadest meaning as understood in the art and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to a certain type or location.

[0177] In the context of this disclosure, when referring to an amino acid sequence, the term "conservative substitution" means that the original amino acid is replaced by a new amino acid that does not substantially change the chemical, physical and / or functional properties of the antibody or fragment, such as its binding affinity to 4Ig-B7H3. In particular, common conservative substitutions of amino acids are well known in the art.

[0178] As used herein, the term "knob-into-hole" technology refers to the amino acid pairing of two polypeptides in vitro or in vivo by introducing a steric protrusion (knob) into one polypeptide and a hole or cavity (hole) into the other polypeptide at the interface where the two polypeptides interact. For example, knob-into-hole has been introduced into the Fc:Fc binding interface of antibodies, C L :C H Interface, or V H / V L The knob-in-hole interface is a structure that is formed by a knob-in-hole structure and a plurality of different heavy chains that are bound to each other. The knob-in-hole structure is a structure that is formed by a knob-in-hole structure and a plurality of different heavy chains that are bound to each other. The knob-in-hole structure is a structure that is formed by a knob-in-hole structure and a plurality of different heavy chains that are bound to each other. The knob-in-hole structure is a structure that is formed by a knob-in-hole structure and a plurality of different heavy chains that are bound to each other. The knob-in-hole structure is a structure that is formed by a knob-in-hole structure and a plurality of different heavy chains that are bound to each other. The knob-in-hole structure is a structure that is formed by a knob-in-hole structure and a plurality of different heavy chains that are bound to each other.

[0179] The term "knob" as used herein in the context of "knob-hole" technology refers to an amino acid change that introduces a protrusion (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0180] The term "hole" as used herein in the context of a "knob-hole" refers to an amino acid change that introduces a pocket or cavity (hole) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0181] An example of an algorithm suitable for determining percentages of sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short word lengths W in the query sequence that, when aligned with the same word length in the database sequence, match or satisfy some positive threshold score T. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as the starting value for searching to find longer HSPs containing them. The word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using 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 hit in each direction is stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score approaches zero or lower 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 a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 by default and compares both chains. For amino acid sequences, the BLAST program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) 50, expectation (E) 10, M=5, N=-4, and a comparison of both strands.

[0182] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0183] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 444-453 (1970), which has been incorporated into the GAP program in the GCG software package, using either the BLOSUM62 matrix or the PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0184] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0185] In the context of nucleic acid, the term "operably connected" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably connected to a coding sequence if it stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system. Typically, the promoter transcriptional regulatory sequence operably connected to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (e.g., enhancers) do not need to be physically adjacent to or next to the coding sequence that they enhance their transcription.

[0186] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-4Ig-B7H3 multispecific antibody as described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. The excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0187] The compositions disclosed herein can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusible solutions. A suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0188] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject to treat a disease, or at least one clinical symptom of a disease or disorder, is sufficient to affect the treatment of the disease, disorder or symptom. A "therapeutically effective amount" can vary with the antibody, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the disease, disorder, and / or the symptoms of the disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated. The appropriate amount in any given case will be apparent to those skilled in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combination subject used to effectively treat the disease, disorder or condition.

[0189] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the therapeutic conditions or disorders described in this disclosure. Such administration encompasses the co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses co-administration in multiple containers or in separate containers (e.g., capsules, powders, and liquids) of each active ingredient. The powder and / or liquid can be redissolved or diluted to a desired dosage before administration. In addition, such administration also encompasses the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In any case, the therapeutic regimen will provide the beneficial effects of the drug combination in terms of treating the conditions or disorders described herein.

[0190] As used herein, the phrase "in combination with" means that the anti-4Ig-B7H3 antibody is administered to the subject simultaneously with, immediately before, or immediately after administration of the additional therapeutic agent. In certain embodiments, the anti-4Ig-B7H3 antibody is administered as a co-formulation with the additional therapeutic agent. Equivalent

[0191] It should be understood that although the invention has been described in conjunction with the detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0192] It should be understood that one, some, any or all of the features of the various embodiments described herein may be combined to form additional embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. Examples Example 1. Generation of mouse anti-B7H3 monoclonal antibodies immunity

[0193] To generate antibodies against B7H3, a cohort of five transgenic mice engineered to produce human variable regions and rat constant regions were immunized with the extracellular binding domain (ECD) of the human 4Ig-B7H3 antigen. Each cohort was subjected to an immunization strategy that included a unique combination of human B7H3 antigen, dose, injection route, and adjuvant. A total of five animals in one cohort were immunized. Animals were immunized at different times between 0 and 100 days. To monitor the immune response, titrated sera were screened by ELISA after 2-6 immunizations, typically over 30-100 days. Sera were screened for antibodies (Ab) that bind to the B7H3 antigen. The B7H3-specific antibody response was measured in each animal, and animals with sufficient anti-B7H3 titers were selected for a final booster immunization over a period of 4 days. Hybridoma fusion and screening

[0194] Lymphoid organs, including spleen and lymph nodes, were isolated from mice immunized as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells derived from SP2 / 0 via PEG-based fusion. Conventional 1640 medium (Gibco) supplemented with HAT was used. TM) The resulting cells were plated in 96-well cell culture plates to select hybridomas. After 10-13 days of culture medium and growth medium replacement, hybridoma culture supernatants were collected from each well and screened to identify wells with secreted B7H3-specific antibodies. All supernatants were initially screened for human ECD-4Ig-B7H3-hFc. The binding of antibodies to human ECD-4Ig-B7H3-hFc was measured by ELISA. The supernatants from 5 hybridoma fusions were screened for B7H3 antibodies. Briefly, 2 μg / mL human ECD-4Ig-B7H3-hFc was coated in a 96-well ELISA plate, and 50 μl of hybridoma culture supernatant was incubated with human ECD-4Ig-B7H3-hFc for 30-60 minutes. The plates were then washed and incubated with an anti-rat IgG Fc secondary Ab conjugated to HRP. After incubation and washing, the plates were developed with HRP substrate and the absorbance was measured.

[0195] Hybridomas from positive wells were transferred to 24-well plates with fresh medium and grown for 2-3 days before being screened again by flow cytometry to confirm antibody binding to human 4Ig-B7H3 and cyno B7H3 overexpressing cell lines.

[0196] Antibody binding to human B7H3-overexpressing cells and cyno B7H3-overexpressing cells was measured by FACS. Briefly, 100 μl of hybridoma culture supernatant was incubated with human 4Ig-B7H3 or cyno B7H3-overexpressing cells for 30-60 minutes, washed, and incubated with an anti-IgG Fc secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry. Subcloning and sequence analysis

[0197] Selected anti-B7H3 Ab-secreting hybridomas were subcloned once or twice to ensure monoclonality. Briefly, positive hybridoma clones were subcloned by limiting dilution. After 7-10 days, culture supernatants were screened by ELISA and flow cytometry was performed as previously described to confirm human and cyno B7H3 antigen binding. Stable hybridoma subclones were cultured in vitro for cell cryopreservation and antibody VH and VL gene cloning and sequencing.

[0198] The subcloned anti-B7H3 Ab-secreting hybridomas were lysed with lysis buffer. The mRNA-containing lysates were then transferred to a 96-well plate and synthesized using Super Script III First-Strand Synthesis SuperMix according to the manufacturer's instructions. TM mRNA isolation, cDNA synthesis, and DNA sequencing were performed (Invitrogen).

[0199] The sequence of the obtained human antibody BGA-3726 is listed in Table 7. Example 2. Determination of binding kinetics and affinity of anti-B7H3 antibodies by SPR

[0200] Purify the anti-B7H3 mAb BGA-3726 and analyze it using BIAcore TM The binding kinetics were characterized by SPR assay using a T-200 (GE Life Sciences). Briefly, anti-mouse IgG Fc antibody was immobilized on an activated CM5 biosensor chip (Cat. No. BR100530, GE Life Sciences). Purified antibody was flowed over the chip surface and captured by the anti-mouse IgG Fc antibody. Serial dilutions of human ECD-4Ig-B7H3-hFc were then flowed over the chip surface, and the changes in the surface plasmon resonance signal were analyzed using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences) to calculate the association rate (k). on ) and dissociation rate (k off ). The equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on The binding affinity profiles of anti-B7H3 mAbs are shown in Tables 1 and Figure 1 Shown in. Table 1. Binding kinetics of BGA-3726 Ab number antigen ka(1 / Ms) kd(1 / s) KD(M) BGA-3726 Human B7H3 3.63E+05 4.49E-06 1.24E-11 Example 3. Determination of the binding affinity of anti-B7H3 antibodies to B7H3 expressed on stable cell lines

[0201] The binding affinity of anti-B7H3 mAbs to human 4Ig-B7H3 and cyno B7H3 overexpressing cell lines was determined by FACS. Briefly, human or cyno B7H3 overexpressing cells were incubated with serially diluted purified antibodies, washed, and incubated with anti-mouse IgG secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry. The binding affinity profiles of anti-B7H3 mAbs are shown in Tables 2-3 and Figure 2 A- Figure 2 Shown in B. Table 2. Cellular binding affinity of BGA-3276 to human B7-H3 Ab number cell lines EC50 (nM) BGA-3726 Human B7H3 1.91 Table 3. Cellular binding affinity of BGA-3276 to cyno B7-H3 Ab number cell lines EC50 (nM) BGA-3726 Cyno B7H3 1.32 Example 4. Determination of non-specific binding of anti-B7H3 antibodies

[0202] The nonspecific binding activity of the anti-B7H3 mAb BGA-3276 was determined by FACS. Briefly, Daudi cells that do not express B7H3 were incubated with purified BGA-3276, washed, and incubated with an anti-rat IgG secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry. The binding MFI is shown in Table 4. BGA-3276 did not show any nonspecific binding to B7H3-negative cells. Table 4. MFI of BGA-3276 binding to Daudi Ab number NK92mi's MFI BGA-3726 4.15 mIgG 16.00 Example 5. Engineering of anti-human B7H3 mAb BGA-3726

[0203] BGA-3726 (SEQ ID NOs: 1-10) is a human antibody composed of fully human Vh and Vk regions. To improve its developability, BGA-3726 was re-engineered to remove potential post-translational modifications (PTMs) and optimize biophysical stability for human therapeutic use.

[0204] BGA-3726 and antibodies engineered from BGA-3726 were constructed as human full-length antibodies using in-house developed expression vectors containing the constant regions of human IgG1 variants (SEQ ID NO: 31) and kappa chains, respectively, with easily adaptable subcloning sites. Expression and preparation of BGA-3726 and its engineered antibodies were achieved by co-transfection of heavy chain and corresponding light chain constructs into 293G cells (in-house developed) and purification using a protein A chromatography column. The purified antibodies were concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C.

[0205] Based on the BGA-3726 original clone, several single mutations were carried out in CDR to remove potential post-translational modification (PTM) sites. Potential PTM sites include potential oxidation sites W34, W96 and W98 (Kabat numbering) in HCDR and potential deamidation site N29 (NG) in LCDR1. Use primers and site-directed mutagenesis kit (catalog number (Cat. No.) FM111-02, Quanshijin Company (TransGen), Beijing, China) containing mutations at specific positions to carry out all mutations. Expected mutation was verified by sequencing analysis. These BGA-3726 variant antibodies, namely BGA-4826 comprising W34Y in HCDR1 (SEQ ID Nos: 11, 2-6, 12, 8, 13, 10), BGA-4498 comprising W96Y in HCDR3 (SEQ ID Nos: 1, 2, 14, 4-6, 15, 8, 16, 10), BGA-4269 comprising W98Y in HCDR3 (SEQ ID Nos: 1, 2, 17, 4-6, 18, 8, 19, 10), BGA-4380 comprising N29Q in LCDR1 (SEQ ID Nos: 1-3, 20, 5-6, 7, 21, 9, 22), and BGA-4265 comprising G30A in LCDR1 (SEQ ID Nos: 1-3, 23, 5-6, 7, 24, 9, 25), were tested in SPR binding assays.

[0206] In summary, engineered versions of humanized monoclonal antibodies, namely BGA-6938 (SEQ ID Nos: 11, 2, 14, 23, 5-6, 26, 24, 27, 25), which is based on BGA-3726 and contains W34Y in HCDR1, W96Y in HCDR3, W98Y in HCDR3, and G30A in LCDR1, and BGA-5488 (SEQ ID Nos: 11, 2, 28, 23, 5-6, 29, 24, 30, 25), which is based on BGA-3726 and contains W34Y in HCDR1, W96Y in HCDR3, W98Y in HCDR3, and G30A in LCDR1, were derived from the mutagenesis process described above and were characterized in detail. The results showed that BGA-3726, BGA-6938, and BGA-5488 had comparable binding affinities to recombinant B7H3.

[0207] For affinity determination, antibodies were captured on an anti-human Fc surface and used for affinity determination based on surface plasmon resonance (SPR) technology. The results of the SPR-measured binding profiles of anti-B7H3 antibodies to the ECD of human 4Ig-B7H3 (Sinobiological, catalog number: 11188-H08H) are summarized in Table 5. BGA-6938 (SEQ ID NO: 11, 2, 14, 23, 5-6, 26, 24, 27, 25) and BGA-5488 (SEQ ID NO: 11, 2, 28, 23, 5-6, 29, 24, 30, 25) had comparable binding affinities with dissociation constants of 0.443 nM and 1.11 nM, respectively, which are comparable to BGA-3726. Table 5. Comparison of the binding affinity of BGA-3726 and its engineered antibodies to B7H3 by SPR

[0208] To evaluate the binding activity of BGA-6938 and BGA-5488 to native B7H3 on live cells, NK92mi cells were engineered to overexpress human 4Ig-B7H3. Live NK92mi / B7H3 cells were seeded in 96-well plates and incubated with serial dilutions of BGA-3726, BGA-6938, and BGA-5488. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The EC values ​​for dose-dependent binding to native human B7H3 were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. 50 Value. Figure 3 A- Figure 3 B and shown in Table 6. Engineered antibodies BGA-6938 and BGA-5488 retained the binding affinity of the parental clone BGA-3726 to native B7H3. Table 6. Comparison of EC50 of BGA-3726 and its humanized antibodies on NK92mi / B7H3 cells by FACS. Table 7. Antibody sequences Example 6. Cell Binding and Internalization of Humanized Ab Combine

[0209] Binding assays were performed to evaluate the affinity of the antibodies. Wild-type IgG1 / κ forms of BGA-6938 and MABX-9001a (humanized anti-B7H3 IgG1 monoclonal antibody for DS-7300a, Yamato et al., Mol. Cancer Ther. 2022 1; 21(4): 635-646) and human IgG antibodies were included in the evaluation. H358 cells were detached from the culture flask using trypsin-EDTA (Cat. No. 25200056, Thermo Fisher Scientific) and washed with binding buffer (PBS containing 2% FBS, pH 7.2). The washed cells were added to a 96-well plate containing the antibody at a density of approximately 100,000 cells in 0.2 mL of binding buffer. In the competition reaction with the cells, the final concentration of the primary antibody was varied, starting from 500 nM and then decreasing in 1:5 dilutions to obtain 11 concentrations. The cells and antibody mixture were incubated on ice for 1 hour. After incubation for 1 hour, the cells were washed twice with binding buffer to separate free antibodies. The washed cells were further labeled with fluorescently labeled anti-human Fc (Catalog No.: 109-605-190, Jackson ImmunoResearch) and incubated on ice for 1 hour. After incubation for 1 hour, the cells were washed twice with binding buffer to separate free antibodies. The fluorescence signal was detected using LSRFortessa (BD). The binding curves of BGA-6938 and MABX-9001a and human IgG antibodies on H358 are shown in Figure 2. Figure 4 Based on the binding results, BGA-6938 showed higher binding affinity to B7H3 compared to MABX-9001a. Internalization

[0210] A desirable property of antibodies conjugated to ADCs is the ability to be internalized into cells. pHrodo was performed according to the manufacturer's protocol. TM The internalization activity of BGA-6938 and MABX-9001a was determined using the Zenon TM pHrodo TM Primary antibodies were labeled with iFL IgG labeling reagent (Catalog No. Z25612, Thermo Fisher Scientific) and RPMI medium. 100,000 H358 cells were suspended in 0.2 mL of antibody-pHrodo and incubated at 37°C. After incubation for 1 hour, 4 hours, 8 hours, and 24 hours, the cells were washed twice with binding buffer (PBS containing 2% FBS, pH 7.2) to separate free antibodies. The ELISA kit was used with an LSRFortessa with PE channel. TM (BD) Detection of fluorescence signal. Fluorescence signal intensity indicates internalization activity. The curves of BGA-6938, MABX-9001a and human IgG antibody on H358 are Figure 5 Based on the internalization results, BGA-6938 showed increased internalization activity compared to MABX-9001a. Example 7. Cell killing activity

[0211] To evaluate the ADC cell killing activity of BGA-6938 and MAbX-9001a, target cells H1650, H441, and H1048 (all lung cancer cell lines) were plated at 1,000 cells per well in 96-well 3D cell culture plates (Catalog No. 6055330, Perkin Elmer) in 100 μl of RPMI-1640 plus 10% FBS medium. After 24 hours, another 50 μl of medium containing serial dilutions of BGA-6938-GGFG-DXd (DAR8) and MABX-9001a-GGFG-DXd (DAR8 or DAR4) concentrations was added to three replicate wells. For the structure of MABX-9001a-GGFG-DXd, see Yamato et al., Mol Cancer Ther 2022 1; 21(4): 635-646. Figure 2 A. After 6 days, cell viability was determined using CellTiter-Glo Luminescent Cell Viability Reagent (G7572; Promega Corporation) and Tecan Spark. Kill curves were analyzed using GraphPad Prism 9 and analyzed in Figure 6Compared with MABX-9001a-GGFG-DXd(DAR8), BGA-6938-GGFG-DXd(DAR8) showed comparable killing activity in H1650 and H441, and increased killing activity in H1048. Example 8. Binding of B7H3 scFv Protein to H358 Tumor Cell Line

[0212] To evaluate the binding ability of the scFv of BGA-6938 on the surface of live H358 tumor cells, the scFv protein was purified by affinity chromatography followed by size exclusion chromatography (see Table 8). 5 H358 cells were seeded in 96-well plates. A dose response curve was generated by adding serially diluted scFv proteins (3.38 pM to 200 nM) to the cells, and bound scFv was detected using the His-tag antibody iFluor 488 (Cat. No. A01800). TM The MFI of each cell population was measured systematically. A four-parameter logistic model was used to determine the EC50 and MFImax of each scFv. The results are shown in Tables 9 and Figure 7 The ScFv of BGA-6938 showed an EC50 of 2.047 nM for the B7H3 antigen on H358 tumor cells. Table 8. B7H3 scFv protein sequences Table 9: Dose-dependent binding of scFv fragments on the H358 cell line Antibody EC50 (nM) MFI scFv of BGA-6938 2.047 782469 Example 9. Epitope mapping of anti-B7H3 antibodies

[0213] To investigate the binding epitope of the anti-B7H3 mAb BGA-6938, truncated human B7-H3 domains were generated by fusing each of the Ig-like domains from the extracellular region of human B7H3 (SEQ ID NO: 80), namely IgV1 (amino acids 29-139 of SEQ ID NO: 80), IgC1 (amino acids 145-238 of SEQ ID NO: 80), IgV2 (amino acids 243-357 of SEQ ID NO: 80), and IgC2 (amino acids 363-456 of SEQ ID NO: 80), along with its transmembrane and intracellular domains. The truncated versions of B7H3 were also fused to an N-terminal FLAG tag. Figure 8The resulting truncated human B7H3 construct shown in A contains an N-terminal FLAG tag followed by an Ig-like domain and the B7H3 C-terminal domain, including the transmembrane and intracellular domains. DNA encoding the truncated version of B7H3 was cloned into the pcDNA 3.4 vector.

[0214] ExpiCHO was transfected with plasmids containing these truncated B7H3 constructs. TM The cells were incubated with 100 nM purified BGA-6938 and a reference antibody DS-7300 (US2022 / 0064312 A1) from Daiichi Sankyo Co., Ltd. The binding of BGA-6938 and DS-7300 to different B7H3 truncated forms was evaluated by detection using Alexa Fluor 647 rabbit anti-human IgG (Cat. No.: 309-605-008, Jackson ImmunoResearch). After incubation of the transfected cells with anti-FLAG mAb (Cat. No.: A01809, Genscript), the expression of each construct was verified by detecting the N-terminal FLAG tag. Figure 8 The data in B show that BGA-6938 specifically binds to the B7H3 V1 and V2 domains and does not bind to the B7H3 C1 and C2 domains. In contrast, the reference antibody DS-7300 binds to the C1 and C2 domains of B7H3 and does not bind to the V1 and V2 domains ( Figure 8 B). Thus, BGA-6938 and DS-7300 have non-overlapping epitopes.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, comprising: (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising: (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 20, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; or (viii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 28; and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: (i) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24; (ii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8; (iii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8; (iv) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8; (v) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8; (vi) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:21; (vii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24; or (viii) the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29, and the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

24.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids have been inserted, deleted or substituted in SEQ ID NOs: 26 and 24, SEQ ID NOs: 7 and 8, SEQ ID NOs: 12 and 8, SEQ ID NOs: 15 and 8, SEQ ID NOs: 18 and 8, SEQ ID NOs: 7 and 21, SEQ ID NOs: 7 and 24, and SEQ ID NOs: 29 and 24.

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein: (i) the heavy chain variable region comprises SEQ ID NO: 26, and the light chain variable region comprises SEQ ID NO: 24; (ii) the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 8; (iii) the heavy chain variable region comprises SEQ ID NO: 12, and the light chain variable region comprises SEQ ID NO: 8; (iv) the heavy chain variable region comprises SEQ ID NO: 15, and the light chain variable region comprises SEQ ID NO: 8; (v) the heavy chain variable region comprises SEQ ID NO: 18, and the light chain variable region comprises SEQ ID NO: 8; (vi) the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 21; (vii) the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 24; or (viii) the heavy chain variable region comprises SEQ ID NO: 29, and the light chain variable region comprises SEQ ID NO:

24.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment or a F(ab')2 fragment.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody or antigen-binding fragment thereof comprises an scFv comprising a VH having the amino acids of SEQ ID NO: 26 and a VL having the amino acids of SEQ ID NO: 24, optionally the VH and VL are connected via an amino acid linker, optionally the amino acid linker is any sequence of SEQ ID NO: 35 to SEQ ID NO:

77.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the amino acid linker is SEQ ID NO:

77.

8. The antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof comprises a scFv having the amino acid sequence of SEQ ID NO:

32.

9. A multispecific antibody or antigen-binding fragment thereof, comprising at least a first antigen-binding domain that specifically binds to human 4Ig-B7H3, wherein the first antigen-binding domain is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; and At least a second antigen binding domain that specifically binds to a second human tumor-associated antigen (TAA). 10 . The multispecific antibody or antigen-binding fragment thereof according to claim 9 , wherein the multispecific antibody is a bispecific antibody.

11. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 9-10, further comprising an amino acid linker, wherein the amino acid linker is any sequence of SEQ ID NO: 35 to SEQ ID NO:

77.

12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3 or IgG4 subclass and / or a light chain constant region of the kappa or lambda type.

13. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1 subclass and a light chain constant region of the kappa type.

14. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

15. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or is aglycosylated or is hypofucosylated.

16. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an added bisecting GlcNac structure.

17. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

18. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin via a cytotoxin linker.

19. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein: (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 80); and / or (2) The antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 80).

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

21. A method for treating cancer, comprising administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, or the pharmaceutical composition according to claim 20 to a patient in need thereof.

22. The method of claim 21, wherein the cancer is 4Ig-B7H3 positive.

23. The method of any one of claims 21-22, wherein the cancer is colorectal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, kidney cancer, lung cancer, or esophageal cancer.

24. The method of claim 23, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

25. The method of claim 24, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.

26. The method of claim 23, wherein the esophageal cancer is esophageal squamous cell carcinoma.

27. The method of any one of claims 21-26, wherein the antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

28. The method of claim 27, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine.

29. The method of claim 27, wherein the therapeutic agent is an immune checkpoint inhibitor.

30. The method of claim 27, wherein the therapeutic agent is an anti-PD-1 antibody.

31. The method of claim 30, wherein the anti-PD1 antibody is tislelizumab.

32. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 19.

33. A vector comprising the nucleic acid according to claim 32.

34. A host cell comprising the nucleic acid of claim 32 or the vector of claim 33.

35. A process for producing an antibody or an antigen-binding fragment thereof, the process comprising culturing the host cell according to claim 34 and recovering the antibody or antibody fragment from the culture.

36. An antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, wherein: (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 80); and / or (2) The antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 80).

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