Anti-B7H3 antibodies and methods of use thereof

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 a cytotoxin, pharmaceutical compositions comprising the above antibodies or antigen-binding fragments, and uses of the antibodies, multispecific antibodies, or compositions for treating diseases such as cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Application No. PCT / CN2022 / 143248, filed December 29, 2022, which is incorporated herein by reference in its entirety.

[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 with other modalities, such as a second tumor-associated antigen (TAA), immune checkpoint, or immune stimulator, to construct antibody-drug conjugates (ADCs), or to form fusion proteins. Finally, the anti-human 4Ig-B7H3 antibodies disclosed herein can be used to treat various cancers. [Background technology]

[0003] B7H3 or B7-H3 (CD276 or B7RP-2) is a type I transmembrane protein identified in 2001 from a dendritic cell (DC) cDNA library (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 pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas human B7H3 contains one or two identical pairs 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 short and lacks 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, which is cleaved from the membrane by proteinases or caused by alternative splicing (Zhang et al., (2008) Immunology. 123(4):538-546).

[0004] B7H3, a member of the B7 protein family, shares 20–27% amino acid identity with other B7 family ligands, but its receptor(s) remain unknown (Chapoval et al., (2001) Nat. Immunol. 2(3):269–274). To date, the molecular mechanisms by which B7H3 participates in immune evasion remain elusive, and its function in T cell-mediated adaptive immunity remains controversial. Although originally identified as a T cell costimulatory molecule (Chapoval et al., (2001) Nat. Immunol. 2(3):269-274; Zhang et al., (2004) Acta Biochim. Biophys. Sin. 36(6):430-436), subsequent studies have suggested that it primarily 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). On the other hand, the crystal structure of mouse B7H3 revealed that the FG loop is important for mB7H3-mediated inhibition of T cell proliferation (Vigdorovich et al., (2013) Structure. 21(5):707-717). Notably, NSCLC with high B7H3 expression is associated with low numbers of tumor-infiltrating lymphocytes and resistance to anti-PD1 therapy, suggesting a role for B7H3 in immune evasion. Therefore, B7H3-targeted therapy in combination with anti-PD-1 / PD-L1 antibody therapy is a promising approach for B7H3-expressing NSCLC (Altan et al., (2017) Clin. Cancer. Res. 23(17):5202-5209).

[0005] In addition to immune regulation, B7H3 also has intrinsic tumor-promoting functions. TCGA analysis revealed that B7H3 expression correlates with the EGFR / PI3K / AKT pathway, the MAPK pathway, and the EMT process across cancer types. Ectopic 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). Accordingly, overexpression of B7H3 correlates with poor prognosis and adverse clinical outcomes in many types of cancer (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. 20(1):28-3).

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

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

[0008] The present disclosure includes antibodies and antibody fragments thereof specific to human 4Ig-B7H3. Additionally, the antibodies and antibody fragments disclosed herein can be used to construct multispecific antibodies with other modalities, such as a second tumor-associated antigen (TAA), immune checkpoint, or immune stimulator, or to construct antibody-drug conjugates (ADCs), or to form fusion proteins. The 4Ig-B7H3 antibody can potentially be used alone or in combination with other therapeutic agents 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] The present disclosure encompasses the following embodiments.

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

[0012] (i) the heavy chain variable region comprises an amino acid sequence 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24; The above antibody or antigen-binding fragment thereof.

[0013] The above antibody or antigen-binding fragment thereof, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids 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, or SEQ ID NOs: 29 and 24 have been inserted, deleted, or substituted.

[0014] (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. The above antibody or antigen-binding fragment thereof.

[0015] The antibody or antigen-binding fragment thereof, which is a monoclonal antibody, a human recombinant 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 comprises an scFv comprising a VH having the amino acid sequence of SEQ ID NO: 26 and a VL having the amino acid sequence of SEQ ID NO: 24, wherein optionally, the VH and VL are connected via an amino acid linker, and optionally, the amino acid linker is any of the sequences of SEQ ID NOs: 35 to 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 comprises an 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 and at least a second antigen-binding domain that specifically binds to a second human tumor-associated antigen (TAA), wherein the first antigen-binding domain is an antibody or antigen-binding fragment thereof described herein.

[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 described above, further comprising an amino acid linker, wherein the amino acid linker has a sequence set forth in any one of SEQ ID NOs: 35 to 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 subclass.

[0024] 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 is reduced or aglycosylated, or hypofucosylated.

[0026] The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises increased bisected GlcNac structures.

[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] (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29 to 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 to 357 of human 4Ig-B7H3 (SEQ ID NO: 80). The above antibody or antigen-binding fragment thereof.

[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 to 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 to 456 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0031] A pharmaceutical composition comprising an 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 of cancer treatment an effective amount of an 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 colon cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, renal cancer, lung cancer, or esophageal carcinoma.

[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 carcinoma is esophageal squamous cell carcinoma.

[0038] The above 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 vector described herein.

[0046] A process for producing an antibody or antigen-binding fragment thereof, comprising culturing a 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, (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29 to 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 to 357 of human 4Ig-B7H3 (SEQ ID NO: 80). The above antibody or antigen-binding fragment thereof.

[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 to 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 to 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 and high affinity to human 4Ig-B7H3.

[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 explanation of the drawings]

[0051] [Figure 1] This figure shows the affinity measurement of purified B7H3 mAb BGA-3726 (mouse chimera) for the human B7H3 antigen by SPR. Specifically, the sensorgram shows the change in binding as a signal emitted from the surface of a glass chip. The glass chip is a plasmon resonance surface bound to BGA-3726. The anti-BGA-3726 antibody was captured with an anti-mouse IgG Fc antibody and immobilized on the chip. After immobilization, serial dilutions of human or cynomolgus monkey B7H3 ectodomain were passed over the chip. Since B7H3 is the target of BGA-3726, the degree of binding is visualized as response units (RU) (Y-axis), which are proportional to binding over time (in seconds) (X-axis). Therefore, the light gray line represents the serial dilutions of human B7H3 protein, and the dark gray line represents the fitted curve corresponding to various concentrations of human B7H3. The dotted line represents the maximum concentration of B7H3 protein. The results were used to calculate the association rate (Kon) and dissociation rate (Koff) for BGA-3295. [Figure 2] (A) and (B) show the binding affinity of purified anti-4Ig-B7H3 mAb BGA-3726 to cells overexpressing human 4Ig-B7H3 by FACS assay. (B) show the binding affinity of purified anti-4Ig-B7H3 mAb BGA-3726 to cells overexpressing cynomolgus monkey B7H3 by FACS assay. [Figure 3]Figure 1 shows a comparison of the binding affinity of recombinant antibodies derived from BGA-3726 to cells overexpressing human 4Ig-B7H3 by FACS assay. (A) shows the dose-dependent binding curve of BGA-3726 to cells overexpressing human 4Ig-B7H3. (B) shows the dose-dependent binding curves of BGA-6938 and BGA-5488 to cells overexpressing human 4Ig-B7H3. [Figure 4] Figure 1 shows a comparison of the binding affinity of BGA-6938 and MABX-9001a to the B7H3-positive tumor cell line H358 by FACS assay. A hIgG isotype Ab was used as a non-binding control. [Figure 5] Figure 1 shows a comparison of the internalization activity of BGA-6938 and MABX-9001a toward B7H3-positive tumor cells H358 by FACS assay at the indicated time points. hIgG isotype Ab was used as a non-binding control. [Figure 6] Figure 1 shows the cell killing activity of BGA-6938-GGFG-DXd(DAR8), MABX-9001a-GGFG-DXd(DAR8), and MABX-9001a-GGFG-DXd(DAR4) in H1650, H441, and H1048 cell models. Iso-GGFG-DXd(DAR8) was used as a non-targeting control. [Figure 7] 1 shows the binding affinity of the scFv of BGA-6938 to H358 cells. [Figure 8] Figure 1 shows epitope mapping of BGB-6938. A shows the construct design of truncated human B7H3. B shows epitope identification of BGB-6938 by domain truncation and comparison of the epitope with DS-7300. DETAILED DESCRIPTION OF THE INVENTION

[0052] The present disclosure provides anti-human 4Ig-B7H3 antibodies and antigen-binding fragments thereof. The present disclosure also provides antibodies with desirable binding affinity, desirable internalization (e.g., increased internalization rate), and other desirable attributes. Anti-human 4Ig-B7H3 antibodies can be used to construct multispecific antibodies with other modalities, such as a second tumor-associated antigen (TAA), immune checkpoint, or immune stimulator, to construct antibody-drug conjugates (ADCs), or to fuse with other domains to form fusion proteins. Furthermore, anti-human 4Ig-B7H3 antibodies and constructs thereof can be used to treat cancer and related diseases.

[0053] I. Anti-4Ig-B7H3 antibody 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 prepared as described below.

[0054] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain having 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 (Table 7). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibodies or antigen-binding fragments comprise an HCDR having the 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 antibodies comprise one, two, three, or more HCDRs having the amino acid sequence of any of the HCDRs listed in Table 7.

[0055] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to human 4Ig-B7H3, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VL domain having the 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 antibodies or antigen-binding fragments comprise an LCDR having the 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 antibodies comprise one, two, three, or more LCDRs having the amino acid sequence of any of the LCDRs listed in Table 7.

[0056] In one embodiment, an 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.

[0057] In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises (a) a heavy chain variable region comprising one or more complementarity determining regions (HCDRs) 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 comprising one or more complementarity determining regions (LCDRs) 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.

[0058] In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs): 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 comprising three complementarity determining regions (LCDRs): 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.

[0059] In another embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 comprises: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 14; an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 1 and / or (b) a heavy chain variable region comprising three complementarity determining regions (HCDRs), wherein the HCDR1 comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 11, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 28; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), wherein the HCDR1 comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6; an LCDR1 comprises an amino acid sequence of SEQ ID NO: 20, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6; or an LCDR1 comprises the amino acid sequence of SEQ ID NO: 23, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0060] In one embodiment, an antibody or antigen-binding fragment 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) set forth in SEQ ID NO: 26; (ii) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 7; (iii) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 12; (iv) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 15; (v) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 18. or (vi) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region (VH) set forth in SEQ ID NO: 29; and / or (i) LCDR1 (light chain complementarity determining region 1), LCDR2, and LCDR3 from the light chain variable region (VL) set forth in SEQ ID NO: 24; (ii) LCDR1, LCDR2, and LCDR3 from the light chain variable region (VL) set forth in SEQ ID NO: 21; or (iii) LCDR1, LCDR2, and LCDR3 from the light chain variable region (VL) set forth in SEQ ID NO: 8.

[0061] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 is an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3, according to the Kabat definition: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; or (viii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6.

[0062] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (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.

[0063] In one embodiment, the antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising an amino acid sequence 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. Includes:

[0064] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids 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 are inserted, deleted, or substituted.

[0065] In one embodiment, the antibody or antigen-binding fragment thereof (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 Includes:

[0066] Other antibodies or antigen-binding fragments thereof of the present disclosure have been altered but still comprise amino acids having at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identity within the CDR regions compared to the CDR regions disclosed in Table 7. In some embodiments, other antibodies or antigen-binding fragments thereof of the present disclosure comprise amino acid alterations (insertions, deletions, or substitutions, optionally conservative amino acid substitutions) where no more than one, two, three, four, or five amino acids have been altered within the CDR regions compared to the CDR regions set forth in the sequences set forth in Table 7, while maintaining binding specificity and affinity.

[0067] Other antibodies of the disclosure include those in which the amino acids, or nucleic acids encoding the amino acids, have been altered in the variable regions (e.g., framework regions of the variable regions) but still 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% percent identity with the variable region sequences set forth in Table 7, while maintaining binding specificity / affinity, and optionally, the corresponding sequences of the CDRs are unchanged. In some embodiments, other antibodies of the disclosure comprise changes in 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 are changed in the variable regions (e.g., framework regions of the variable regions) compared to the variable regions set forth in the sequences set forth in Table 7, while maintaining binding specificity / affinity, and optionally, the corresponding sequences of the CDRs are unchanged.

[0068] In another embodiment, the present disclosure provides human 4Ig-B7H3 at 1 x 10 -6 M~1×10 -11 Binding affinity (K D In another embodiment, the anti-4Ig-B7H3 antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 at about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, about 1 x 10 -10 M, or approximately 1 x 10 -11 Binding affinity (K D ) to join them.

[0069] The present disclosure also provides nucleic acid sequences encoding the VH or VL of antibodies that specifically bind to human 4Ig-B7H3, which nucleic acid sequences can be optimized for expression in mammalian cells.

[0070] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-4Ig-B7H3 antibodies listed in Table 7. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with (e.g., competitively inhibit in a statistically significant manner) the antibodies listed in Table 7 in binding assays. The ability of a test antibody to inhibit the binding of an antibody of the present disclosure and its antigen-binding fragment to 4Ig-B7H3 indicates that the test antibody can compete with that antibody or its antigen-binding fragment for binding to 4Ig-B7H3. Without being bound by any one theory, such antibodies may bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on 4Ig-B7H3 as the competing antibody or antigen-binding fragment thereof. In certain embodiments, an antibody that binds to the same epitope on 4Ig-B7H3 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0071] 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 to 139 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0072] 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 to 357 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0073] 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 the IgV1 domain of human 4Ig-B7H3. 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 the IgV2 domain of human 4Ig-B7H3. 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 binds to both the IgV1 and IgV2 domains of human 4Ig-B7H3.

[0074] 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 does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 145 to 238 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0075] 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 does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 363 to 456 of human 4Ig-B7H3 (SEQ ID NO: 80).

[0076] 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 does not bind to the IgC1 domain of human 4Ig-B7H3. 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 does not bind to the IgC2 domain of human 4Ig-B7H3. 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 does not bind to either the IgC1 domain or the IgC2 domain of human 4Ig-B7H3.

[0077] 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.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a human recombinant antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0079] In one embodiment, the antibody or antigen-binding fragment thereof is in an scFv format comprising, in N-terminal to C-terminal orientation, VH-VL or VL-VH. In some embodiments, the VH and VL are connected via an amino acid linker 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 listed in Table 7. In some embodiments, the VL is any one of the VLs listed 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 of SEQ ID NOs:35-77. In one embodiment, the amino acid linker is SEQ ID NO:77.

[0080] In one embodiment, the antibody or antigen-binding fragment thereof comprises an scFv having the amino acid sequence of SEQ ID NO: 32. In another embodiment, the antibody or antigen-binding fragment thereof comprises an scFv of SEQ ID NO: 32.

[0081] II. Anti-4Ig-B7H3 multispecific antibody In one embodiment, the anti-4Ig-B7H3 antibodies disclosed herein can be used to construct multispecific antibodies with other modalities such as a second human tumor-associated antigen (TAA), immune checkpoint, or immune stimulator, with 4Ig-B7H3 functioning as the first TAA.

[0082] In one embodiment, the anti-4Ig-B7H3 antibody disclosed herein can be incorporated into an anti-4Ig-B7H3xTAA multispecific antibody, where the TAA is an antibody or fragment thereof directed against any human tumor-associated antigen. The antibody molecule is a multispecific antibody molecule, e.g., comprising multiple antigen-binding domains, where 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, trispecific, or tetraspecific antibody.

[0083] In one embodiment, a multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. A bispecific antibody comprises a first antigen-binding domain that specifically binds human 4Ig-B7H3 and a second antigen-binding domain that specifically binds to a second TAA. This includes bispecific antibodies comprising 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, a bispecific antibody comprises an antigen-binding fragment, which can be a Fab, F(ab'), Fv, single-chain Fv (scFv), or single-domain antibody.

[0084] In one embodiment, a multispecific antibody of the disclosure is administered to a second human TAA and / or human 4Ig-B7H3 at a dose of 1×10 -6 M~1×10 -10 M, or even 1 x 10 -11Binding affinity (K D In another embodiment, the multispecific antibody of the disclosure binds to the second human TAA and / or human 4Ig-B7H3 at about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, about 1 x 10 -10 M, or approximately 1 x 10 -11 Binding affinity (K D ) to join them.

[0085] 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) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; or (viii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6. and the second antigen-binding domain specifically binds to a second human TAA.

[0086] 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.

[0087] Previous work (Coloma and Morrison Nature Biotech. 15:159-163 (1997)) described engineered tetravalent bispecific antibodies by fusing DNA encoding a single-chain anti-dansyl antibody Fv (scFv) to the C-terminus (CH3-scFv) or hinge (hinge-scFv) of an anti-dansyl antibody IgG3. The present disclosure provides multivalent antibodies (e.g., tetravalent antibodies) having at least two antigen-binding domains, which can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibodies herein contain three to eight, preferably four, antigen-binding domains that specifically bind to at least two antigens.

[0088] In one embodiment, the multispecific antibody is a bispecific antibody. In another embodiment, the multispecific antibody further comprises an amino acid linker described herein, such as any one of SEQ ID NOs: 35 to 77.

[0089] III. Anti-human 4Ig-B7H3 antibody conjugated to a cytotoxin 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.

[0090] cytotoxin Cytotoxins or cytotoxic agents include any agent detrimental to cell growth, viability, or proliferation, including, but not limited to, tubulin-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-(2chloroethyl)-1,2-dimethanesulfonylhydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitin, aminopterin, anguidine, anthracyclines, and anthramycin. Antihistamines (AMC), auristatins, bleomycin, busulfan, butyric acid, calicheamicins (e.g., calicheamicin γ1), camptothecin, carminomycin, carmustine, cemadotin, cisplatin, colchicine, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, dacarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxyanthracene dione, disorazole, dolastatins (e.g., dora Statins 10), doxorubicin, duocarmycin, echinomycin, eleutherobin, emetine, epothilone, esperamicin, estramustine, ethidium bromide, etoposide, fluorouracil, geldanamycin, gramicidin D, glucocorticoids, irinotecan, kinesin spindle protein (KSP) inhibitors, leptomycin, leurocin, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercaptopurine, methopterin, methotrexate Examples of antihistamines include sartan, mithramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxin, procaine, propranolol, pteridine, puromycin, pyrrolobenzodiazepines (PBDs), rhizoxin, streptozotocin, tallysomycin, taxol, tenoposide, tetracaine, thioepachlorambucil, tomaymycin, topotecan, tubulysin, vinblastine, vincristine, vindesine, vinorelbine, and derivatives of any of the foregoing.

[0091] Cytotoxin Linker A cytotoxin linker, or linker for an ADC, is any group or moiety that links, connects, or binds an antibody or antigen-binding protein described herein to a therapeutic moiety, such as 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; Ducrry, 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.

[0092] Suitable binding agent or cytotoxin linkers for the antibody conjugates described herein are those that are stable enough to take advantage of the circulating half-life of the antibody while simultaneously being able to release their payload after antigen-mediated internalization of the conjugate. The linker may be cleavable or non-cleavable. Cleavable linkers include those that are cleaved by intracellular metabolism, such as hydrolysis, reduction, or enzymatic cleavage, after internalization. Non-cleavable linkers include those that release the attached payload after internalization by lysosomal degradation of the antibody. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically unstable linkers, enzymatically cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or include peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, malcaproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.

[0093] Any cytotoxin linker molecule or linker technology known in the art can be used to make or construct the ADC of the present disclosure.In certain embodiments, the cytotoxin 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, for example, linkers comprising or consisting of GGFC, MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide moiety in a protease-cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide moiety 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. Further examples of 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 the references cited therein, the contents of which are incorporated by reference in their entireties.

[0094] In certain embodiments, the cytotoxin linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, e.g., capable of releasing at least the payload portion in the presence of an enzyme or at a particular pH range or value. In some embodiments, the linker comprises an enzyme-cleavable moiety. Exemplary enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester bonds, hydrazones, and disulfide bonds. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0095] In some embodiments, the cytotoxin linker comprises a non-cleavable moiety.

[0096] Suitable cytotoxin linkers also include, but are not limited to, those chemically bonded to a single linking agent, such as two cysteine ​​residues of an antibody, which can serve to mimic the disulfide bonds of an antibody that are disrupted as a result of the conjugation process.

[0097] In some 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 some 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, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of the amino acids are attached to a side chain group, as described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the cytotoxin linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine.

[0098] IV. Human 4Ig-B7H3-targeting fusion protein Anti-human 4Ig-B7H3 antibodies can be used to fuse with other proteins or other functional domains to form fusion or chimeric proteins.

[0099] In some embodiments, the anti-human 4Ig-B7H3 antibody is fused to an immune checkpoint, immune stimulator, cytokine, or second TAA, either directly or indirectly via an amino acid linker described herein.

[0100] In one embodiment, the anti-human 4Ig-B7H3 antibody is fused to a functional domain or receptor subunit that may serve to transduce signals from the scFv and confer antibody specificity to immune cells such as T cells, NK cells, as well as other effector cells.

[0101] In some embodiments, the anti-human 4Ig-B7H3 antibody is used to construct a chimeric antigen receptor (CAR). More information on CAR construction can be found in Guedan et al., (2019) Mol. Ther. Methods Clin. Dev. 12:145-156.

[0102] Functional domains or receptor subunits include transmembrane domains, hinge regions, intracellular signaling domains, costimulatory domains, and the like.

[0103] V. Other Modifications Constant region and Fc region The heavy chain constant region can be the wild-type sequence of a heavy chain constant region derived from the IgG1, IgG2, IgG3, or IgG4 subclass. The light chain constant region can be the wild-type sequence of a light chain derived from a kappa or lambda subclass. In one embodiment, the heavy chain constant region is the wild-type sequence of a constant region derived from an IgG1 subclass. The light chain constant region is the wild-type sequence of a light chain derived from a kappa subclass. 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, containing the mutations E116P, L117A, L118A, G119del, and P212A. These amino acid mutations correspond to E233P, L234A, L235A, G236del, and P329A in the EU numbering system.

[0104] In one embodiment, the Fc region can be a wild-type Fc region of the subclass IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 or IgG4 Fc domain with reduced effector function.

[0105] In one embodiment, the antibody or antigen-binding fragment thereof comprises an Fc domain with extended half-life. In another embodiment, the antibody or antigen-binding fragment thereof comprises an IgG1 Fc domain into which YTE mutations (M252Y / S254T / T256E (EU numbering) as described in US7658921) located in CH2 of the IgG Fc region have been introduced.

[0106] In another embodiment, the antibodies of the disclosure have potent Fc-mediated effector function, wherein the antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells.

[0107] In yet another embodiment, the Fc region is altered by substituting 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 substituted with a different amino acid residue, resulting in an antibody with altered affinity for an effector ligand while retaining 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 approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0108] In another embodiment, one or more amino acid residues can be substituted 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 by Idusogie et al.

[0109] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues include, but are not limited to, the heavy chain constant regions of IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the kappa isotype, as described by Jefferis et al. (2009) MAbs.1:332-338.

[0110] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance 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. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., (2001) J. Biol. Chem. 276:6591-6604).

[0111] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody has no or reduced glycosylation). Altering the glycosylation can, for example, increase the affinity of the antibody for its antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can increase the affinity of the antibody for its antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0112] Additionally or alternatively, antibodies can be generated with altered types of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNAc structures). Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such glycosylation 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 for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked sugars, resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (see also Umana et al., (1999) Nat. Biotech. 17:176-180).

[0113] In another aspect, when reduced ADCC is desired, the human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al., (2010) Mabs. 2:181-189). However, native IgG4 has been found to be less stable under stress conditions, such as in acidic buffers or at elevated temperatures (Angal, S. (1993) Mol. Immunol. 30:105-108; Dall'Acqua, W. et al., (1998) Biochemistry. 37:9266-9273; Aalberse et al., (2002) Immunol. 105:9-19). Reduced ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form antibody halves, which generates bispecific antibodies in vivo through a process called "Fab arm exchange" (Van der Neut Kolfschoten M, et al., (2007) Science. 317:1554-157). Mutation of serine to proline at position 228 (EU numbering system) appears to inhibit IgG4 heavy chain separation (Angal, S. (1993) Mol. Immunol. 30:105-108; Aalberse et al., (2002) Immunol. 105:9-19).Some amino acid residues in the hinge and gamma Fc region have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., (1991) Proc. Natl. Acad. Sci. USA. 88:9036-9040; Mukherjee, J. et al., (1995) FASEB J. 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). Furthermore, some IgG4 isoforms that occur rarely in the human population may also result in different physicochemical properties (Brusco, A. et al., (1998) Eur. J. Immunogenet. 25:349-55; Aalberse et al., (2002) Immunol. 105:9-19). To generate multispecific antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc regions of human IgG4 and introduce several alterations. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83 to 88 of U.S. Pat. No. 8,735,553 by Li et al.

[0114] In another embodiment, an antibody of the disclosure comprises a human IgG4 Fc domain with S228P and / or R409K substitutions (according to the EU numbering system).

[0115] Amino Acid Linker It is understood that the presence or absence of an amino acid linker has minimal effect on the activity of an antibody or protein of the disclosure.

[0116] It is also understood that the domains and / or regions of the polypeptide chains of an antibody or protein can be separated by linker regions of various lengths. In some embodiments, antigen-binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3, or the entire Fc region by linker regions. For example, VL1-CL-(linker)VH2-CH1. Such linker regions can contain a random assortment of amino acids or a limited set of amino acids. Such linker regions can be flexible or rigid (see US 2009 / 0155275).

[0117] Multispecific antibodies have been synthesized using dimerization devices such as leucine zippers (Kostelny et al., (1992) J. Immunol. 148:1547-53; de Kruifetal J. (1996) Biol. Chem. 271:7630-4) and Ig C / CH1 domains (Muller et al., (1998) FEBS Lett. 422:259-64), with or without the use of flexible linkers; 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., (1996) Bio / Technology (NY). 14:192-6); al., (2000) J. Immunol. 165:7050-7); and miniantibody format (Packet et al., (1992) Biochemistry 31:1579-84; Packet et al., (1993) Bio / Technology. 11:1271-7), constructed by genetically fusing two single-chain Fv (scFv) or Fab fragments (Mallender et al., (1994) J. Biol. Chem. 269:199-206; Mallender et al., (1995) Proc. Natl. Acad. Sci. USA 92:7021-5; Zapata et al., (1995) Protein Eng. 8.1057-62).

[0118] 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 of their 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 included in the linker region.In another embodiment, the linker is selected from the group consisting of 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:43), GGGGSGS (SEQ ID NO:44), GGGGSGGS (SEQ ID NO:45), GGGGSGGGGS (SEQ ID NO:46), GGGSGGGGSGGGGS (SEQ ID NO:47), AKTTPKL EEGEFSEAR (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), RADAAAAAGGPGS (SEQ ID NO: 57), RADAAAA(G4S)4 (SEQ ID NO: 58). 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 ID NO: 68), AKTTPPSVTPLAP (SEQ ID NO: 69), AKTT AP (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 WO2007 / 024715).

[0119] Dimerization-specific amino acids In one embodiment, the multivalent antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change results in "knobs-into-holes" interactions, increasing the likelihood of correct multivalent antibody assembly. The dimerization-specific amino acid can be in the CH1 domain or the CL domain, or a combination thereof. Dimerization-specific amino acids are used to pair CH1 domains with other CH1 domains (CH1-CH1) and CL domains with other CL domains (CL-CL), and can be found in at least WO2014082179, WO2015181805 family, and WO2017059551. The dimerization-specific amino acid can be in the Fc domain or combined with a 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.

[0120] antibody production Antibodies and antigen-binding fragments thereof can be produced by any means 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 suitable host cell known in the art, such as a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc.

[0121] The present disclosure further provides polynucleotides encoding the antibodies or proteins described herein, e.g., polynucleotides encoding heavy chain variable regions or light chain variable regions comprising the complementarity determining regions described herein. In some embodiments, 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 to 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 embodiments, 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 to a polynucleotide selected from SEQ ID NO:10, SEQ ID NO:22, or SEQ ID NO:25.

[0122] The present disclosure also provides polynucleotides that encode the scFvs described herein, for example, polynucleotides that encode the amino acid sequence of SEQ ID NO:32.

[0123] The polynucleotides of the present disclosure can encode the variable region sequences of the multispecific antibodies described herein. They can also encode both the variable and constant regions of the multispecific antibodies. In another embodiment, the polynucleotides of the present disclosure can encode the amino acid sequences of the fusion proteins described herein.

[0124] In some embodiments, the polynucleotides described herein can be codon-optimized for expression in a host cell, e.g., a eukaryotic cell, more particularly a mammalian cell (e.g., a CHO cell).

[0125] The present disclosure also provides expression vectors and host cells for producing the antibodies herein. The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of antibodies or antigen-binding fragments. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be increased by incorporating enhancers appropriate for the cell system used (see, e.g., Scharf et al., (1994) Results Probl. Cell Differ. 20:125; Bittner et al., (1987) Meth. Enzymol., 153:516). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0126] Host cells for harboring and expressing antibody chains can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be made, typically containing expression control sequences compatible with the host cell (e.g., an origin of replication). Additionally, a variety of well-known promoters are available, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems derived from phage lambda. Promoters typically control expression (optionally with operator sequences) and contain ribosome binding site sequences for initiating and completing transcription and translation. Other microbes, such as yeast, can also be used to express antibodies. Insect cells can also be used in conjunction with baculovirus vectors. In another embodiment, 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 harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is reviewed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences such as an origin of replication, a promoter, an enhancer (see, for example, Queen et al., (1986) Immunol. Rev. 89:49-68), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences.These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or regulatable. Useful promoters include the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0127] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for detecting 4Ig-B7H3. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of 4Ig-B7H3 in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In other embodiments, such tissues include normal and / or cancerous tissues that express 4Ig-B7H3 at higher levels than other tissues.

[0128] In one aspect, the present disclosure provides a method for detecting the presence of 4Ig-B7H3 in a biological sample. In certain aspects, the method includes contacting the biological sample with an anti-4Ig-B7H3 antibody under conditions that allow binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample can include, but is not limited to, a urine, tissue, sputum, or blood sample.

[0129] Also included are methods for diagnosing disorders associated with 4Ig-B7H3 expression. In certain embodiments, the methods include contacting test cells with an anti-4Ig-B7H3 antibody; determining (quantitatively or qualitatively) the expression level of 4Ig-B7H3 expressed by the test cells by detecting binding of the anti-4Ig-B7H3 antibody to the 4Ig-B7H3 polypeptide; and comparing the expression level by the test cells with the 4Ig-B7H3 expression level in control cells (e.g., normal cells of the same tissue origin as the test cells or non-4Ig-B7H3-expressing cells), wherein a higher level of 4Ig-B7H3 expression in the test cells compared to the control cells indicates the presence of a disorder associated with 4Ig-B7H3 expression.

[0130] Treatment method The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for treating 4Ig-B7H3-associated disorders or diseases. In one aspect, the 4Ig-B7H3-associated disorder or disease is cancer. In some embodiments, the cancer is 4Ig-B7H3-positive.

[0131] In one aspect, the present disclosure provides a method of treating cancer. In certain aspects, the method comprises administering an effective amount of an anti-4Ig-B7H3 antibody or antigen-binding fragment, or a 4Ig-B7H3 antibody-containing multispecific antibody to a patient in need thereof. In another aspect, the present disclosure provides an anti-4Ig-B7H3 antibody or antigen-binding fragment, or multispecific antibody, or pharmaceutical composition for use in treating cancer. In another aspect, the present disclosure provides use of an anti-4Ig-B7H3 antibody or antigen-binding fragment, a multispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0132] Cancers include, but are not limited to, colon cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, renal cancer, lung cancer, or esophageal carcinoma. In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In another embodiment, the non-small cell lung cancer is squamous non-small cell lung cancer. In another embodiment, the esophageal cancer is esophageal squamous cell carcinoma.

[0133] The antibodies or antigen-binding fragments disclosed herein can be administered by any suitable means, for example, parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration if localized treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0134] The antibodies or antigen-binding fragments of the present disclosure can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical professionals. The antibodies are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above.

[0135] For the prevention or treatment of disease, the appropriate dosage of an antibody or antigen-binding fragment of the disclosure will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0136] Combination therapy In one embodiment, the anti-4Ig-B7H3 antibodies of the present disclosure, or multispecific antibodies comprising anti-4Ig-B7H3 antibodies, can be used in combination with other therapeutic agents. Other therapeutic agents that can be used in combination with the 4Ig-B7H3 antibodies of the present disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel formulations; (e.g., Abraxane®), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, cephalosporin ... cefoside, 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, oncolytics, fatumumab, RO5072759, LFB-R603), CD52-targeted agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19-targeted agents (e.g., MEDI-551, MOR2 08), 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).

[0137] The anti-4Ig-B7H3 antibody of the present disclosure can be used in combination with other therapeutic agents, such as other immune checkpoint antibodies. Such immune checkpoint antibodies may include anti-PD1 antibodies. Anti-PD-1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab, or nivolumab. Tislelizumab (SEQ ID NOs: 22 and 23 in Table 7) is disclosed in US Pat. No. 8,735,553. Pembrolizumab (formerly known as MK-3475), disclosed in US Pat. Nos. 8,354,509 and 8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab is approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is undergoing clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. US8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma.

[0138] In one embodiment, the present disclosure provides the use of an anti-4Ig-B7H3 antibody or a multispecific antibody containing an anti-4Ig-B7H3 antibody in combination with an anti-PD-1 antibody (such as tislelizumab or another anti-PD-1 antibody described above) in the manufacture of a medicament for the treatment of cancer, such as the cancers described above. In another embodiment, the present disclosure provides a combination of an anti-4Ig-B7H3 antibody or a multispecific antibody containing an anti-4Ig-B7H3 antibody with an anti-PD-1 antibody (such as tislelizumab or another anti-PD-1 antibody described above) for use in the treatment of cancer, such as the cancers described above.

[0139] Combination therapy is intended to mean, refer to, and include any one of the following: the simultaneous administration of such combination therapy to a patient in need of treatment when such components are formulated together into a single dosage form that releases said components to the patient substantially simultaneously; the substantially simultaneous administration of such a combination to said patient in need of treatment, where such components are formulated separately from one another in separate dosage forms that are taken by said patient at substantially the same time and whereby said components are released to the patient at substantially the same time; Sequential administration of such combination therapy to said patient in need of treatment, where such components are formulated in separate dosage forms separate from one another and taken by said patient sequentially with a significant time interval between each administration, such that the components are released to the patient at substantially different times; and Sequential administration of such a combination to a patient in need of treatment, where such components are formulated together into a single dosage form that releases such components in a controlled manner, and releases them simultaneously, sequentially, and / or overlappingly to the patient at simultaneous and / or different times, and each portion is administered by the same or different route.

[0140] Pharmaceutical Composition Also provided are compositions, such as pharmaceutical preparations, comprising anti-4Ig-B7H3 antibodies or antigen-binding fragments thereof (including multispecific antibodies), or polynucleotides comprising sequences encoding the antibodies or antigen-binding fragments. In certain embodiments, the compositions comprise one or more anti-4Ig-B7H3 antibodies or antigen-binding fragments, or one or more polynucleotides comprising sequences encoding one or more anti-4Ig-B7H3 antibodies or antigen-binding fragments. These compositions can further comprise suitable carriers, e.g., pharmaceutically acceptable excipients such as buffers, which are well known in the art.

[0141] Pharmaceutical formulations of the anti-4Ig-B7H3 antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions 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)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., 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 dextrins; 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0142] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0143] 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, e.g., films, or microcapsules.

[0144] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0145] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art.

[0146] As used in this specification, including the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0147] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0148] As used herein, the term "anti-cancer agent" refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0149] 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.

[0150] As used herein, the terms "administration," "administering," "treating," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, or diagnostic agent or composition with such animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell and, when a fluid is in contact with the cell, contact of a reagent with a fluid. The terms "administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., of a cell, with a reagent, diagnostic agent, binding compound, or with another cell. The term "subject" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., a rat, mouse, dog, cat, rabbit), and most preferably a human. In one aspect, treating any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0151] The term "subject" in the context of this disclosure is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein).

[0152] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen through non-covalent forces at multiple sites. Generally, the more interactions, the stronger the affinity.

[0153] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing 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 consists 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 consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability 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 the amino terminus to the 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 can 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 (C1q) of the classical complement system.

[0154] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies, human recombinant antibodies, single-chain antibodies (scFv), single-domain antibodies, Fab fragments, Fab' fragments, or F(ab')2 fragments. Antibodies can be of 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 (ADCs). In addition, antibodies include derivatives thereof by directly or indirectly binding to another agent (another drug or antibody) or by forming a complex with another agent.

[0155] The term "chimeric antibody" refers to molecules created with domains from different species, i.e., by fusing the variable domains of an antibody from one host species (e.g., mouse, rabbit, llama, etc.) with fused constant domains from another species (e.g., human).

[0156] In some embodiments, anti-4Ig-B7H3 antibodies comprise at least one antigen-binding site, at least the variable region. In some embodiments, anti-4Ig-B7H3 antibodies comprise antigen-binding fragments derived from the 4Ig-B7H3 antibodies described herein. In some embodiments, anti-4Ig-B7H3 antibodies are isolated or recombinant. In some embodiments, anti-4Ig-B7H3 antibodies also include multispecific antibodies that target 4Ig-B7H3 as at least one arm and other antigen(s) as another arm(s).

[0157] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies with different amino acid sequences within the variable domains, particularly the complementarity-determining regions (CDRs), which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those of skill in the art. See, for example, Kohler et al., (1975) Nature. 256:495-497; U.S. Patent 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 be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography methods well known to those skilled in the art.

[0158] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Human heavy chains are further classified as α, δ, ε, γ, or μ, and antibody isotypes are defined 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 approximately 12 or more amino acids, and heavy chains also contain a "D" region of approximately 10 amino acids.

[0159] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0160] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both 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 locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (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 (see "IMGT" numbering scheme).The definition of antigen-binding sites is also described in: Ruiz et al., (2000) Nucleic Acids Res. 28:219-221; and Lefranc, MP, (2001) Nucleic Acids Res. 29:207-209; MacCallum et al., (1996) J. Mol. Biol. 262:732-745; and Martin et al., (1989) Proc. Natl. Acad. Sci. USA, 86:9268-9272; Martin et al., (1991) Methods Enzymol. 203:121-153; and Rees et al., in Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in 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). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, 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.In IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0161] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region comprises amino acid residues from a "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, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., which define antibody CDR regions by sequence. See also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, which define antibody CDR regions by structure. The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0162] Unless otherwise indicated, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, 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'), and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules, e.g., single-chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

[0163] As used herein, an antibody "specifically binds" to a target protein means that the antibody exhibits preferential binding to that target relative to other proteins, although this specificity does not require absolute binding specificity. The terms "specifically bind" or "selectively bind" an antibody are used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, and refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biologics, such as a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one aspect, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0164] As used herein, an "antigen-binding domain" comprises at least six CDRs and specifically binds to an epitope (or, in the context of a single-domain antibody, three CDRs). 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. Multispecific antibodies can be bispecific, trispecific, tetraspecific, etc., with an antigen-binding domain directed to each specific epitope. Multispecific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) comprising multiple antigen-binding domains, for example, two, three, four, or more antigen-binding domains that specifically bind to a first epitope and two, three, four, or more antigen-binding domains that specifically bind to a second epitope.

[0165] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0166] The terms "humanized" or "humanized antibody" refer to forms of antibodies that contain sequences from non-human (e.g., mouse, rabbit, llama, etc.) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). Where necessary to distinguish a humanized antibody from its rodent parent antibody, the name of the antibody clone will be prefixed with "hum," "hu," "Hu," or "h." Humanized forms of rodent / camelid antibodies generally contain the same CDR sequences of the rodent parent antibody, but can contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0167] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with 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. 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 with 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 framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that comprise variable regions. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The 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 the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., (2000) J. Mol. Biol. 296:57-86.

[0168] “Equilibrium dissociation constant (K D The term "dissociation rate constant (kd, time -1 ) to the association rate constant (ka, time -1 , M -l ) The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8 Less than m, e.g., about 10-9 Less than M or 10 -10 M or less, and in some embodiments, about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0169] The terms "cancer" or "tumor" as used herein have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. In the context of this disclosure, cancer is not limited to any particular type or location.

[0170] In the context of this disclosure, the term "conservative substitution," when referring to an amino acid sequence, means replacing an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to 4Ig-B7H3. In particular, common conservative changes of amino acids are well known in the art.

[0171] As used herein, the term "knob-into-hole" technology refers to amino acids that direct pairing of two polypeptides either in vitro or in vivo by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide at the interface where they interact. For example, knob-into-hole technology can be used to direct pairing of two polypeptides, either in vitro or in vivo, ... L :C H Interface, or V H / V LIn some embodiments, knob-into-hole amino acids have been introduced into the VH or VL region to ensure proper pairing of two different heavy chains together during the production of multispecific antibodies. For example, multispecific antibodies with knob-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. Knob-into-hole technology can also be used with VH or VL regions to ensure proper pairing.

[0172] As used herein, the term "knob," in the context of "knob-into-hole" technology, refers to an amino acid change that introduces a knob into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0173] As used herein, the term "hole," in the context of "knob-into-hole," refers to an amino acid change that introduces a socket or hole in a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0174] An example of an algorithm suitable for determining percent 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 analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as initiating points for searches to find longer HSPs containing them. Word hits are extended outward along both ends of each sequence for as long as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value; if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and both strands are compared.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0175] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, 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 sequences of nucleotides or amino acids will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with 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.

[0176] 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 a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, 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 a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0177] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0178] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous to or located in close proximity to the coding sequence whose transcription they enhance.

[0179] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-4Ig-B7H3 multispecific antibody 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 and absorption delaying agents, and the like that are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0180] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Suitable forms depend on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable and infusible solutions. One suitable mode of administration is parenteral administration (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.

[0181] As used herein, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of ordinary skill in the art and can also be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined agents to effectively treat the disease, disorder, or condition.

[0182] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in this disclosure. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes co-administration in multiple containers or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration also includes the use of various therapeutic agents in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effects of the drug combination in treating the condition or disorder described herein.

[0183] As used herein, the term "in combination with" means that the anti-4Ig-B7H3 antibody is administered to a subject simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent. In certain embodiments, the anti-4Ig-B7H3 antibody is administered as a combination with an additional therapeutic agent.

[0184] equivalent While the present invention has been described with reference to its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0185] It is 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 be apparent to those skilled in the art. [Example]

[0186] Example 1. Generation of mouse anti-B7H3 monoclonal antibodies immunization To generate antibodies against B7H3, a cohort of five transgenic mice engineered to produce human variant and rat constant regions was immunized with the extracellular binding domain (ECD) of human 4Ig-B7H3 antigen. Each cohort received a unique immunization regimen, including a unique combination of human B7H3 antigen, dose, injection route, and adjuvant. A total of five animals were immunized per cohort. Animals were immunized over various time periods, ranging from 0 to 100 days. To monitor the immune response, titrated sera were screened by ELISA, usually after two to six immunizations over a period of 30 to 100 days. Sera were screened for antibody (Ab) binding to the B7H3 antigen. B7H3-specific antibody responses were measured in each animal, and animals with sufficient anti-B7H3 titers were selected for a final boost four days later.

[0187] Hybridoma fusion and screening Lymphoid organs, including spleens and lymph nodes, were isolated from immunized mice as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells derived from SP2 / 0 using PEG-based fusion. For hybridoma selection, the resulting cells were plated in 96-well cell culture plates using standard 1640 medium (Gibco™) supplemented with HAT. After 10–13 days of culture and growth medium changes, hybridoma culture supernatants were collected from individual wells and screened to identify wells that secreted B7H3-specific antibodies. All supernatants were initially screened against human ECD-4Ig-B7H3-hFc. Antibodies binding to human ECD-4Ig-B7H3-hFc were measured by ELISA. Supernatants from five hybridoma fusions were screened for B7H3 antibodies. Briefly, 2 μg / mL of human ECD-4Ig-B7H3-hFc was coated in a 96-well ELISA plate, and 50 μl of hybridoma culture supernatant was co-incubated with human ECD-4Ig-B7H3-hFc for 30–60 min. This was then washed and incubated with an anti-rat IgG Fc secondary Ab conjugated to HRP. After incubation and washing, the plate was developed with HRP substrate, and absorbance was measured.

[0188] Hybridomas from positive wells were transferred to 24-well plates in fresh culture medium, grown for 2–3 days, and then screened again by flow cytometry to confirm antibodies binding to cell lines overexpressing human 4Ig-B7H3 and cynomolgus monkey B7H3.

[0189] Antibody binding to cells overexpressing human B7H3 and cells overexpressing cynomolgus B7H3 was measured by FACS. Briefly, 100 μl of hybridoma culture supernatant and cells overexpressing human 4Ig-B7H3 or cynomolgus B7H3 were co-incubated for 30–60 min, washed, and incubated with an anti-IgG Fc secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

[0190] Subcloning and sequence analysis Selected anti-B7H3 Ab screening 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 described above to confirm human and cynomolgus monkey B7H3 antigen binding. Stable hybridoma subclones were cultured in vitro, and the cells were cryopreserved for gene cloning and sequencing of antibody VH and VL.

[0191] After subcloning, the anti-B7H3 Ab-secreting hybridomas were lysed with cell lysis buffer, and the mRNA-containing lysates were then transferred to a 96-well plate for mRNA isolation, cDNA synthesis, and DNA sequencing using SuperScript III First-Strand Synthesis SuperMix™ (Invitrogen) according to the manufacturer's instructions.

[0192] The sequence of the resulting human antibody BGA-3726 is listed in Table 7.

[0193] Example 2. Measurement of binding kinetics and affinity of anti-B7H3 antibodies by SPR Anti-B7H3 mAb BGA-3726 was purified and its binding kinetics was characterized by SPR assay using a BIAcore™ T-200 (GE Life Sciences). Briefly, anti-mouse IgG Fc antibody was immobilized on an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences). The 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 surface plasmon resonance signal were analyzed. Using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences), the association rate (kon ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity profiles of the anti-B7H3 mAbs are shown in Table 1 and Figure 1. [Table 1]

[0194] Example 3. Determination of binding affinity of anti-B7H3 antibodies to B7H3 expressed in stable cell lines The binding affinity of anti-B7H3 mAbs to cell lines overexpressing human 4Ig-B7H3 and cynomolgus B7H3 was measured by FACS. Briefly, cells overexpressing human or cynomolgus B7H3 were incubated with serially diluted purified antibodies, washed, and incubated with an 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 Figures 2A-B. [Table 2] [Table 3]

[0195] Example 4. Measurement of non-specific binding of anti-B7H3 antibody The nonspecific binding activity of anti-B7H3 mAb BGA-3276 was measured by FACS. Briefly, Daudi cells, which 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 exhibit any nonspecific binding to B7H3-negative cells. [Table 4]

[0196] Example 5. Recombination of anti-human B7H3 mAb BGA-3726 BGA-3726 (SEQ ID NOs: 1-10) is a human antibody containing fully human Vh and Vk regions. BGA-3726 was re-engineered to remove potential post-translational modifications (PTMs) and to optimize biophysical stability for therapeutic use in humans.

[0197] BGA-3726 and antibodies recombinant from BGA-3726 were constructed in a full-length human antibody format using an in-house developed expression vector containing the constant regions of a human IgG1 variant (SEQ ID NO: 31) and kappa chain, respectively, with adaptable subcloning sites. Expression and preparation of BGA-3726 and its recombinant antibodies were achieved by cotransfection of the heavy chain construct and the corresponding light chain construct into 293G cells (developed in-house) and purification using a Protein A column. The purified antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C in a freezer.

[0198] Based on the original clone of BGA-3726, several single mutations were made within the CDRs to remove potential post-translational modification (PTM) sites. Potential PTM sites included potential oxidation sites W34, W96, and W98 (Kabat numbering) in the HCDR and a potential deamidation site N29 (NG) in LCDR1. All mutations were made using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog number FM111-02, TransGen, Beijing, China). The desired mutations were confirmed by sequencing analysis. These BGA-3726 variant antibodies, BGA-4826 (SEQ ID NOs: 11, 2-6, 12, 8, 13, 10) containing W34Y in HCDR1, BGA-4498 (SEQ ID NOs: 1, 2, 14, 4-6, 15, 8, 16, 10) containing W96Y in HCDR3, BGA-4269 (SEQ ID NOs: 1, 2, 17, 4-6, 18, 8, 19, 10) containing W98Y in HCDR3, BGA-4380 (SEQ ID NOs: 1-3, 20, 5-6, 7, 21, 9, 22) containing N29Q in LCDR1, and BGA-4265 (SEQ ID NOs: 1-3, 23, 5-6, 7, 24, 9, 25) containing G30A in LCDR1, were tested in SPR binding assays.

[0199] In summary, recombinant versions of the humanized monoclonal antibodies BGA-6938 (SEQ ID NOs: 11, 2, 14, 23, 5-6, 26, 24, 27, 25), which are based on BGA-3726 and contain W34Y in HCDR1, W96Y in HCDR3, and G30A in LCDR1, and BGA-5488 (SEQ ID NOs: 11, 2, 28, 23, 5-6, 29, 24, 30, 25), which are based on BGA-3726 and contain W34Y in HCDR1, W96Y in HCDR3, W98Y in HCDR3, and G30A in LCDR1, were derived from the above-mentioned mutation process and characterized in detail. The results showed that BGA-3726, BGA-6938, and BGA-5488 have comparable binding affinities to recombinant B7H3.

[0200] For affinity measurements, antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The binding profiles of anti-B7H3 antibodies to the ECD of human 4Ig-B7H3 (Sinobiological, catalog: 11188-H08H) measured by SPR are summarized in Table 5. BGA-6938 (SEQ ID NOs: 11, 2, 14, 23, 5-6, 26, 24, 27, 25) and BGA-5488 (SEQ ID NOs: 11, 2, 28, 23, 5-6, 29, 24, 30, 25) have comparable binding affinities with dissociation constants of 0.443 nM and 1.11 nM, respectively, which are comparable to the binding affinity of BGA-3726. [Table 5]

[0201] To assess the binding activity of BGA-6938 and BGA-5488 to native B7H3 in live cells, NK92mi cells were engineered to overexpress human 4Ig-B7H3. Live NK92mi / B7H3 cells were seeded into 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. EC2 values ​​for dose-dependent binding to human native B7H3 were calculated. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figures 3A-3B and Table 6, recombinant antibodies BGA-6938 and BGA-5488 retained the binding affinity of the parent clone BGA-3726 for native B7H3. [Table 6] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10]

[0202] Example 6. Cell binding and internalization of humanized Abs join Binding assays were performed to evaluate antibody affinity. Wild-type IgG1 / kappa format BGA-6938, MABX-9001a (a humanized anti-B7H3 IgG1 monoclonal antibody used against 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 removed from flasks using trypsin-EDTA (catalog no. 25200056, ThermoFisher) and washed with binding buffer (PBS containing 2% FBS, pH 7.2). The washed cells were added to 96-well plates containing antibodies at a density of approximately 100,000 cells in 0.2 mL of binding buffer. The final concentration of primary antibody in the competition reaction with cells was varied, starting at 500 nM and then decreasing at 1:5 dilutions over 11 concentrations. The mixture of cells and antibodies was incubated on ice for 1 hour. After 1 hour of incubation, the cells were washed twice with binding buffer to separate the free antibody. The washed cells were further labeled with fluorescently labeled anti-human Fc (Cat. No.: 109-605-190, Jackson ImmunoResearch) and incubated on ice for 1 hour. After 1 hour of incubation, the cells were washed twice with binding buffer to separate the free antibody. Fluorescent signals were detected using an LSR Fortessa (BD). The binding curves for BGA-6938 and MABX-9001a, as well as human IgG antibodies, at H358 are shown in Figure 4. Based on the binding results, BGA-6938 showed higher binding affinity for B7H3 compared to MABX-9001a.

[0203] internalization One of the desirable attributes of antibodies for conjugation to ADCs is their ability to be internalized into cells. The internalization activity of BGA-6938 and MABX-9001a was measured using the pHrodo™ assay (catalog number: Z25612, ThermoFisher) according to the manufacturer's protocol. Primary antibodies were labeled with Zenon™ pHrodo™ iFL IgG Labeling Reagent (catalog number: Z25612, ThermoFisher) and RPMI medium. 100,000 H358 cells were suspended and incubated with antibody-pHrodo in 0.2 mL at 37°C. After incubation for 1, 4, 8, and 24 hours, the cells were washed twice with binding buffer (PBS containing 2% FBS, pH 7.2) to separate free antibody. Fluorescence signals were detected using an LSRFortessa™ (BD) with a PE channel. The fluorescence signal intensity represents the internalization activity. The curves for BGA-6938, MABX-9001a, and the human IgG antibody at H358 are shown in Figure 5. Based on the internalization results, BGA-6938 showed increased internalization activity compared to MABX-9001a.

[0204] Example 7. Cell killing activity To investigate 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 100 μl of RPM1-1640 + 10% FBS medium in a 96-well 3D cell culture plate (Cat. No. 6055330, Perkin Elmer). After 24 hours, an additional 50 μl of medium and serial dilutions of BGA-6938-GGFG-DXd (DAR8) and MAbX-9001a-GGFG-DXd (DAR8 or DAR4) were added to triplicate wells. For the structure of MABX-9001a-GGFG-DXd, see Figure 2A in Yamato et al., Mol Cancer Ther 2022 1;21(4):635-646. After 6 days, cell viability was measured using CellTiter-Glo Luminescent Cell Viability Reagent (G7572; Promega Corporation) and a Tecan Spark. Killing curves were analyzed using GraphPad Prism 9 and are shown in Figure 6. BGA-6938-GGFG-DXd(DAR8) showed comparable killing activity in H1650 and H441, and increased killing activity in H1048, compared to MABX-9001a-GGFG-DXd(DAR8).

[0205] Example 8. Binding of B7H3 scFv protein in the H358 tumor cell line To assess the binding activity of the scFv of BGA-6938 on the surface of live H358 tumor cells, the scFv protein (see Table 8) was purified by affinity chromatography followed by size exclusion chromatography. For the assay, 2 x 10 5H358 cells were seeded in a 96-well plate. 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). The MFI of each cell population was measured using the Satorius iQue3™ system. The EC50 and MFImax of each scFv were determined using a four-parameter logistic model. The results are shown in Table 9 and Figure 7. The BGA-6938 scFv showed an EC50 of 2.047 nM against the B7H3 antigen in H358 tumor cells. [Table 8] [Table 9]

[0206] Example 9. Epitope mapping of anti-B7H3 antibodies To investigate the binding epitope of the anti-B7H3 mAb BGA-6938, we generated domain-truncated human B7H3 by fusing each Ig-like domain 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), to its transmembrane and intracellular domains. The truncated versions of B7H3 were also fused to an N-terminal FLAG tag. The resulting truncated human B7H3 construct, shown in Figure 8A, contains an N-terminal FLAG tag followed by the Ig-like domains, including the transmembrane and intracellular domains, and the B7H3 C-terminal domain. DNA encoding the truncated versions of B7H3 was cloned into the pcDNA3.4 vector.

[0207] Plasmids containing these truncated B7H3 constructs were used to transfect ExpiCHO™ cells for transient protein expression, followed by incubation with 100 nM purified BGA-6938 and the reference antibody DS-7300 (Daiichi, US2022 / 0064312A1). Binding of BGA-6938 and DS-7300 to the various B7H3 truncated forms was assessed by detection with Alexa Fluor 647 rabbit anti-human IgG (catalog: 309-605-008, Jackson ImmunoResearch). Expression of each construct was verified by detection of the FLAG tag at the N-terminus after incubation of transfected cells with anti-FLAG mAb (catalog: A01809, Genscript). The data in Figure 8B show that BGA-6938 specifically binds to the B7H3 V1 and V2 domains, but not to the B7H3 C1 and C2 domains. In contrast, the reference antibody DS-7300 binds to the C1 and C2 domains of B7H3, but not to the V1 and V2 domains (Figure 8B). 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, (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) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; or (viii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO:

6. The antibody or antigen-binding fragment thereof, comprising:

2. (i) the heavy chain variable region comprises an amino acid sequence 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24; The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof described in claim 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids 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 have been inserted, deleted, or substituted.

4. (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. An antibody or antigen-binding fragment thereof according to any one of the preceding claims.

5. 10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, which is a monoclonal antibody, a human recombinant 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 of claim 5, wherein the antibody or antigen-binding fragment thereof comprises an scFv comprising a VH having the amino acid sequence of SEQ ID NO: 26 and a VL having the amino acid sequence of SEQ ID NO: 24, and optionally the VH and VL are connected via an amino acid linker, and optionally the amino acid linker is any sequence from SEQ ID NO: 35 to SEQ ID NO:

77.

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

77.

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

32.

9. 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 according to any one of claims 1 to 8; at least a second antigen-binding domain that specifically binds to a second human tumor-associated antigen (TAA); 1. A multispecific antibody or antigen-binding fragment thereof comprising:

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 to 10, further comprising an amino acid linker, wherein the amino acid linker is any of the sequences of SEQ ID NOs: 35 to 77.

12. 10. The antibody or antigen-binding fragment thereof of 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. 10. The antibody or antigen-binding fragment thereof of 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 subclass.

14. 10. The antibody or antigen-binding fragment thereof of 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. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is reduced or aglycosylated or hypofucosylated.

16. 10. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises increased bisected GlcNac structures.

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

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

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

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

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

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

23. The method of any one of claims 21 to 22, wherein the cancer is colon cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, renal cancer, lung cancer, or esophageal carcinoma.

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

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

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

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

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

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

31. 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 of any one of claims 1 to 19.

33. A vector comprising the nucleic acid of claim 32.

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

35. 35. A process for producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of 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, (1) the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29 to 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 to 357 of human 4Ig-B7H3 (SEQ ID NO: 80). The antibody or antigen-binding fragment thereof.

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