Anti-B7H3 antibody and method of use

Abstract

This invention provides an antibody or its antigen-binding fragment that binds to human 4Ig-B7H3.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of PCT Application No. PCT / CN2023 / 082099, entitled "Anti - B7H3 Antibodies and Methods of Use", filed on March 17, 2023, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to antibodies and antigen - binding fragments that specifically bind to human 4Ig - B7H3.

Background Art

[0003] B7H3 or B7 - H3 is a type I transmembrane protein identified from a dendritic cell (DC) cDNA library in 2001 (Chapoval, Ni et al. 2001). It is also called CD276, B7RP - 2, etc. As part of the B7 immunoregulatory family, B7H3 contains two isoforms, 4Ig - B7H3 and 2Ig - B7H3, in humans, and 4Ig - B7H3 is the major isoform expressed in malignant cells (Steinberger, Majdic et al. 2004). The extracellular domain of mouse B7H3 is composed of a pair of immunoglobulin variable (IgV) - like domains and an immunoglobulin constant (IgC) - like domain, while human B7H3 contains one or two identical pairs due to exon duplication (Sun, Richards et al. 2002, Steinberger, Majdic et al. 2004). The intracellular domain of B7H3 is short and has no known signaling motifs (Picarda, Ohaegbulam et al. 2016). In addition to the transmembrane form, B7H3 can exist in a soluble form, which is cleaved from membrane B7H3 by proteases or caused by alternative splicing (Zhang, Hou et al. 2008).

[0004] B7H3, belonging to the B7 protein family, shares 20-27% amino acid identity with other B7 family ligands, but its receptor(s) remain unclear (Chapoval, Ni et al. 2001). To date, the molecular mechanisms by which B7H3 is involved in immune evasion remain unexplained, and its function in T cell-mediated adaptive immunity is still controversial. It was initially identified as a T cell costimulatory molecule (Chapoval, Ni et al. 2001, Zhang, Chen et al. 2004), but later studies have suggested that it primarily plays a role in immunosuppression (Suh, Gajewska et al. 2003, Prasad, Nguyen et al. 2004, Fukushima, Sumi et al. 2007, Veenstra, Flynn et al. 2015). On the other hand, crystal structure analysis of mouse B7H3 has revealed that the FG loop is important for inhibiting mB7H3-mediated T cell proliferation (Vigdorovich, Ramagopal et al. 2013). In particular, non-small cell lung cancer (NSCLC) with high B7H3 expression is associated with only a small number of tumor-infiltrating lymphocytes and exhibits high resistance to anti-PD1 therapy, suggesting a role of B7H3 in immune evasion. Therefore, B7H3-targeted therapy combined with anti-PD-1 / PD-L1 antibody therapy is a promising approach for NSCLC expressing B7H3 (Altan, Pelekanou et al. 2017).

[0005] In addition to immune regulation, B7H3 also possesses unique tumor-promoting functions. TCGA analysis revealed that B7H3 expression correlates with the EGFR / PI3K / AKT pathway, MAPK pathway, and EMT process across all cancer types, and that abnormal B7H3 expression promotes tumor metastasis, angiogenesis, glycolysis, and drug resistance (Tekle, Nygren et al. 2012, Liu, Zhang et al. 2015, Lee, Martin-Orozco et al. 2017, Flem-Karlsen, Fodstad et al. 2018, Liu, Zhang et al. 2019, Lai, Sun et al. 2020). Therefore, B7H3 overexpression is associated with poor prognosis and poor clinical outcomes in many cancer types (Crispen, Sheinin et al. 2008, Bachawal, Jensen et al. 2015, Fan, Zhu et al. 2016, Song, Shi et al. 2016, Wu, Zhao et al. 2016, Benzon, Zhao et al. 2017).

[0006] Numerous studies have reported that B7H3 is highly overexpressed in a wide range of solid tumors and tumor vascular systems, but its expression is low and limited in normal tissues (Loo, Alderson et al. 2012, Seaman, Zhu et al. 2017, Du, Hirabayashi et al. 2019, Yamato, Hasegawa et al. 2022), making it an attractive target for cancer treatment. The majority of assets targeting B7H3 are in the early stages of development. Y-Mabs is developing omburtamabs with radionuclides including I131 or Lu177, with its primary asset being I131-omburtamab, which targets brain tumors. Enoblituzumab is a Phase I / II Fc-enhanced B7H3 antibody, but when combined with an anti-PD1 antibody, it showed only limited antitumor efficacy in anti-PD1 / PD-L1 resistant patients. B7H3 ADCs, including MGC018 from MacroGenics and DS-7300 from Daiichi Sankyo, demonstrated preliminary antitumor activity and a favorable safety profile with good tolerability in a Phase I trial.

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

[0008] This disclosure relates to an antibody that specifically binds to human 4Ig-B7H3 and its antigen-binding fragment.

[0009] In embodiments, this disclosure relates to an antibody or its antigen-binding fragment that specifically binds to human 4Ig-B7H3, comprising: (1) A heavy chain variable region (VH) including (a) HCDR1 (heavy chain complementarity determination region 1) of SEQ ID NO: 7, (b) HCDR2 of SEQ ID NO: 8, and (c) HCDR3 of SEQ ID NO: 9, or (2) A heavy chain variable region including (a) HCDR1 of SEQ ID NO: 12, (b) HCDR2 of SEQ ID NO: 13, and (c) HCDR3 of SEQ ID NO: 14.

[0010] In the embodiment, the antibody or its antigen-binding fragment includes the following: (1) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 10, (2) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 20, (3) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 24, (4) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 26, (5) Heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 30 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, (6) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 32, (7) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 34, (8) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 36, (9) Heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 40 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, (10) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 42, (11) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 44, (12) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 46, (13) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 48, (14) Heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 50 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, (15) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 15, (16) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 52, (17) A heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 54 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, or (18) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 56.

[0011] In the embodiment, the antibody or its antigen-binding fragment has one, two, three, four, five, six, seven, eight, nine, or ten amino acids inserted, deleted, or substituted in at least one of the following: SEQ ID NOs: 10, SEQ ID NOs: 20, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 30, SEQ ID NOs: 32, SEQ ID NOs: 34, SEQ ID NOs: 36, SEQ ID NOs: 40, SEQ ID NOs: 42, SEQ ID NOs: 44, SEQ ID NOs: 46, SEQ ID NOs: 48, SEQ ID NOs: 50, SEQ ID NOs: 15, SEQ ID NOs: 52, SEQ ID NOs: 54, and SEQ ID NOs: 56.

[0012] In the embodiment, the antibody or its antigen-binding fragment includes the following: (1) Heavy chain variable region including sequence number 10, (2) Heavy chain variable region including sequence number 20, (3) Heavy chain variable region including sequence number 24, (4) The heavy chain variable region containing SEQ ID NO: 26, (5) The heavy chain variable region containing SEQ ID NO: 30, (6) The heavy chain variable region containing SEQ ID NO: 32, (7) The heavy chain variable region containing SEQ ID NO: 34, (8) The heavy chain variable region containing SEQ ID NO: 36, (9) The heavy chain variable region containing SEQ ID NO: 40, (10) The heavy chain variable region containing SEQ ID NO: 42, (11) The heavy chain variable region containing SEQ ID NO: 44, (12) The heavy chain variable region containing SEQ ID NO: 46, (13) The heavy chain variable region containing SEQ ID NO: 48, (14) The heavy chain variable region containing SEQ ID NO: 50, (15) The heavy chain variable region containing SEQ ID NO: 15, (16) The heavy chain variable region containing SEQ ID NO: 52, (17) The heavy chain variable region containing SEQ ID NO: 54, or (18) The heavy chain variable region containing SEQ ID NO: 56.

[0013] In an embodiment, the antibody or its antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a heavy-chain antibody (HcAb), or a VHH.

[0014] In an embodiment, the multispecific antibody or its antigen-binding fragment is at least a first antigen-binding domain that specifically binds to a first human tumor antigen (TAA), wherein the first TAA is human 4Ig-B7H3, and the first antigen-binding domain comprises an antibody or its antigen-binding fragment disclosed herein, the first antigen-binding domain, and at least a second antigen-binding domain that specifically binds to a second human TAA.

[0015] In an embodiment, the multispecific antibody or its antigen-binding fragment is a bispecific antibody.

[0016] In an embodiment, the multispecific antibody or its antigen-binding fragment contains an amino acid linker, and the amino acid linker is any one of the sequences of SEQ ID NO: 79 to SEQ ID NO: 121.

[0017] In an embodiment, the antibody or its antigen-binding fragment contains a heavy chain constant region of a subclass of IgG1, IgG2, IgG3, or IgG4, and / or a light chain constant region of kappa type or lambda type.

[0018] In an embodiment, the antibody or its antigen-binding fragment contains a heavy chain constant region of the IgG1 subclass.

[0019] In an embodiment, the antibody or its antigen-binding fragment has antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0020] In an embodiment, the antibody or its antigen-binding fragment is hypoglycosylated, or non-glycosylated, or hypofucosylated.

[0021] In an embodiment, the antibody or its antigen-binding fragment contains an increased bisecting GlcNAc structure.

[0022] The present disclosure also relates to a VHH, which specifically binds to human 4Ig-B7H3.

[0023] In an embodiment, the VHH includes the following: (1) (a) A heavy chain variable region (VH) containing HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 7, (b) HCDR2 of SEQ ID NO: 8, and (c) HCDR3 of SEQ ID NO: 9, or (2) (a) HCDR1 of SEQ ID NO: 12, (b) HCDR2 of SEQ ID NO: 13, and (c) HCDR3 of SEQ ID NO: 14-containing heavy chain variable region.

[0024] In an embodiment, the VHH includes the following: (1) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 10, (2) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 20, (3) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 24, (4) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 26, (5) Heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 30 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, (6) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 32, (7) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 34, (8) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 36, (9) Heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 40 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, (10) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 42, (11) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 44, (12) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 46, (13) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 48, (14) Heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 50 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, (15) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 15, (16) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 52, (17) A heavy chain variable region containing an amino acid sequence that is at least identical to sequence number 54 by 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, or (18) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 56.

[0025] In this embodiment, in VHH, one, two, three, four, five, six, seven, eight, nine, or ten amino acids are inserted, deleted, or substituted in at least one of the following: SEQ ID NOs: 10, 20, 24, 26, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 15, 52, 54, and 56.

[0026] In this embodiment, VHH includes: (1) Heavy chain variable region including sequence number 10, (2) Heavy chain variable region including sequence number 20, (3) Heavy chain variable region including sequence number 24, (4) Heavy chain variable region including sequence number 26, (5) Heavy chain variable region including sequence number 30, (6) Heavy chain variable region including sequence number 32, (7) Heavy chain variable region including sequence number 34, (8) Heavy chain variable region including sequence number 36, (9) Heavy chain variable region including sequence number 40, (10) Heavy chain variable region including sequence number 42, (11) Heavy chain variable region including sequence number 44, (12) Heavy chain variable region including sequence number 46, (13) Heavy chain variable region including sequence number 48, (14) Heavy chain variable region including sequence number 50, (15) Heavy chain variable region including sequence number 15, (16) Heavy chain variable region including sequence number 52, (17) Heavy chain variable region including sequence number 54, or (18) Heavy chain variable region including sequence number 56.

[0027] In embodiments, a multispecific antibody or its antigen-binding fragment comprises at least a first antigen-binding domain that specifically binds to a first human tumor antigen (TAA), wherein the first TAA is human 4Ig-B7H3 and the first antigen-binding domain includes VHH as disclosed herein, and at least a second antigen-binding domain that specifically binds to a second human TAA.

[0028] In this embodiment, the multispecific antibody or its antigen-binding fragment is a bispecific antibody.

[0029] In the embodiment, the multispecific antibody or its antigen-binding fragment includes an amino acid linker, and the amino acid linker is one of the sequences of SEQ ID NOs. 79 to 121.

[0030] In the embodiments, the pharmaceutical composition comprises an antibody or its antigen-binding fragment disclosed herein, or VHH, and a pharmaceutically acceptable carrier.

[0031] In embodiments, this disclosure relates to isolated nucleic acids encoding antibodies or antigen-binding fragments thereof disclosed herein.

[0032] In embodiments, this disclosure relates to vectors comprising nucleic acids disclosed herein.

[0033] In embodiments, this disclosure relates to a host cell comprising a nucleic acid or vector disclosed herein.

[0034] In embodiments, the present disclosure relates to a process for generating an antibody or an antigen-binding fragment thereof, the process comprising culturing host cells and recovering the antibody or antibody fragment disclosed herein from the culture.

[0035] In the embodiment, the anti-human 4Ig-B7H3 antibody or its antigen-binding fragment has at least one of the following characteristics: (1) Specific binding to human 4Ig-B7H3 and high affinity, (2) It is a humanized antibody with a low immunogenicity risk to humans, and (3) It has good purity and is easy to purify.

[0036] In embodiments, the present disclosure provides anti-human 4Ig-B7H3 VHH that is highly humanized, exhibits a low immunogenicity risk to humans, maintains specific binding and high affinity to human 4Ig-B7H3, and / or exhibits high purity and is easily purified. [Brief explanation of the drawing]

[0037] [Figure 1] This shows the binding of BGA-026 and BGA-056 to the B7H3 antigen by ELISA. [Figure 2] Flow cytometry demonstrates the binding of BGA-026 and BGA-056 to HEK293 cells. [Figure 3] Flow cytometry demonstrates the binding of BGA-026 chimeras and BGA-2042 to H358 cells. [Figure 4]Flow cytometry demonstrates the binding of BGA-056 chimeras and BGA-174 to H358 cells. [Figure 5A] Flow cytometry demonstrates the binding of VHH1_C001 and VHH1_C002 to the B7H3-positive cancer cell line H358. [Figure 5B] Flow cytometry demonstrates the binding of VHH1_C001 and VHH1_C002 to the B7H3-negative cancer cell line MDA-MB-453. [Figure 6A] This shows the binding of VHH1_C001 and VHH1_C002 to 4-Ig-B7H3 and 2Ig-B7H3 via surface plasmon resonance (SPR). [Figure 6B] This shows the binding of VHH1_C001 and VHH1_C002 to 4-Ig-B7H3 and 2Ig-B7H3 via surface plasmon resonance (SPR). [Figure 6C] This shows the binding of VHH1_C001 and VHH1_C002 to 4-Ig-B7H3 and 2Ig-B7H3 via surface plasmon resonance (SPR). [Figure 6D] This shows the binding of VHH1_C001 and VHH1_C002 to 4-Ig-B7H3 and 2Ig-B7H3 via surface plasmon resonance (SPR). [Figure 7] Epitope binning for VHH1_C001 and VHH1_C002 is shown. [Modes for carrying out the invention]

[0038] This disclosure relates to anti-human 4Ig-B7H3 antibodies and their antigen-binding fragments. The disclosed antibodies have desirable binding affinity and other desirable properties, such as a low risk of immunogenicity in humans. Anti-human 4Ig-B7H3 antibodies and antibody fragments can be used to construct multispecific antibodies with additional functions, such as binding to a second human tumor-associated antigen (TAA), immune checkpoint inhibition, or immunostimulation; to construct antibody-drug conjugates (ADCs); or to fuse with other domains to form fusion proteins. Furthermore, anti-4Ig-B7H3 antibodies and their constructs, or pharmaceutical compositions containing them, can be used to treat cancers and related disorders that express 4Ig-B7H3.

[0039] definition Unless otherwise defined below or elsewhere in this document, all other technical and scientific terms used herein have the meanings generally understood by those skilled in the art.

[0040] As used herein, including in the attached claims, singular words such as "a," "an," and "the" include their corresponding plural referents unless otherwise explicitly stated in the context.

[0041] The term "or" is used to mean "and / or" unless otherwise specified in the context, and is interchangeable with it.

[0042] Unless otherwise specified or evident from the context, the term “about” as used herein means a value or composition that falls within the tolerance range of a particular value or composition determined by those skilled in the art, which depends in part on how the value or composition is measured or determined, i.e., on the limits of the measuring system. For example, “about” may mean within or greater than one standard deviation, according to the convention of the art. “About” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% more or less than the stated value. For example, about 5 mg may contain any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms may mean a value of up to one order of magnitude or up to five times the stated value. Where specific values ​​or compositions are provided in this disclosure, unless otherwise specified, the meaning of “approximately” should be assumed to be within the tolerance range of those specific values ​​or compositions.

[0043] The term "human 4Ig-B7H3" refers to the 4Ig isoform of the type I transmembrane protein B7H3 in humans. The nucleic acid sequence of human 4Ig-B7H3 is described in Sequence ID No. 1, based on GenBank sequence accession number: NM_001024736.1. The amino acid sequence of human 4Ig-B7H3 is Sequence ID No. 2.

[0044] As used herein, the terms “administer” and “administer” mean, when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Cell therapy includes contact of a reagent with cells, as well as contact of a reagent with a fluid where the fluid is in contact with cells.

[0045] The terms “subject” or “patient” as used herein include any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, primates), and most preferably a human (e.g., a patient having or at risk of having one of the disorders described herein).

[0046] In one aspect, “treating” any disease or disorder means improving the disease or disorder (i.e., delaying, preventing, or reducing the onset of at least one of the disease or its clinical symptoms). In another aspect, “treating,” “treating,” or “treatment” means mitigating or improving at least one physical parameter, including one that may not be identifiable by the patient. In yet another aspect, “treating,” “treating,” or “treatment” means modulating the disease or disorder either physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both.

[0047] As used herein, the term "affinity" 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 at numerous sites via forces other than covalent bonds. Generally, the more interactions there are, the stronger the affinity.

[0048] As used herein, the term “antibody” (“Ab”) refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a more reversible and specific manner than covalent bonding. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists 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 consists 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 hypervariable regions called complementarity-determining regions (CDRs), and interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four framework regions (FRs), arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody 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 (Clq) of the classical complement system.

[0049] The location of the CDR and framework region can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001), Chothia and Lesk, J.Mol.Biol., 196:901-917 (1987), Chothia et al., Nature, 342:877-883 (1989), Chothia et al., J.Mol.Biol., 227:799-817 (1992), Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997), Lefranc, M.-P., The See Immunologist, 7, 132-136 (1999) and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003).

[0050] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies and human-modified antibodies. 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).

[0051] The term "chimeric antibody" refers to a molecule created by fusing a variable domain of an antibody derived from a different species, specifically from a domain derived from one host species (e.g., mouse, rabbit, llama, etc.), with a constant domain of an antibody derived from another species (e.g., human).

[0052] In some embodiments, the anti-4Ig-B7H3 antibody comprises at least one antigen-binding site, which may be at least a variable region. In some embodiments, the anti-4Ig-B7H3 antibody comprises an antigen-binding fragment derived from the 4Ig-B7H3 antibody described herein. In some embodiments, the anti-4Ig-B7H3 antibody is either isolated or recombinant. In some embodiments, the anti-4Ig-B7H3 antibody also encompasses a multispecific antibody that targets 4Ig-B7H3 as at least one arm and other antigens as other arms.

[0053] In this specification, the terms “monoclonal antibody” or “mAb” or “Mab” refer to a substantially homogeneous group of antibodies, i.e., the antibody molecules within that group have identical amino acid sequences, except for the possibility of naturally occurring mutations that may exist in small amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a number of different antibodies with different amino acid sequences within their variable domains, particularly within their CDRs, which are often specific to different epitopes. The modifier “monoclonal” characterizes the antibody as being obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497, U.S. Patent No. 4,376,110, Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988, and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein may be any immunoglobulin class such as IgG, IgM, IgD, IgE, IgA, and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas that produce monoclonal antibodies can be cultured in vitro or in vivo. High-titer monoclonal antibodies can be obtained through in vivo generation. In this case, cells from individual hybridomas are intraperitoneally injected into mice, such as Balb / c mice, stimulated with pristine, to generate ascites containing high concentrations of the desired antibody. From such ascites, or from the culture supernatant, isotype IgM or IgG monoclonal antibodies can be purified using column chromatography methods well known to those skilled in the art.

[0054] Generally, the basic structural unit of an antibody is a tetramer. Each tetramer contains two identical polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The carboxyl-terminus of the heavy chain can define a constant region primarily involved in effector function. Typically, human light chains are classified into kappa and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and the isotypes of the antibody are defined as IgA, IgD, IgE, IgG, and IgM, respectively.

[0055] Within the light and heavy chains, the variable and constant regions are linked by "J" regions consisting of approximately 12 or more amino acids, and the heavy chain also contains "D" regions consisting of approximately 10 amino acids.

[0056] The variable region of each light chain / heavy chain (VL / VH) pair forms the antibody binding site. Therefore, intact antibodies generally have two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0057] Typically, both the 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). CDRs are usually aligned by the framework regions, enabling binding to specific epitopes. Generally, from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (FR3), CDR-3 (CDR3), and FR-4 (FR4). The location of the CDR and framework region can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J.Mol.Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J.Mol.Biol., 227:799-817 (1992); Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997); ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (see "IMGT" numbering procedure).The definition of antigen-binding sites is also described in the following: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J.Mol.Biol., 262:732-745 (1996); and Martin et al., Proc.Natl.Acad.Sci.USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); 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), while 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), while the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the definitions of CDRs by Kabat and Chothia, the CDR consists 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 region are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), while the CDR amino acid residues in the VL region are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR region of an antibody can be determined using the program IMGT / DomainGap Align.

[0058] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable region includes amino acid residues from the "CDR" (e.g., LCDR1, LCDR2, and LCDR3 of the light chain variable domain, and HCDR1, HCDR2, and HCDR3 of 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. (Defining the CDR region of antibodies by sequence), and also see: Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (Defining the CDR region of antibodies by structure). The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0059] Unless otherwise specified, “antigen-binding fragment” means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which it is bound by a full-length antibody, for example, a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, e.g., single-chain Fv(ScFv), nanobodies (or VHH antibodies), multispecific antibodies formed from antibody fragments, and bicyclic peptides (Hurov, K. et al., 2021. Journal for ImmunoTherapy of Cancer, 9(11)).

[0060] As used herein, "specifically binding" of an antibody or antigen-binding antibody fragment to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target compared to other proteins, but this specificity does not require absolute binding specificity. "Specific" or "selective" binding reactions determine, for example, the presence of an antigen in a heterogeneous population of proteins and other biologics in a biological sample, blood, serum, plasma, or tissue sample. Thus, under specific, designated immunoassay conditions, the antibody or its antigen-binding fragment specifically binds at least twice as much to a particular antigen compared to background levels, and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, the antibody or its antigen-binding fragment specifically binds at least ten times as much to a particular antigen compared to background levels of binding, and does not specifically bind in significant amounts to other antigens present in the sample.

[0061] As used herein, the “antigen-binding domain” comprises at least six CDRs (or, in the case of a single-domain antibody, three CDRs) and specifically binds to an epitope. 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 also comprises at least three CDRs that specifically bind to a second epitope. A multispecific antibody can be bispecific, triplicate, quadruplicate, etc., with antigen-binding domains directed to each specific epitope. A multispecific antibody can be polyvalent (e.g., a bispecific quadruple antibody) and contain 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. The "antigen-binding domain" of a single-chain antibody, such as a heavy-chain antibody, or VHH, contains an antigen-binding domain that specifically binds to an epitope without pairing with further variable domains. The binding site of a single variable domain of an immunoglobulin is formed by a single VH / VHH or VL domain.

[0062] As used herein, the terms “VHH domain,” “VHH antibody,” “VHH antibody fragment,” and “VHH” (also known as nanobody) originally referred to the antigen-binding domain of heavy-chain-only antibodies produced by camelids (naturally occurring antibodies lacking a light chain, C. Hamers-Casterman et al., Nature, volume 363, pages 446-448 (1993)), and are distinct from the heavy-chain variable domain (VH domain) of conventional tetrameric antibodies. VHH antibodies retain the immunoglobulin fold of conventional tetrameric antibodies, with only three hypervariable loops, CDR1, CDR2, and CDR3, binding to their target. Many VHHs bind to their targets with similar affinity to conventional full-size antibodies and may possess other properties superior to conventional full-size antibodies.

[0063] In this specification, the term "human antibody" means an antibody containing only human immunoglobulin protein sequences. Human antibodies may contain mouse glycans if they are produced in mice, mouse cells, or mouse cell-derived hybridomas. Similarly, "mouse antibody" or "rat antibody" means an antibody containing only mouse immunoglobulin protein sequences or an antibody containing only rat immunoglobulin protein sequences, respectively.

[0064] The terms "humanized" or "humanized antibody" refer to a form of antibody that contains sequences from non-human (e.g., mouse, rabbit, llama, etc.) antibodies, in addition to sequences from human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all hypervariable loops in the variable domains correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulins. When it is necessary to distinguish humanized antibodies from parent (e.g., rodent) antibodies, the prefix "hum," "hu," "Hu," or "h" is added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequence as the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, improve the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0065] 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. A corresponding human germline sequence may 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. A corresponding human germline sequence may be the framework region only, the complementarity-determining region only, the framework and complementarity-determining regions, the variable region, or any other combination of the sequence or subsequence. Sequence identity can be determined by aligning the two sequences using the methods described herein, for example, BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region may also be derived from such a human sequence, e.g., a human germline sequence, or a variant version of a human germline sequence, or from an antibody containing a consensus framework sequence derived from human framework sequence analysis, as described, for example, in Knappik et al., J.Mol.Biol.296:57-86, 2000.

[0066] The terms “equilibrium dissociation constant,” “KD,” or “M” refer to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, Ml). The equilibrium dissociation constant can be measured using any method known in the art. The antibodies of this disclosure generally have an equilibrium dissociation constant less than about 10⁻⁷ or less than 10⁻⁸ M, for example, less than about 10⁻⁹ M or less than 10⁻¹⁰ M, less than 10⁻¹¹ M, less than 10⁻¹² M, or less than 10⁻¹³ M.

[0067] As used herein, the terms “cancer” or “tumor” have the broadest meaning as understood in the art and refer to a physiological condition in mammals typically characterized by uncontrolled cell proliferation. In the context of this disclosure, cancer is not limited to any particular type or location.

[0068] In the context of this disclosure, where referring to an amino acid sequence, the term “conservative substitution” means the substitution of 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, such as its binding affinity to 4Ig-B7H3. Common conservative substitutions of amino acids are well known in the art.

[0069] As used herein, the term “knob-into-hole” technology refers to an amino acid that directs the pairing of two polypeptides together, either in vitro or in vivo, by introducing a spatial bulge (knob) into one polypeptide and a socket or cavity (hole) into the other polypeptide (at the interface where they interact). For example, knob-into-holes are introduced at the Fc:Fc binding interface, CL:CHI interface, or VH / VL interface of an antibody (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, knob-into-holes ensure the correct pairing of two different heavy chains during the production of multispecific antibodies. For example, multispecific antibodies having knob-into-hole amino acids within their Fc regions may further contain a single variable domain linked to each Fc region, or different heavy chain variable domains that pair with similar or different light chain variable domains. The knob-into-hole technique can also be used in VH or VL regions to ensure correct pairing.

[0070] Examples of algorithms suitable for determining sequence identity and sequence similarity percentages include the BLAST algorithm, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W within the query sequence, which are words that match or satisfy a threshold score T that would have a positive value if aligned with words of the same length in the database sequence. T is called the neighbor word score threshold. These first hit neighbor words serve as a value to initiate a search for longer HSPs that contain them. Word hits are extended toward both ends of each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a matching pair of residues, always >0) and N (penalty score for mismatched residues, always <0). For amino acid sequences, the cumulative score is calculated using a score matrix. Word hits are stopped in each direction if the cumulative alignment score falls by X from the maximum attainable value; if the cumulative score becomes zero or less due to the accumulation of one or more negative score 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 alignment. The BLASTN program (for nucleotide sequences) uses 11 word lengths (W), 10 expected values ​​(E), M=5, N=-4, and two-strand comparison as defaults.For amino acid sequences, the BLAST program uses a word length of 3, an expected value of 10 (E), and a BLOSUM62 score matrix of 50 (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignment (B), expected value of 10 (E), M=5, N=-4, and double-strand comparison as defaults.

[0071] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that a match between two sequences of nucleotides or amino acids occurs by chance. For example, a nucleic acid is considered 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.

[0072] The percentage of 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). This algorithm is incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), incorporated into the GAP program of the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0073] The term “nucleic acid” is used herein in the same sense as the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof, either in single-chain or double-chain form. This term encompasses nucleic acids containing known nucleotide analogs or modified skeletal residues or bonds, which include synthetic nucleic acids, naturally occurring nucleic acids, and naturally occurring nucleic acids, which have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0074] In relation to nucleic acids, the term "functionally linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to a functional relationship between a transcriptional regulatory sequence and a transcription sequence. For example, a promoter or enhancer sequence is functionally linked to a coding sequence if it stimulates or modulates the transcription of that coding sequence in a suitable host cell or other expression system. Generally, promoters and transcriptional regulatory sequences that are functionally linked to a transcription sequence are physically contiguous to the transcription sequence; i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequence in which they enhance transcription.

[0075] In some embodiments, the Disclosure provides compositions comprising the anti-4Ig-B7H3 antibody described herein, for example, pharmaceutically acceptable compositions, formulated with at least one pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically acceptable excipient” includes all physiologically compatible solvents, dispersions, isotonic agents, and absorption retarders, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or dermal administration (e.g., by injection or infusion).

[0076] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and liquid, semi-solid, and solid dosage forms such as suppositories. The appropriate form depends on the intended method of administration and therapeutic use. One appropriate method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, antibodies are administered by intravenous infusion or injection. In certain embodiments, antibodies are administered by intramuscular or subcutaneous injection.

[0077] As used herein, the term “therapeutic dose” refers to the amount of antibody sufficient to produce such treatment for a disease, disorder, or symptom when administered to a subject to treat the disease, disorder, or symptom, or to treat at least one of the clinical symptoms of the disease or disorder. “Therapeutic dose” may vary depending on the antibody, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the symptoms of the disease, disorder, and / or the age of the subject being treated, and / or the weight of the subject being treated. The appropriate dose in any given case may be obvious to those skilled in the art or can be determined by customary experimentation. In the case of combination therapy, “therapeutic dose” refers to the total amount of the combination components.

[0078] The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder. Such administrations include co-administration of these therapeutic agents in a substantially simultaneous manner. Such administrations also include co-administrations in multiple or separate containers or formulations (e.g., capsules, powders, and liquids) for each active ingredient. Powders and / or liquids may be reconstituted or diluted to the desired dose before administration. Furthermore, “combination therapy” includes the use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In any case, the treatment regimen provides the beneficial effect of drug combination in the treatment of the conditions or disorders described herein.

[0079] As used herein, the expression "in combination with" means that the anti-4Ig-B7H3 antibody is administered to the 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. Anti-4Ig-B7H3 antibody [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Table 1-18] [Table 1-19] [Table 1-20] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0080] This disclosure provides an antibody or its antigen-binding fragment that specifically binds to human 4Ig-B7H3 (i.e., an anti-human 4Ig-B7H3 antibody or its antigen-binding fragment). The antibody or antigen-binding fragment may be produced as described below.

[0081] In one embodiment, the anti-human 4Ig-B7H3 antibody or antibody fragment (e.g., antigen-binding fragment) includes a heavy chain variable region (VH) domain having at least one amino acid sequence from among SEQ ID NOs: 10, 20, 24, 26, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 15, 52, 54, or 56 (Tables 1-2). In another embodiment, the anti-human 4Ig-B7H3 antibody or antigen-binding fragment includes an HCDR having one amino acid sequence from any of the heavy chain complementarity-determining regions (HCDRs) listed in Tables 1-2. In one embodiment, the anti-human 4Ig-B7H3 antibody or antigen-binding fragment includes one, two, three, or more HCDRs having one of the amino acid sequences from any of the HCDRs listed in Tables 1-2.

[0082] In one embodiment, an antibody or antigen-binding fragment that specifically binds to human 4Ig-B7H3 includes one or more complementarity-determining regions (CDRs) comprising an amino acid sequence selected from SEQ ID NOs. 7, SEQ ID NOs. 8, and SEQ ID NOs. 9, or an amino acid sequence selected from SEQ ID NOs. 12, SEQ ID NOs. 13, and SEQ ID NOs. 14.

[0083] In one embodiment, the anti-human 4Ig-B7H3 antibody or its antigen-binding fragment comprises (i) HCDR1, HCDR2, and HCDR3 derived from VH as described in SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 15, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56.

[0084] In another embodiment, an antibody or antigen-binding fragment that specifically binds to human 4Ig-B7H3 includes a heavy chain variable region comprising three HCDRs, according to Kabat numbering: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0085] In another embodiment, an antibody or antigen-binding fragment that specifically binds to human 4Ig-B7H3 includes a heavy chain variable region comprising three HCDRs, according to Kabat numbering: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 14.

[0086] In one embodiment, the antibody or antigen-binding fragment of the present disclosure includes a heavy chain variable region comprising (a) at least one amino acid sequence from among SEQ ID NOs: 10, SEQ ID NOs: 20, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 30, SEQ ID NOs: 32, SEQ ID NOs: 34, SEQ ID NOs: 36, SEQ ID NOs: 40, SEQ ID NOs: 42, SEQ ID NOs: 44, SEQ ID NOs: 46, SEQ ID NOs: 48, SEQ ID NOs: 50, SEQ ID NOs: 15, SEQ ID NOs: 52, SEQ ID NOs: 54, or SEQ ID NOs: 56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one or more of SEQ ID NOs: 10, SEQ ID NOs: 20, SEQ ID NOs: 24, SEQ ID NOs: 26, SEQ ID NOs: 30, SEQ ID NOs: 32, SEQ ID NOs: 34, SEQ ID NOs: 36, SEQ ID NOs: 40, SEQ ID NOs: 42, SEQ ID NOs: 44, SEQ ID NOs: 46, SEQ ID NOs: 48, SEQ ID NOs: 50, SEQ ID NOs: 15, SEQ ID NOs: 52, SEQ ID NOs: 54, and SEQ ID NOs: 56.

[0087] Other antibodies or antigen-binding fragments of the Disclosure contain amino acids that are modified but have at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity in the CDR region compared to the CDR regions disclosed in Tables 1-2. In some embodiments, the CDR region includes amino acid changes (insertions, deletions, or substitutions (which may be conservative amino acid substitutions)) in which the CDR region has changed one, two, three, four, or five or fewer amino acids while maintaining binding specificity and affinity compared to the CDR regions disclosed in Tables 1-2.

[0088] Other antibodies or antigen-binding fragments of the Disclosure include those in which amino acids, or nucleic acids encoding amino acids, are altered within a variable region (e.g., a framework region of the variable region), but with binding specificity / affinity maintained, and having at least 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity percentage with the sequences of the variable regions disclosed in Tables 1-2. In some embodiments, the corresponding sequences of the CDRs remain unchanged. In some embodiments, the antibody or its antigen-binding fragment retains binding specificity / affinity, while the variable region (e.g., the framework region of the variable region) undergoes changes of 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 or fewer amino acids compared to the variable region disclosed in Tables 1-2. In some embodiments, the corresponding sequence of the CDR remains unchanged.

[0089] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 and comprises VH including (a) HCDR1 of SEQ ID NO: 7, (b) HCDR2 of SEQ ID NO: 8, and (c) HCDR3 of SEQ ID NO: 9, wherein amino acids P26, F37, and A94 are retained within the framework region.

[0090] In some embodiments, the disclosure provides anti-human 4Ig-B7H3 antibodies or antigen-binding fragments thereof, or variants thereof, as disclosed in Table 1, in which HCDR1, HCDR2, and HCDR3 remain unchanged and amino acids P26, F37, and A94 are retained.

[0091] In some embodiments, the disclosure provides an antibody or antigen-binding fragment that specifically binds to human 4Ig-B7H3 and includes a heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to at least one of SEQ ID NOs: 10, 20, 24, 26, 30, 32, 34, 36, 40, 42, 44, 46, 48, or 50, and retains amino acids P26, F37, and A94 within a framework region.

[0092] In another embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human 4Ig-B7H3 with a binding affinity (KD) of 1 × 10⁻⁶ M to 1 × 10⁻¹¹ M. In another embodiment, an anti-4Ig-B7H3 antibody or antigen-binding fragment thereof binds to human 4Ig-B7H3 with a binding affinity (KD) of about 1 × 10⁻⁶ M, about 1 × 10⁻⁷ M, about 1 × 10⁻⁸ M, about 1 × 10⁻⁹ M, or about 1 × 10⁻¹¹ M.

[0093] This disclosure also provides nucleic acid sequences encoding VH or VL of antibodies that specifically bind to human 4Ig-B7H3. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0094] This disclosure also provides antibodies and their antigen-binding fragments that bind to the same epitopes as the anti-4Ig-B7H3 antibodies described in Tables 1-2. Therefore, additional antibodies and their antigen-binding fragments can be identified in binding assays based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with the antibodies described in Tables 1-2. The ability of test antibodies to inhibit the binding of the antibodies and their antigen-binding fragments of this disclosure to 4Ig-B7H3 demonstrates that the test antibody can compete with that antibody or its antigen-binding fragment for binding to 4Ig-B7H3. Such antibodies can bind to the same or related (e.g., structurally similar or spatially proximal) epitopes on 4Ig-B7H3 as those of the competing antibody or its antigen-binding fragment, without being constrained by any one theory. In certain embodiments, the antibody that binds to the same epitopes on 4Ig-B7H3 as those of the antibodies or their antigen-binding fragments of this disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0095] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a VHH.

[0096] Anti-4Ig-B7H3 multispecific antibody In one embodiment, the anti-4Ig-B7H3 antibody or its antigen-binding fragment disclosed herein can be used to construct a multispecific antibody having additional functions, such as binding to a second human tumor-associated antigen (TAA), immune checkpoint inhibition, or immunostimulation. In some examples, 4Ig-B7H3 functions as a first TAA.

[0097] In one embodiment, the 4Ig-B7H3 antibody or antigen-binding fragment disclosed herein can be incorporated into an anti-4Ig-B7H3xTAA multispecific antibody, where TAA is an antibody or fragment against any human tumor-associated antigen (TAA). For example, if it contains several antigen-binding domains, the antibody is a multispecific antibody molecule in which at least one antigen-binding domain sequence specifically binds to 4Ig-B7H3 and a second antigen-binding domain sequence specifically binds to TAA. In various embodiments, the multispecific antibody contains a third, fourth, or fifth antigen-binding domain. In various embodiments, the multispecific antibody is a bispecific, triplicate, or quadruplicate antibody. In each embodiment, the multispecific antibody contains at least one anti-4Ig-B7H3 antigen-binding domain and at least one anti-TAA antigen-binding domain.

[0098] 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 to human 4Ig-B7H3 and a second antigen-binding domain that specifically binds to a second TAA. A bispecific antibody also comprises a second heavy-chain variable domain and a second light-chain variable domain that specifically binds to a second TAA, as well as a first heavy-chain variable domain and / or a first light-chain variable domain that specifically binds to human 4Ig-B7H3. In another embodiment, a bispecific antibody comprises an antigen-binding fragment of an antibody that specifically binds to 4Ig-B7H3 and an antigen-binding fragment that specifically binds to a TAA. If the bispecific antibody comprises an antigen-binding fragment, the antigen-binding fragment can be Fab, F(ab')2, Fv, single-chain Fv(scFv), or a single-domain antibody.

[0099] In some embodiments, the multispecific antibodies of the Disclosure bind to a second human TAA and / or human 4Ig-B7H3 with a binding affinity (KD) of 1 × 10⁻⁶ M to 1 × 10⁻¹¹ M. In some embodiments, the multispecific antibodies of the Disclosure bind to a second human TAA and / or human 4Ig-B7H3 with a binding affinity (KD) of approximately 1 × 10⁻⁶ M, approximately 1 × 10⁻⁷ M, approximately 1 × 10⁻⁸ M, approximately 1 × 10⁻⁹ M, approximately 1 × 10⁻¹⁰ M, or approximately 1 × 10⁻¹¹ M.

[0100] In one embodiment, the disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain that specifically binds to human 4Ig-B7H3 comprises an anti-human 4Ig-B7H3 antibody or antigen-binding fragment described herein, and a second antigen-binding domain that specifically binds to a second human TAA.

[0101] In another embodiment, the disclosure provides a multispecific antibody or antigen-binding fragment thereof, wherein a first antigen-binding domain that specifically binds to human 4Ig-B7H3 comprises at least one of SEQ ID NOs: 10, 20, 24, 26, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 15, 52, 54, or 56, and a second antigen-binding domain specifically binds to a second human TAA.

[0102] Previous experiments (Coloma and Morrison Nature Biotech. 15:159-163 (1997)) described a tetravalent bispecific antibody manipulated by fusing DNA encoding a single-chain anti-dansyl antibody Fv (scFv) after the C-terminus (CH3-scFv) or after the hinge (hinge-scFv) of an IgG3 anti-dansyl antibody. This disclosure provides a polyvalent antibody (e.g., a tetravalent antibody) having at least two antigen-binding domains, which can be readily generated by recombinant expression of the nucleic acid encoding the polypeptide chain of the antibody. The polyvalent antibodies described herein comprise 3 to 8, preferably 4, antigen-binding domains that specifically bind to at least two antigens.

[0103] 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 one of SEQ ID NOs. 79 to 121.

[0104] Anti-human 4Ig-B7H3 antibody conjugated with cytotoxin An antibody-drug conjugate (ADC) can be constructed using the anti-human 4Ig-B7H3 antibody or its antigen-binding fragment according to this disclosure. 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.

[0105] cytotoxin Cytotoxins or cytotoxic agents include any agent that is harmful to cell growth, viability, or proliferation, and this includes, but is 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 this disclosure include, for example, 1-(2-chloroethyl)-1,2-dimethanesulfonylhydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-dien-2,6-diin-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitins, aminopterin, anguidin, anthracycline, and anthramycin (AMC). Auristatins, bleomycin, busulfan, butyrate, calicheamicins (e.g., calicheamicin gamma 1), camptothecin, carminomycins, carmustine, semadin, cisplatin, colchicine, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxyanthracine dione, disorazoles, dorastatin (e.g., dorastatin 10), dox Sorbicin, duocalmycin, echinomycins, eloiterobins, emetine, epothyrons, esperamycin, estramustines, ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D, glucocorticoids, irinotecans, kinesin spindle protein (KSP) inhibitors, leptomycins, leurosines, lidocaine, lomustine (CCNU), metansinoids, mechloretamine, melphalan, mercatopurines, metopterins, This includes methotrexate, mitramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxins, procaine, propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PBDs), rhizoxins, streptozotocin, thalisomycins, taxol, tenoposide, tetracaine, thioepachlorambucil, tomaimycins, topotecans, tubulsin, vinblastine, vincristine, vindesine, vinorelbines, and any derivatives of the above.

[0106] Cytotoxin linker A cytotoxin linker or ADC linker is any group or portion that links, connects, or binds the antibody or antigen-binding fragment described herein to a therapeutic portion, such as a cytotoxic drug. 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; and *Antibody-Drug Conjugates*; Wang, J., Shen, W.-C, and Zaro, JL, Eds.; Springer International Publishing, 2015, the contents of which are incorporated herein by reference in their entirety.

[0107] In general, suitable conjugates or cytotoxic linkers for the antibody conjugates described herein are stable enough to utilize the circulating half-life of the antibody and, at the same time, allow for payload release after the internalization of the antigen-mediated conjugate. The linker may be cleavable or incleavable. Cleavable linkers include those that are cleaved by intracellular metabolism following internalization (e.g., hydrolysis, reduction, or enzymatic cleavage). Incleavable linkers include those that release the bound payload via lysosomal degradation of the antibody following internalization. Suitable linkers include, but are not limited to, acid-unstable linkers, hydrolysis-unstable linkers, enzymatically cleavable linkers, reduction-unstable linkers, self-destructive linkers, and incleavable linkers. Suitable linkers include, but are not limited to, peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, malcaproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units, or those containing these.

[0108] The ADCs of this disclosure can be fabricated or constructed using any cytotoxic linker molecule or linker technology known in the art. In certain embodiments, the cytotoxic linker is a cleavable linker. According to other embodiments, the linker is an incleavable linker. Examples of linkers that may be used in the context of this disclosure include, for example, GGFG, MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine) dipeptide moieties in protease-cleavable linkers, ala-phe (alanine-phenylalanine) dipeptide moieties in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl-4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate), and linkers comprising or comprising their variants and combinations. Examples of additional linkers that may be used in the context of this disclosure are provided, for example, in US7,754,681 and Ducry, Bioconjugate Chem., 2010, 27:5-13, and the references cited therein, the contents of which are incorporated herein by reference in their entirety.

[0109] In certain embodiments, the cytotoxin linker is stable under physiological conditions. In certain embodiments, the linker is cleavable and can release at least the payload portion, for example, in the presence of an enzyme or within a specific pH range or value. In some embodiments, the linker includes an enzymatically cleavable portion. Exemplary enzymatically cleavable portions include, but are not limited to, peptide bonds, ester bonds, hydrazones, and disulfide bonds. In some embodiments, the linker includes a cathepsin-cleavable linker.

[0110] In some embodiments, the cytotoxin linker includes an inclementable portion.

[0111] Suitable cytotoxin linkers include, but are not limited to, single binders, such as those chemically bonded to two cysteine ​​residues of an antibody. Such linkers can mimic the role of the antibody's disulfide bond, which is disrupted as a result of the conjugation process.

[0112] In some embodiments, the cytotoxin linker comprises one or more amino acids. Preferred amino acids include natural, unnatural, standard, non-standard, protein constituent, protein non-constitutive, 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 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.

[0113] Fusion protein targeting human 4Ig-B7H3 The anti-human 4Ig-B7H3 antibody or its antigen-binding fragment disclosed herein can be fused with other proteins or other functional domains to form a fusion protein or chimeric protein.

[0114] In some embodiments, an anti-human 4Ig-B7H3 antibody or its antigen-binding fragment is fused with an immune checkpoint, immunostimulator, cytokine, or second TAA, either directly or indirectly via an amino acid linker as described herein.

[0115] In one embodiment, an anti-human 4Ig-B7H3 antibody or its antigen-binding fragment is fused with a functional domain or receptor subunit that can convert the signal from scFv / VHH and confer antibody specificity to immune cells such as T cells or NK cells, as well as other effector cells.

[0116] In some embodiments, a chimeric antigen receptor (CAR) is constructed using an anti-human 4Ig-B7H3 antibody or its antigen-binding fragment. Further information on the CAR constructs can be found in Guedan et al., Mol Ther Methods Clin Dev 2019;12:145-156.

[0117] Functional domains or receptor subunits include, for example, transmembrane domains, hinge regions, intracellular signaling domains, and costimulatory domains.

[0118] Steady-state and Fc region modification The heavy chain constant region can be the wild-type sequence of the heavy chain constant region derived from a subclass of IgG1, IgG2, IgG3, or IgG4. The light chain constant region can be the wild-type sequence of the light chain derived from a kappa or lambda type. In one embodiment, the heavy chain constant region is the wild-type sequence of the constant region derived from IgG1. The light chain constant region is the wild-type sequence of the light chain derived from a kappa chain. In one embodiment, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 18. In another embodiment, the heavy chain constant region includes the mutants E233P, L234A, L235A, G236del, and P329A.

[0119] In one embodiment, the Fc region may be a wild-type Fc region of a subclass of IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody or its antigen-binding fragment contains an Fc domain of IgG1 or IgG4 with reduced effector function.

[0120] In one embodiment, the antibody or its antigen-binding fragment includes an extended half-life Fc domain. In another embodiment, the antibody or its antigen-binding fragment includes the Fc domain of IgG1, into which a YTE mutation (M252Y / S254T / T256E (EU numbering) as described in US7658921) is introduced at CH2 of the IgG Fc region.

[0121] In another embodiment, the antibody of this disclosure has a potent effector function mediated by Fc, and the antibody mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against target cells.

[0122] In various embodiments, the Fc region is modified 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 different amino acid residues, resulting in an antibody that has a modified affinity for the effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand with modified affinity may be, for example, the Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patents 5,624,821 and 5,648,260 (both by Winter et al.).

[0123] In another embodiment, one or more amino acid residues may be substituted with one or more different amino acid residues so that the antibody has modified C1q binding and / or reduced or absent complement-dependent cell-mediated cytotoxicity (CDC). This technique is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0124] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to immobilize complement. This approach is described, for example, in Bodmer et al.'s published WO94 / 29351. In certain embodiments, one or more amino acids of the antibody or its antigen-binding fragment of the present disclosure are replaced with one or more allotype amino acid residues for the IgG1 subclass and kappa isotype. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant regions of the light chains of kappa isotypes, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0125] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the antibody's ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or to increase the antibody's affinity for the Fcγ receptor. This approach is described, for example, in Presta's publication WO00 / 42072. Furthermore, 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., J. Biol. Chem. 276:6591-6604, 2001).

[0126] In yet another embodiment, the glycosylation of an antibody is modified. For example, a non-glycosylated antibody can be produced (i.e., the antibody lacks glycosylation or has reduced glycosylation). By modifying glycosylation, for example, the affinity of the antibody for an "antigen" can be increased. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, by making one or more amino acid substitutions, one or more variable region framework glycosylation sites are removed, thereby removing glycosylation at that site. Such nonglycosylation can increase the affinity of the antibody for an antigen. Such approaches are described, for example, in U.S. Patents 5,714,350 and 6,350,861 by Co et al.

[0127] Additionally or alternatively, antibodies with altered glycosylation types can be produced (e.g., low-fucosylated antibodies with reduced fucosyl residue levels, or antibodies with increased bisecting GlcNac structures). Such modified glycosylation patterns have been shown to enhance the ADCC activity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with modified glycosylation pathways. Cells with modified glycosylation pathways have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with modified glycosylation. For example, Hang et al. (EP1,176,195) describe a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, and therefore antibodies expressed in such cell lines exhibit low fucosylation. Presta's publication WO03 / 035835 describes the Lecl3 cell line, a variant CHO cell line with reduced ability to bind fucose to Asn(297)-linked carbohydrates, which also results in reduced fucosylation of antibodies expressed in its host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s WO99 / 54342 describes a cell line manipulated to express glycoprotein-modified glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), so that antibodies expressed in the manipulated cell line show increased bisecting GlcNac structure, leading to increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0128] In another aspect, when reduction of ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to possess only moderate ADCC and little CDC effector function (Moore GL, et al., 2010 MAbs, 2:181-189). However, natural IgG4 has been found to be less stable in acidic buffers or under stress conditions such as elevated temperature (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 binding the antibody to IgG4 Fc manipulated 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 less desirable intrinsic properties of IgG4 is its ability to dynamically separate its two heavy chains in solution to form half an antibody, which in vivo generates a bispecific antibody via a process called "Fab arm exchange" (Van der Neut Kolfschoten M., et al., 2007 Science, 317:1554-157). A serine-to-proline mutation at position 228 (EU numbering system) appeared to inhibit the separation of the IgG4 heavy chain (Angal, S. 1993 Mol Immunol, 30:105-108, Aalberse et al., 2002 Immunol, 105:9-19).Some amino acid residues in the hinge and γFc region have been reported to affect antibody interactions with the Fcγ receptor (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, K. Let 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, several IgG4 isoforms that occur rarely in the human population can also induce different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To produce antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc region of human IgG4 and introduce several modifications. These modified IgG4 Fc molecules can be found in Sequence IDs 83-88 of U.S. Patent No. 8,735,553 by Li et al.

[0129] In another embodiment, the antibody of this disclosure comprises the Fc domain of human IgG4 having the S228P and / or R409K substitution (according to the EU numbering system).

[0130] Amino acid linker The domains and / or regions of the polypeptide chains of the bispecific tetravalent antibodies or constructs disclosed herein may be separated by linker regions of varying lengths. In some embodiments, antigen-binding domains are separated from each other by linker regions from the entire CL, CH1, hinge, CH2, CH3, or Fc region. For example, a polypeptide chain may contain the sequence VL1-CL-(linker)VH2-CH1, VH-linker-VL. Such linker regions may contain random amino acid classifications or limited sets of amino acids. Such linker regions may be flexible or rigid (see US2009 / 0155275). The inclusion of amino acid linkers has little to no effect on the activity of the antibodies disclosed herein.

[0131] Multispecific antibodies can be dimerized via dimerization mechanisms such as leucine zippers (Kostelny et al., J.Immunol. 1992 148:1547-53, de Kruifetal J.Biol.Chem. 1996 271:7630-4) and Ig C / CH1 domains (Muller et al., FEBS Lett. 422:259-64), into diabodies (Holliger et al., (1993) Proc.Nat.Acad.Sci.USA. 1998 90:6444-8, Zhu et al., Bio / Technology (NY) 1996 14:192-6), Fab-scFv fusion (Schoonjans et al., J.Immunol. 2000 165:7050-7), and mini-antibody formats (Pack et al., Biochemistry). Fv (scFv) or Fab fragments are constructed by genetically fusing two single-stranded Fv (scFv) or Fab fragments with or without a flexible linker, as described by Pack et al., Bio / Technology 1993 11:1271-7 (Mallender et al., J. Biol. Chem. 1994 269:199-206, Mack et al., Proc. Natl. Acad. Sci. USA. 1995 92:7021-5, Zapata et al., Protein Eng. 1995 8.1057-62).

[0132] The multispecific antibodies and constructs disclosed herein may include a linker region between one or more of the antigen-binding domain, CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain, or Fc region, comprising 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. In some embodiments, the linker region is composed of the amino acids glycine and serine.The linker is the sequence GS (sequence number 79), GGS (sequence number 80), GSG (sequence number 81), SGG (sequence number 82), GGG (sequence number 83), GGGS (sequence number 84), SGGG (sequence number 85), GGGGS (sequence number 86), GGGGSGS (sequence number 87), GGGGSGS (sequence number 88), GGGGSGGS (sequence number 89), GGGGSGGGGS (sequence number 90), GGGGSGGGGSGGGGS (sequence number 91), AKTTPKLEEGEFSEAR (sequence number Column number 92), AKTTPKLEEGEFSEARV (sequence number 93), AKTTPKLGG (sequence number 94), SAKTTPKLGG (sequence number 95), AKTTPKLEEGEFSEARV (sequence number 96), SAKTTP (sequence number 97), SAKTTPKLGG (sequence number 98), RADAAP (sequence number 99), RADAAPTVS (sequence number 100), RADAAAAGGPGS (sequence number 101), RADAAAA(G4S)4 (sequence number 102), SAKTTP (distribution Column number 103), SAKTTPKLGG (SEQ ID NO: 104), SAKTTPKLEEGEFSEARV (SEQ ID NO: 105), ADAAP (SEQ ID NO: 106), ADAAPTVSIFPP (SEQ ID NO: 107), TVAAP (SEQ ID NO: 108), TVAAPSVFIFPP (SEQ ID NO: 109), QPKAAP (SEQ ID NO: 110), QPKAAPSVTLFPP (SEQ ID NO: 111), AKTTPP (SEQ ID NO: 112), AKTTPPSVTPLAP (SEQ ID NO: 113), AKTTAP (Sequence ID: 103), SAKTTPKLGG (SEQ ID NO: 104), SAKTTPKLEEGEFSEARV (SEQ ID NO: 105), ADAAP (SEQ ID NO: 106), ADAAPTVSIFPP (SEQ ID NO: 107), TVAAP (SEQ ID NO: 108), TVAAPSVFIFPP (SEQ ID NO: 109), QPKAAP (SEQ ID NO: 110), QPKAAPSVTLFPP (SEQ ID NO: 111), AKTTPP (SEQ ID NO: 112), AKTTPPSVTPLAP (SEQ ID NO: 113), AKTTAP (Sequence ID NO: 103), SAKTTPKLGG (SEQ ID NO: 104), SAKTTPKLEEGEFSEARV (SEQ ID NO: 105), ADAAP (SEQ ID NO: 106), ADAAPTVSIFPP (SEQ ID NO: 107), TVAAP (SEQ ID NO: 108), TVAAPSVFIFPP (SEQ ID NO: 109), QPKAAP (SEQ ID NO: 110), QPKAAPSVTLFPP (SEQ ID NO: 111 This may include numbers 114), AKTTAPSVYPLAP (sequence number 115), ASTKGP (sequence number 116), ASTKGPSVFPLAP (sequence number 117), GENKVEYAPALMALS (sequence number 118), GPAKELTPLKEAKVS (sequence number 119), and GHEAAAVMQVQYPAS (sequence number 120), GGGGSGGGGSGGGGSGGGGS (sequence number 121) or any combination thereof (see WO2007 / 024715).

[0133] Dimerization-specific amino acids In one embodiment, a polyvalent antibody or construct includes at least one dimerization-specific amino acid change. This dimerization-specific amino acid change may result in a "knob-into-hole" interaction and may increase the likelihood of the desired polyvalent antibody being correctly assembled. The dimerization-specific amino acid may be within a CH1 domain, a CL domain, or a combination thereof. Suitable dimerization-specific amino acids used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL) can be found in at least the disclosures WO2014082179, WO2015181805, and WO2017059551. The dimerization-specific amino acid may also be within an Fc domain and may be combined with a dimerization-specific amino acid within a CH1 or CL domain. In one embodiment, the disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair.

[0134] antibody generation Anti-B7H3 antibodies and their antigen-binding fragments 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 may be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0135] This disclosure further provides polynucleotides encoding antibodies as described herein, for example, polynucleotides encoding a variable region or segment of a heavy chain or light chain containing a complementarity-determining region as 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 with a polynucleotide selected from Tables 1-2.

[0136] The polynucleotides of this disclosure can encode variable region sequences of anti-B7H3 antibodies. They can also encode both the variable and constant regions of antibodies. Some of the polynucleotide sequences encode polypeptides containing both the heavy and light chain variable regions of one of the exemplified anti-B7H3 antibodies.

[0137] In some embodiments, the polynucleotides described herein are codon-optimized for expression in host cells, eukaryotic cells, and more specifically mammalian cells (e.g., CHO cells).

[0138] This disclosure also provides expression vectors and host cells for generating anti-4Ig-B7H3 antibodies. The choice of expression vector depends on the host cells in which the vector is intended to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably ligated to a polynucleotide encoding an anti-4Ig-B7H3 antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent the expression of the insertion sequence outside of controlled induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Culture of transformed organisms can be grown under non-inducible conditions without biasing the population towards coding sequences in which the expression product is better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for the efficient expression of anti-4Ig-B7H3 antibodies or antigen-binding fragments. These elements typically include the ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by incorporating the appropriate enhancer into the cell line being used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, an SV40 enhancer or a CMV enhancer can be used to increase expression in mammalian host cells.

[0139] The host cells for harboring and expressing the anti-4Ig-B7H3 antibody vector may be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of this disclosure. Other suitable microbial hosts include rods such as Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors containing expression regulatory sequences (e.g., origins of replication) adapted to the host cell can typically be constructed. Furthermore, there are many well-known promoters available, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or phage-lambda-derived promoter systems. Promoters typically contain (optionally, operator sequences) ribosome-binding site sequences for controlling expression and initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express the anti-4Ig-B7H3 polypeptide. Insect cells can also be used in combination with baculovirus vectors.

[0140] In other embodiments, mammalian host cells are used to express and produce the anti-4Ig-B7H3 polypeptide of this disclosure. For example, these may be hybridoma cell lines expressing endogenous immunoglobulin genes, or mammalian cell lines having exogenous expression vectors. These include any normal non-immortal, 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 cultures for polypeptide expression is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Mammalian host cell expression vectors may contain expression regulatory sequences such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Appropriate promoters may be constitutive, cell type-specific, stage-specific, and / or tunable or modifiable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (e.g., the human early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0141] Detection and diagnostic methods The antibodies or antigen-binding fragments of this 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 “detection” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues. In other embodiments, such tissues include normal tissues and / or cancerous tissues that express 4Ig-B7H3 at higher levels than other tissues.

[0142] In one embodiment, the present disclosure provides a method for detecting the presence of 4Ig-B7H3 in a biological sample. In a particular embodiment, the method includes contacting a biological sample with an anti-4Ig-B7H3 antibody or its antigen-binding fragment under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, urine, tissue, sputum, or blood.

[0143] The method also includes a method for diagnosing disorders associated with 4Ig-B7H3 expression. In certain embodiments, the method includes contacting test cells with an anti-4Ig-B7H3 antibody and detecting the binding of the anti-4Ig-B7H3 antibody to the 4Ig-B7H3 polypeptide to determine the level of 4Ig-B7H3 expression (either quantitatively or qualitatively) expressed by the test cells, and comparing the expression level in the test cells with the 4Ig-B7H3 expression level in control cells (e.g., normal cells or non-4Ig-B7H3 expressing cells of the same tissue origin as the test cells), where a higher level of 4Ig-B7H3 expression in the test cells compared to control cells indicates the presence of a disorder associated with 4Ig-B7H3 expression.

[0144] Pharmaceutical compositions and preparations Furthermore, compositions are provided comprising a pharmaceutical formulation containing an anti-4Ig-B7H3 antibody, its antigen-binding fragment, a multispecific antibody, or a polynucleotide containing a sequence encoding an anti-4Ig-B7H3 antibody, its antigen-binding fragment, or a multispecific antibody. These compositions may further comprise a suitable carrier, for example, a pharmaceutically acceptable excipient containing a buffer well known in the art.

[0145] The pharmaceutical formulations of anti-4Ig-B7H3 antibodies or their 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 a desired degree of purity with one or more optionally selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphoric acid, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as 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 approximately 10 residues) polypeptides, and proteins. Examples of pharmaceutically acceptable carriers herein include, but are not limited to, hydrophilic polymers such as serum albumin, gelatin, or immunoglobulins, polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other hydrocarbons including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and uses, including rHuPH20, are described in U.S. Patents US7,871,607 and 2006 / 0104968.In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0146] An exemplary lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0147] Sustained-release formulations can be prepared. Suitable examples of sustained-release preparations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody, where these matrices are in the form of molded articles, such as films or microcapsules.

[0148] Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0149] equivalent While the anti-human 4Ig-B7H3 antibody and its antigen-binding fragment have been described in connection with their detailed description, it should be understood that the foregoing description is illustrative and does not limit the scope of the invention as defined by the attached claims. Other embodiments, advantages, and modifications are included in the following claims.

[0150] It will be understood that one, some, any, or all of the characteristics of the various embodiments disclosed herein may be combined to form further embodiments of the Disclosure. These and other embodiments of the Disclosure will be apparent to those skilled in the art. [Examples]

[0151] Example 1. Production of anti-huB7H3 VHH antibody B7H3 recombinant protein for phage campaign and binding assays To discover VHH antibodies against human B7H3, several recombinant proteins were designed, expressed, and subjected to phage panning and screening. The cDNA coding region of full-length human 4Ig-B7H3 (hu 4Ig-B7H3) was ordered based on the B7H3 GenBank sequence (accession number: NM_001024736.1, gene from Sinobio, catalog: HG11188-M, referred to herein as SEQ ID NO: 1). Briefly, the coding region of the extracellular domain (ECD) of hu 4Ig-B7H3, consisting of amino acids (AA) 29-466 (SEQ ID NO: 2), was PCR amplified. The coding regions of human IgG1 (huIgG1)Fc (SEQ ID NOs. 3-4) and mouse IgG2a (mIgG2a)Fc (SEQ ID NOs. 5-6) were amplified by PCR and then conjugated with the ECD of hu 4Ig-B7H3 (SEQ ID NOs. 58) by ligation to produce the huIgG1 Fc fusion protein hu 4Ig-B7H3 ECD-huIgG1 and the mIgG2a Fc fusion protein hu 4Ig-B7H3 ECD-mIgG2a. Next, the PCR products were cloned into pcDNA3.1-based expression vectors (Invitrogen, Carlsbad, CA, USA) to obtain recombinant huIgG1 Fc fusion protein expression plasmids and mIgG2a Fc fusion protein expression plasmids. For the generation of recombinant fusion proteins, the plasmids were transiently transfected into a HEK293-based mammalian cell expression system (developed in-house) and cultured for 5-7 days in a CO2 incubator equipped with a rotary shaker. The supernatant containing the recombinant proteins was collected and clarified by centrifugation. The recombinant proteins were purified using a Protein A column (catalog: 17127901, GE Life Sciences) or Ni-NTA agarose (catalog: R90115, Invitrogen). All recombinant proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots in a freezer at -80°C.

[0152] Lama immunization and phage library construction One llama was immunized with hu4Ig-B7H3 ECD-mIgG2a. Two weeks after the fourth immunization, llama PBMCs were collected for RNA extraction using standard techniques (P. Chomczynski, et al., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Analytical Biochem 162(1)1987 156-159).

[0153] Phage libraries were constructed by reverse transcription and splice overlap extension PCR. PCR products were double digested with NcoI / NotI and ligated to the phagemid vector Pcantab-5E. The repertoire was then transformed into Escherichia coli TG1 bacteria and validated by DNA Sanger sequencing of random clones (>96 clones were analyzed). After a rescue step using the KM13 helper phage, phages were purified directly from the culture supernatant by two precipitations using PEG / NaCl. Following transformation into E. coli bacteria, libraries of size >10⁷ were obtained.

[0154] Phage display panning and screening Phage display selection was performed using a standard protocol for phage display (Silacci et al., (2005) Proteomics, 5, 2340-50; Zhao et al., (2014) PLoS One, 9, e111339). Briefly, in rounds 1 and 2, 10 μg / ml of immobilized hu 4Ig-B7H3 ECD-huIgG1 in immunotubes (catalog 470319, ThermoFisher) was used. For selection in round 3, NK92mi cells overexpressing hu 4Ig-B7H3 (NK92mi / hu 4Ig-B7H3 cells) were used. The immunotubes were blocked in PBS containing 5% milk powder (w / v) for 1 hour. 2.69 × 10¹² (Round 1) and 9.00 × 10¹² (Round 2) phages were depleted with CD40-mIgG2a over 1 hour with PBS containing 5% skim milk, and then incubated with the antigen for 1 hour. For selection in Round 3, cell panning was performed using NK92mi / hu 4Ig-B7H3 cells (NK92mi cells were used as depleted cells). After washing with TBST, bound phages were eluted with 100 mM triethylamine (Sigma-Aldrich). The eluted phages were used to infect metaphase logarithmic E. coli TG1 bacteria and seeded on 2×YT plates supplemented with 2% glucose and 100 μg / ml ampicillin. After selection in Round 3, individual clones were harvested and phage-containing supernatants were prepared using a standard protocol. Anti-hu 4Ig-B7H3 VHH antibodies were screened using phage ELISA.

[0155] In the phage ELISA, Maxisorp immunoplates were coated with antigen and blocked with PBS buffer containing 3% milk powder (w / v). The phage supernatant was added to the wells of the ELISA plate for 1 hour. After washing with TBST, conjugated phages were detected using HRP-conjugated anti-M13 antibody (SinoBiological) and 3,3',5,5'-tetramethylbenzidine substrate (Invitrogen).

[0156] Expression and purification of Fc-fusion VHH antibody Next, using an expression vector developed in-house, anti-hu 4Ig-B7H3 VHH antibodies were constructed in human Fc-fusion VHH antibody format (VHH-Fc). The VHH domain antibody was fused to the N-terminus of human IgG1 Fc (SEQ ID NOs. 17-18). Expression and preparation of the Fc-fusion VHH antibodies were achieved by transfection into 293G cells and purification using a Protein A column (GE Healthcare, 17-5438-02). The purified antibodies were concentrated in PBS and stored in aliquots in a freezer at -80°C.

[0157] Example 2. Characterization of purified anti-hu4Ig-B7H3 VHH antibody In antigen ELISA, Maxisorp immunoplates were coated with the antigen hu 4Ig-B7H3 ECD-mIgG2a or mouse B7H3-his (SinoBiological, 50973-M08H) and blocked with PBS buffer containing 3% BSA (w / v) (blocking buffer). Monoclonal VHH antibody was added to the wells of the ELISA plate for 1 hour. After washing with TBST, conjugated antibodies were detected using HRP-conjugated anti-human IgG antibody (Sigma, A0170) and 3,3',5,5'-tetramethylbenzidine substrate (Invitrogen). Figure 1 shows the analysis of two representative clones (BGA-026 and BGA-056) by ELISA. The results show that BGA-026 (SEQ ID NOs. 7-11) and BGA-056 (SEQ ID NOs. 12-16) bind to hu 4Ig-B7H3 with better affinity compared to mouse B7H3-His (SinoBiological, 50973-M08H).

[0158] To determine affinity, the binding affinity between BGA-026 or BGA-056 and the antigen, human B7H3(ECD)-His(SinoBiological,11188-H08H), was tested using surface plasmon resonance (SPR) technology. The binding profiles of BGA-026 and BGA-056 determined by SPR are summarized in Table 3. Both BGA-026 and BGA-056 showed good binding affinity to human B7H3. [Table 3]

[0159] For cell binding analysis of BGA-026 and BGA-056, HEK293 cells were seeded in 96-well plates and incubated with a diluted series of BGA-026 or BGA-056. Alexa Fluor® 647 anti-human IgG Fc antibody (BioLegend, 410714) was used as a secondary antibody to detect antibody binding to the cell surface. The EC50 values ​​for dose-dependent binding to HEK293 cells were determined by fitting dose-response data using GraphPad Prism. The cell binding profiles for BGA-026 and BGA-056 are summarized in Figure 2 and Table 4. [Table 4]

[0160] The amino acid sequences and DNA sequences of BGA-026 and BGA-056 are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0161] Example 3. Humanization of BGA-026 To humanize BGA-026, we searched for human germline IgG genes that share a high degree of homology with the protein sequence of the BGA-026 variable region by blasting the IMGT human immunoglobulin gene database. The human IGVH 3-23*04 gene, which is frequently present in the human antibody repertoire and has high homology with BGA-026, was selected as the template for humanization.

[0162] Humanized variants were constructed in VHH-human IgG1 Fc (SEQ ID NO: 18) format using a proprietary expression vector with readily compatible subcloning sites. The humanized variants were expressed by transfection of the construct into Expi293F cells and purified using a Protein A column (catalog: 17-5438-02, GE Life Sciences). The purified antibodies were concentrated to 0.5–5 mg / ml in PBS and stored in aliquots in a -80°C freezer.

[0163] Framework swapping based on BGA-026 Humanization was performed by CDR transplantation, preserving important revertant mutations. Humanized VHH antibodies derived from BGA-026 were manipulated in VHH-human IgG1 Fc format using a proprietary expression vector. In the initial round of humanization, mutations from the camelid variable region to human amino acid residues in the framework region were induced by 3D structural analysis. In the first round of humanization, structurally important camelid framework residues (including 9 amino acid residues (P26, R27, Q30, F37, E44, R45, F47, V78, and A94 (Kabat numbering))) were retained to maintain the canonical structure of the CDR, resulting in BGA-154 (Table 9, SEQ ID NOs. 20-21). The binding affinity between BGA-154 and human B7H3(ECD)-His (catalog: 11188-H08H, SinoBiological) was tested using SPR. BGA-154 exhibited good binding affinity comparable to that of the chimeric camelid antibody BGA-026 (Table 6).

[0164] Variants with point mutations based on BGA-154 Single-point mutations based on BGA-154 were performed on the nine revertant mutations listed above, and their significance was investigated (BGA-1541 to BGA-1549, Table 9, SEQ ID NOs. 22 to 39).

[0165] The binding affinity of BGA-1541 to BGA-1549 was tested by SPR using human B7H3(ECD)-His (catalog: 11188-H08H, SinoBiological). The binding affinity is shown in Table 6. SPR analysis of BGA-1541 to BGA-1549 identified three critical reversion sites: P26, F37, and A94. [Table 6]

[0166] BGA-2041 to BGA-2046 were generated by performing combination mutations on the other six amino acid sites while retaining the revert mutations in three amino acids (P26, F37, A94) (Table 7, SEQ ID NOs. 40-51). The binding affinity of BGA-2041 to BGA-2046, as tested by SPR, is shown in Table 7. All of BGA-2041 to BGA-2046 exhibit good binding affinity to human B7H3. SPR analysis of BGA-2041 to BGA-2046 revealed that variants BGA-2041, BGA-2042, and BGA-2046 have affinity equivalent to that of the BGA-026 chimeric antibody.

[0167] Using an expression vector developed in-house, anti-hu 4Ig-B7H3 VHH antibodies were constructed in human Fc-fusion VHH antibody format (VHH-Fc). Expression and preparation of the Fc-fusion VHH antibodies were achieved by transfection into 293G cells and purification using a Protein A column (GE Healthcare, 17-5438-02). The purified antibodies were concentrated in PBS and stored in aliquots in a -80°C freezer. All tested antibodies except BGA-1541, BGA-1544, and BGA-1549 (see Tables 6 and 7) showed good affinity and high purity, and were easily purified. [Table 7]

[0168] Determination of cell binding of humanized variants from BGA-026 The binding activity of BGA-026 chimeric antibody and humanized variant BGA-2042 was evaluated using the H358 non-small cell lung cancer cell line, which naturally expresses high levels of B7H3. H358 cells were seeded in 96-well plates and incubated with a series of diluted BGA-026 chimeric antibodies and humanized variant BGA-2042. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The EC50 values ​​of dose-dependent binding to H358 cells were determined by fitting dose-response data to a 4-parameter logistic model using GraphPad Prism. The data shown in Figure 3 and Table 8 demonstrate that humanized variant BGA-2042 exhibits comparable binding affinity to BGA-026 chimeric antibody and possesses the ability to bind to natural B7H3 on cells. [Table 8] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11] [Table 9-12]

[0169] Example 4. Humanization of BGA-056 To humanize BGA-056, we searched for human germline IgG genes that share a high degree of homology with the protein sequence of the BGA-056 variable region by blasting the IMGT human immunoglobulin gene database. The human IGVH 3-23*01 gene, which is frequently present in the human antibody repertoire and has high homology with BGA-056, was selected as the template for humanization.

[0170] Humanized variants were constructed in VHH-human IgG1 Fc format using a proprietary expression vector with readily compatible subcloning sites. The humanized variants were expressed by transfection of the construct into Expi293F cells and purified using a Protein A column (catalog: 17-5438-02, GE Life Sciences). The purified antibodies were concentrated to 0.5–5 mg / ml in PBS and stored in aliquots in a -80°C freezer.

[0171] Framework swapping based on BGA-056 Humanization was performed by CDR transplantation, preserving important revertant mutations. Humanized VHH antibodies derived from BGA-056 were manipulated in VHH-human IgG1 Fc format using a proprietary expression vector. In the first round of humanization, mutations from the camelid variable region in the framework region to human amino acid residues were induced by 3D structural analysis. In the first round of humanization, structurally important camelid framework residues (including two amino acid residues (Q94 and R103 (Kabat numbering))) were preserved to maintain the canonical structure of the CDR, resulting in BGA-174 (Table 10, SEQ ID NOs. 52-53). BGA-174 exhibits equivalent binding affinity to the chimeric camelid antibody BGA-056 (Table 10), demonstrating the preservation of this property.

[0172] Variants with point mutations based on BGA-174 Single-point mutations based on BGA-174 were performed on the two revertant mutations mentioned above, and their significance was investigated (BGA-1741 and BGA-1742, Table 10, Sequence IDs 54-57). The binding affinity of BGA-1741 and BGA-1742 was tested by SPR. This binding affinity is shown in Table 10. BGA-174, BGA-1741, and BGA-1742 have affinity equivalent to BGA-056.

[0173] Using an expression vector developed in-house, we constructed anti-hu 4Ig-B7H3 VHH antibodies in a human Fc-fusion VHH antibody format (VHH-Fc). Expression and preparation of the Fc-fusion VHH antibodies were achieved by transfection into 293G cells and purification using a Protein A column (GE Healthcare, 17-5438-02). Humanized BGA-174, BGA-1741, and BGA-1742 exhibit high affinity and purity, and are easily purified. [Table 10]

[0174] Determination of cell binding of humanized variants from BGA-056 The binding activity of BGA-056 chimeric antibody and humanized variant BGA-174 was evaluated using H358, a non-small cell lung cancer cell line that naturally expresses high levels of B7H3. H358 cells were seeded in 96-well plates and incubated with a series of diluted BGA-056 chimeric antibodies or humanized variant BGA-174. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The EC50 values ​​of dose-dependent binding to B7H3-expressing cell lines were determined by fitting dose-response data to a 4-parameter logistic model using GraphPad Prism. The data shown in Figure 4 and Table 11 demonstrate that humanized variant BGA-174 exhibits comparable binding affinity to BGA-056 chimeric antibody. [Table 11]

[0175] Determination of the affinity of BGA-174 and BGA-174 His-tagged constructs were prepared by adding a 6×His tag to the C-terminus of BGA-2042 (SEQ ID NO: 42) ("VHH1_C001") and BGA-174 (SEQ ID NO: 52) ("VHH1_C002"). The binding affinity and kinetic constants of the purified VHH fragments were determined by surface plasmon resonance (Biacore 8K) at 25°C. 4Ig-B7H3 (catalog: 11188-H02H) and 2Ig-B7H3 (catalog: B73-H5253) proteins were coupled onto a CM5 Biocore sensor derivatized by amine coupling using an anti-human IgG antibody (catalog: 29234600). The VHH fragments were then flowed at a rate of 40 μl / min. The association of VHH with the B7H3 protein was monitored for 120 seconds, and the dissociation of VHH in buffer 1×HBS (0.01M HEPES (pH 7.4), 0.15M NaCl, 3mM EDTA, 0.05% v / v surfactant P20) was monitored for more than 120 seconds.

[0176] Ka and Kd were determined by fitting the real-time sensorgram to a 1:1 binding model using Biacore Insight Evaluation software. The binding dissociation equilibrium constant (KD) was calculated from the following reaction rates. KD (M) = kd / ka.

[0177] The binding kinetic parameters of the VHH protein to the B7H3 protein are shown in Table 12 and Figures 5A - 5B.

Table 12

[0178] Binding activity of the VHH fragment to native B7H3 To evaluate the binding ability of anti - B7H3 VHH to B7H3 on the surface of live cells, 1 × 106 H358 cells or MDA - MB - 453 cells were seeded in 96 - well plates. To generate the VHH dose - response curve, serially diluted VHH protein (0.2 nM - 200 nM) was added to the cells, and the bound VHH was detected using the His - tag antibody iFluor488 (Catalog number A01800). The mean fluorescence intensity (MFI) of each cell population was measured using the Satorius iQue3 system, and the EC50 and Emax of each VHH were determined using a four - parameter logistic model. The results are shown in Table 13 and Figures 6A - 6D.

Table 13

[0179] Epitope mapping of the humanized VHH fragment To evaluate whether two VHH fragments (VHH1_C001 and VHH1_C002) can compete with each other for binding to their respective epitopes on B7H3, a binding competition assay was performed by the tandem method using Octet® RH96. Briefly, the 4Ig-B7H3 protein (Catalog: 11188-H02H) was captured on the Octet® AHC2 biosensor. Then, the biosensor was immersed in a 100 nM VHH1_C002 fragment for 180 seconds. Subsequently, the biosensor was immersed in a mixed solution of 100 nM VHH1_C002 and 100 nM VHH1_C001. As a control group, VHH1_C001 alone or VHH1_C002 alone was tested. The results are shown in Figure 7. The results indicate that the epitopes of VHH1_C001 and VHH1_C002 partially compete with each other.

Table 14-1

Table 14-2

Table 14-3

Claims

1. An antibody or its antigen-binding fragment that specifically binds to human 4Ig-B7H3, (1) A heavy chain variable region (VH) including (a) HCDR1 (heavy chain complementarity determination region 1) of SEQ ID NO: 7, (b) HCDR2 of SEQ ID NO: 8, and (c) HCDR3 of SEQ ID NO: 9, or (2) The antibody or its antigen-binding fragment, comprising (a) HCDR1 of SEQ ID NO: 12, (b) HCDR2 of SEQ ID NO: 13, and (c) a heavy chain variable region including HCDR3 of SEQ ID NO:

14.

2. (1) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, (2) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 20, (3) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 24, (4) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 26, (5) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 30, (6) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 32, (7) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 34, (8) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 36, (9) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 40, (10) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 42, (11) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 44, (12) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 46, (13) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 48, (14) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 50, (15) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 15, (16) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to sequence number 52, (17) A heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 54, or (18) Heavy chain variable region containing an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 56 The antibody or antigen-binding fragment according to claim 1, comprising:

3. The antibody or antigen-binding fragment according to claim 2, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids are inserted, deleted, or substituted in at least one of SEQ ID NOs: 10, 20, 24, 26, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 15, 52, 54, and 56.

4. (1) Heavy chain variable region including sequence number 10, (2) Heavy chain variable region including sequence number 20, (3) Heavy chain variable region including Sequence ID No. 24, (4) Heavy chain variable region including sequence number 26, (5) Heavy chain variable region including sequence number 30, (6) Heavy chain variable region including sequence number 32, (7) Heavy chain variable region including sequence number 34, (8) Heavy chain variable region including sequence number 36, (9) Heavy chain variable region including sequence number 40, (10) Heavy chain variable region including sequence number 42, (11) Heavy chain variable region including sequence number 44, (12) Heavy chain variable region including sequence number 46, (13) Heavy chain variable region including sequence number 48, (14) Heavy chain variable region including sequence number 50, (15) Heavy chain variable region including sequence number 15, (16) Heavy chain variable region including sequence number 52, (17) Heavy chain variable region including sequence number 54, or (18) Heavy chain variable region including sequence number 56 An antibody or antigen-binding fragment thereof according to any one of the prior claims, comprising:

5. An antibody or antigen-binding fragment thereof according to any one of the prior claims, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-modified antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a heavy-chain antibody (HcAb), or VHH.

6. The antibody or antigen-binding fragment according to claim 5, wherein the antibody is VHH.

7. At least a first antigen-binding domain that specifically binds to a first human tumor antigen (TAA), wherein the first TAA is human 4Ig-B7H3, and the first antigen-binding domain comprises an antibody or antigen-binding fragment described in any one of the prior claims, At least a second antigen-binding domain that specifically binds to a second human TAA, A multispecific antibody or its antigen-binding fragment, including the above.

8. The multispecific antibody or its antigen-binding fragment according to claim 7, wherein the multispecific antibody is a bispecific antibody.

9. A multispecific antibody or antigen-binding fragment according to claim 7 or claim 8, comprising an amino acid linker, wherein the amino acid linker is a sequence of any of SEQ ID NOs. 79 to SEQ ID NOs.

121.

10. An antibody or antigen-binding fragment according to any one of the prior claims, comprising a heavy chain constant region of a subclass of IgG1, IgG2, IgG3, or IgG4, and / or a kappa-type or lambda-type light chain constant region.

11. An antibody or antigen-binding fragment according to any one of the prior claims, comprising a heavy chain constant region of an IgG1 subclass.

12. An antibody or antigen-binding fragment thereof according to any one of the prior claims, having antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC).

13. An antibody or antigen-binding fragment according to any one of the prior claims, which is low-glycosylated, or not glycosylated, or low-fucosylated.

14. An antibody or antigen-binding fragment according to any one of the prior claims, comprising an increased bisecting GlcNac structure.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of the prior claims, and a pharmaceutically acceptable carrier.

16. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14.

17. A vector comprising the nucleic acid described in claim 16.

18. A host cell comprising the nucleic acid described in claim 16 or the vector described in claim 17.

19. A process for generating an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 18, and recovering the antibody or antibody fragment from the culture.

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