B7H3 binder

Abstract

The present invention relates to novel antigen-binding molecules that specifically bind to B7H3. The present invention also relates to chimeric antigen receptors (CARs) and antibody-drug conjugates (ADCs) comprising the antigen-binding molecules. Also provided are uses of the antigen-binding molecules, CARs, and ADCs, and pharmaceutical compositions comprising the antigen-binding molecules, CARs, and ADCs.

Description

Technical Field

[0001] The present invention relates to novel antigen-binding molecules that specifically bind to B7H3. The present invention also relates to chimeric antigen receptors (CARs) and antibody-drug conjugates (ADCs) comprising the antigen-binding molecules. Also provided are uses of the antigen-binding molecules, CARs, and ADCs, as well as pharmaceutical compositions comprising the antigen-binding molecules, CARs, and ADCs.

Background Art

[0002] Immunotherapy in the form of CAR T cell technology has brought complete clinical effects and long-term cures to many patients with B cell malignancies that would otherwise have been refractory. Despite such progress, similar success has not been replicated in solid tumors for several reasons, including the relatively lack of suitable antigen targets and issues of penetration and persistence in the solid tumor environment. Pediatric solid tumors pose further challenges due to the rarity of neoantigens and the immunologically "cold" hostile microenvironment.

[0003] B7-H3 (CD276), a member of the immunoglobulin superfamily and B7 family closely related to PD-L1, has emerged as a potential target for cancer immunotherapy in both solid and liquid malignancies occurring in adults and children. B7-H3 is found in most pediatric solid cancers and tends to be upregulated in high-grade tumors, but is relatively absent in healthy cells.

[0004] B7-H3 exists as alternatively spliced isoforms. In mice, there is a single isoform containing two immunoglobulin domains (2×Ig) C1 and V1. In contrast, human cells can also express a larger C1 / V1 / C2 / V2 (4×Ig), which is a near-exact duplication of 2×Ig. 4×Ig is the dominant isoform in human cells, including cancer cells.

[0005] When initially identified, B7-H3 was thought to be involved in T cell activation. However, over time, a series of evidence has come to indicate that its main role is as an inhibitor of both the innate and adaptive immune systems. The mechanism of action of B7-H3 is not well understood and several receptors are thought to be involved, but no study has finally identified the receptor(s) through which B7-H3 transmits signals. Furthermore, B7-H3 is thought to have a non-immunological role in cancer progression, and high expression has been associated with invasion, metastasis, increased resistance to chemotherapy, and poor prognosis.

[0006] To date, two anti-B7-H3 CAR T cell preparations have been reported and are in clinical trials. Both incorporate single-chain Fv fragments (scFvs) adapted from the monoclonal antibodies MGA271 and 376.96, respectively. Preclinical studies of anti-B7-H3 CAR-T using these two scFvs have shown cytotoxic ability against various solid tumors in vitro and in animal models.

[0007] There is a need for improved treatments for cancers such as solid tumors.

Summary of the Invention

[0008] The inventors have identified novel B7-H3 binders with favorable properties that can be used in cancer treatment, particularly in the treatment of many solid tumors. The inventors constructed a scFv library and then screened for binders by ELISA. A ScFv library was constructed from mice immunized with two domains of the 4×Ig isoform of human B7-H3 (the most proximal domain fused to the most distal domain). When a portion of the scFvs identified in the initial screening was cloned into the scFv-Fc protein format, it showed specific binding to human 4×Ig B7-H3 expressed on the surface of human cells. Furthermore, some of these scFvs were produced as chimeric antibodies, showed specific binding to B7-H3 in ELISA, and showed binding to B7-H3 naturally expressed in a neuroblastoma cell line. The selected anti-human B7-H3 scFvs were cloned, for example, into CAR-T and evaluated for anti-tumor reactivity in cytotoxicity, cytokine, and proliferation assays. Further studies have also been conducted using the antigen-binding molecules (e.g., scFvs) of the present invention in antibody-drug conjugates (ADCs) or multispecific antigen-binding molecules (e.g., bispecific antibodies, bispecific T cell engagers (BiTE), etc.). The antigen-binding molecules (e.g., scFvs) of the present invention exhibit excellent antigen-specific cytotoxicity and cytokine secretion levels.

[0009] Accordingly, in a first aspect, the present invention is an antigen-binding molecule comprising a binding domain that specifically binds to B7H3, wherein the binding domain comprises a heavy chain variable domain and / or a light chain variable domain, the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3, the light chain variable domain comprises light chain complementarity-determining regions (LCDRs) 1, 2, and 3, and the antigen-binding molecule (a) the heavy chain variable domain sequence of SEQ ID NO: 2 and the light chain variable domain sequence of SEQ ID NO: 10; or (b) the heavy chain variable domain sequence of SEQ ID NO: 18 and the light chain variable domain sequence of SEQ ID NO: 26; or (c) the heavy chain variable domain sequence of SEQ ID NO: 34 and the light chain variable domain sequence of SEQ ID NO: 42; or (d) The heavy chain variable domain sequence of SEQ ID NO: 50 and the light chain variable domain sequence of SEQ ID NO: 58; or (e) The heavy chain variable domain sequence of SEQ ID NO: 66 and the light chain variable domain sequence of SEQ ID NO: 74; or (f) The heavy chain variable domain sequence of SEQ ID NO: 82 and the light chain variable domain sequence of SEQ ID NO: 90; or (g) The heavy chain variable domain sequence of SEQ ID NO: 98 and the light chain variable domain sequence of SEQ ID NO: 106; or (h) The heavy chain variable domain sequence of SEQ ID NO: 114 and the light chain variable domain sequence of SEQ ID NO: 122; or (i) The heavy chain variable domain sequence of SEQ ID NO: 130 and the light chain variable domain sequence of SEQ ID NO: 138; or (j) The heavy chain variable domain sequence of SEQ ID NO: 146 and the light chain variable domain sequence of SEQ ID NO: 154; or (k) The heavy chain variable domain sequence of SEQ ID NO: 162 and the light chain variable domain sequence of SEQ ID NO: 170; or (l) The heavy chain variable domain sequence of SEQ ID NO: 178 and the light chain variable domain sequence of SEQ ID NO: 186; or (m) The heavy chain variable domain sequence of SEQ ID NO: 194 and the light chain variable domain sequence of SEQ ID NO: 202; or (n) The heavy chain variable domain sequence of SEQ ID NO: 210 and the light chain variable domain sequence of SEQ ID NO: 218; or (o) The heavy chain variable domain sequence of SEQ ID NO: 226 and the light chain variable domain sequence of SEQ ID NO: 234; or (p) The heavy chain variable domain sequence of SEQ ID NO: 242 and the light chain variable domain sequence of SEQ ID NO: 250; or (q) Provided is an antigen-binding molecule comprising the complementarity determining region (CDR) sequences of the heavy chain variable domain sequence of SEQ ID NO: 258 and the light chain variable domain sequence of SEQ ID NO: 266.

[0010] In a preferred embodiment, the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 66 and the light chain variable domain sequence of SEQ ID NO: 74.

[0011] In a second aspect, the present invention provides an antigen-binding molecule comprising a binding domain that specifically binds to B7H3, wherein the binding domain comprises a heavy-chain variable domain and / or a light-chain variable domain, the heavy-chain variable domain comprises heavy-chain complementarity-determining regions (HCDRs) 1, 2, and 3, and the light-chain variable domain comprises light-chain complementarity-determining regions (LCDRs) 1, 2, and 3, (a) HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, and LCDR3 comprises the sequence of SEQ ID NO: 16; or (b) HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, and LCDR3 comprises the sequence of SEQ ID NO: 32; or (c) HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, and LCDR3 comprises the sequence of SEQ ID NO: 48; or (d) HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, and LCDR3 comprises the sequence of SEQ ID NO: 64; or (e) HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, and LCDR3 comprises the sequence of SEQ ID NO: 80; or (f) HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, and LCDR3 comprises the sequence of SEQ ID NO: 96; or (g) The HCDR1 comprises the sequence of SEQ ID NO: 100, the HCDR2 comprises the sequence of SEQ ID NO: 102, the HCDR3 comprises the sequence of SEQ ID NO: 104, the LCDR1 comprises the sequence of SEQ ID NO: 108, the LCDR2 comprises the sequence of SEQ ID NO: 110, and the LCDR3 comprises the sequence of SEQ ID NO: 112; or (h) The HCDR1 comprises the sequence of SEQ ID NO: 116, the HCDR2 comprises the sequence of SEQ ID NO: 118, the HCDR3 comprises the sequence of SEQ ID NO: 120, the LCDR1 comprises the sequence of SEQ ID NO: 124, the LCDR2 comprises the sequence of SEQ ID NO: 126, and the LCDR3 comprises the sequence of SEQ ID NO: 128; or (i) The HCDR1 comprises the sequence of SEQ ID NO: 132, the HCDR2 comprises the sequence of SEQ ID NO: 134, the HCDR3 comprises the sequence of SEQ ID NO: 136, the LCDR1 comprises the sequence of SEQ ID NO: 140, the LCDR2 comprises the sequence of SEQ ID NO: 142, and the LCDR3 comprises the sequence of SEQ ID NO: 144; or (j) The HCDR1 comprises the sequence of SEQ ID NO: 148, the HCDR2 comprises the sequence of SEQ ID NO: 150, the HCDR3 comprises the sequence of SEQ ID NO: 152, the LCDR1 comprises the sequence of SEQ ID NO: 156, the LCDR2 comprises the sequence of SEQ ID NO: 158, and the LCDR3 comprises the sequence of SEQ ID NO: 160; or (k) The HCDR1 comprises the sequence of SEQ ID NO: 164, the HCDR2 comprises the sequence of SEQ ID NO: 166, the HCDR3 comprises the sequence of SEQ ID NO: 168, the LCDR1 comprises the sequence of SEQ ID NO: 172, the LCDR2 comprises the sequence of SEQ ID NO: 174, and the LCDR3 comprises the sequence of SEQ ID NO: 176; or (l) The HCDR1 comprises the sequence of SEQ ID NO: 180, the HCDR2 comprises the sequence of SEQ ID NO: 182, the HCDR3 comprises the sequence of SEQ ID NO: 184, the LCDR1 comprises the sequence of SEQ ID NO: 188, the LCDR2 comprises the sequence of SEQ ID NO: 190, and the LCDR3 comprises the sequence of SEQ ID NO: 192; or (m) The HCDR1 contains the sequence of SEQ ID NO: 196, the HCDR2 contains the sequence of SEQ ID NO: 198, the HCDR3 contains the sequence of SEQ ID NO: 200, the LCDR1 contains the sequence of SEQ ID NO: 204, the LCDR2 contains the sequence of SEQ ID NO: 206, and the LCDR3 contains the sequence of SEQ ID NO: 208; or (n) The HCDR1 contains the sequence of SEQ ID NO: 212, the HCDR2 contains the sequence of SEQ ID NO: 214, the HCDR3 contains the sequence of SEQ ID NO: 216, the LCDR1 contains the sequence of SEQ ID NO: 220, the LCDR2 contains the sequence of SEQ ID NO: 222, and the LCDR3 contains the sequence of SEQ ID NO: 224; or (o) The HCDR1 contains the sequence of SEQ ID NO: 228, the HCDR2 contains the sequence of SEQ ID NO: 230, the HCDR3 contains the sequence of SEQ ID NO: 232, the LCDR1 contains the sequence of SEQ ID NO: 236, the LCDR2 contains the sequence of SEQ ID NO: 238, and the LCDR3 contains the sequence of SEQ ID NO: 240; or (p) The HCDR1 contains the sequence of SEQ ID NO: 244, the HCDR2 contains the sequence of SEQ ID NO: 246, the HCDR3 contains the sequence of SEQ ID NO: 248, the LCDR1 contains the sequence of SEQ ID NO: 252, the LCDR2 contains the sequence of SEQ ID NO: 254, and the LCDR3 contains the sequence of SEQ ID NO: 256; or (q) The HCDR1 contains the sequence of SEQ ID NO: 260, the HCDR2 contains the sequence of SEQ ID NO: 262, the HCDR3 contains the sequence of SEQ ID NO: 264, the LCDR1 contains the sequence of SEQ ID NO: 268, the LCDR2 contains the sequence of SEQ ID NO: 270, and the LCDR3 contains the sequence of SEQ ID NO: 272, to provide an antigen-binding molecule.

[0012] In a third aspect, the present invention is an antigen-binding molecule comprising a binding domain that specifically binds to B7H3, wherein the binding domain comprises a heavy chain variable domain and / or a light chain variable domain, (a) the heavy chain variable domain contains the sequence of SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and the light chain variable domain contains the sequence of SEQ ID NO: 10 or a sequence having at least 90% identity thereto; or (b) The heavy chain variable domain comprises the sequence of SEQ ID NO: 18 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity thereto; or (c) The heavy chain variable domain comprises the sequence of SEQ ID NO: 34 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 42 or a sequence having at least 90% identity thereto; or (d) The heavy chain variable domain comprises the sequence of SEQ ID NO: 50 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 58 or a sequence having at least 90% identity thereto; or (e) The heavy chain variable domain comprises the sequence of SEQ ID NO: 66 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 74 or a sequence having at least 90% identity thereto; or (f) The heavy chain variable domain comprises the sequence of SEQ ID NO: 82 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 90 or a sequence having at least 90% identity thereto; or (g) The heavy chain variable domain comprises the sequence of SEQ ID NO: 98 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 106 or a sequence having at least 90% identity thereto; or (h) The heavy chain variable domain comprises the sequence of SEQ ID NO: 114 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 122 or a sequence having at least 90% identity thereto; or (i) The heavy chain variable domain comprises the sequence of SEQ ID NO: 130 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 138 or a sequence having at least 90% identity thereto; or (j) the heavy chain variable domain comprises the sequence of SEQ ID NO: 146 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 154 or a sequence having at least 90% identity thereto; or (k) the heavy chain variable domain comprises the sequence of SEQ ID NO: 162 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity thereto; or (l) the heavy chain variable domain comprises the sequence of SEQ ID NO: 178 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 186 or a sequence having at least 90% identity thereto; or (m) the heavy chain variable domain comprises the sequence of SEQ ID NO: 194 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 202 or a sequence having at least 90% identity thereto; or (n) the heavy chain variable domain comprises the sequence of SEQ ID NO: 210 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 218 or a sequence having at least 90% identity thereto; or (o) the heavy chain variable domain comprises the sequence of SEQ ID NO: 226 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 234 or a sequence having at least 90% identity thereto; or (p) the heavy chain variable domain comprises the sequence of SEQ ID NO: 242 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 250 or a sequence having at least 90% identity thereto; or (q) the heavy chain variable domain comprises the sequence of SEQ ID NO: 258 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 266 or a sequence having at least 90% identity thereto, to provide an antigen-binding molecule.

[0013] In a preferred embodiment, the binding domain comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 66 or a sequence having at least 90% identity thereto and / or a light chain variable domain comprising the sequence of SEQ ID NO: 74 or a sequence having at least 90% identity thereto.

[0014] In a fourth aspect, the invention provides a chimeric antigen receptor (CAR) or a chimeric co-stimulatory receptor (CCR) comprising an antigen-binding molecule described herein that specifically binds to B7H3.

[0015] In a fifth aspect, the invention provides a cell comprising a CAR described herein, which is a T cell.

[0016] In a sixth aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding molecule or a CAR described herein.

[0017] In a seventh aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable domain or a light chain variable domain described herein.

[0018] In an eighth aspect, the invention provides an expression vector comprising a nucleic acid molecule described herein.

[0019] In a ninth aspect, the invention provides a host cell comprising a nucleic acid molecule or a vector described herein.

[0020] In a tenth aspect, the invention provides an antibody-drug binder (ADC) comprising an antigen-binding molecule described herein linked to a drug.

[0021] In an eleventh aspect, the invention provides a pharmaceutical composition comprising an antigen-binding molecule, a CAR, a cell comprising a CAR, or an ADC described herein, and optionally a pharmaceutically acceptable carrier.

[0022] In a 12th aspect, the present invention provides a method of treating cancer, comprising administering to a subject in need thereof an antigen-binding molecule, CAR, cell comprising a CAR, ADC, or pharmaceutical composition described herein.

[0023] In a 13th aspect, the present invention provides an antigen-binding molecule, CAR, cell comprising a CAR, ADC, or pharmaceutical composition described herein for use in a method of treating cancer.

[0024] In a 14th aspect, the present invention provides a method of detecting cancer in a subject, comprising contacting a biological sample from the subject with an antigen-binding molecule described herein and detecting the antigen-binding molecule bound to the sample, wherein binding of the antigen-binding molecule to the sample indicates that the subject has cancer, and optionally, the cancer is selected from the group consisting of solid tumor, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma, rhabdomyosarcoma, hematological malignancy, acute myeloid leukemia, desmoplastic small round cell tumor (DSRCT), melanoma, breast cancer, prostate cancer, colon cancer, lung cancer, kidney cancer, or pancreatic cancer, or oral squamous cell carcinoma (SCC). BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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Mode for Carrying Out the Invention

[0026] It should be understood that the various uses of the disclosed products and methods can be adjusted according to the specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments of the invention and are not intended to be limiting. Whether above or below, all publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0027] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen-binding molecule" includes "antigen-binding molecules", etc.

[0028] B7H3 B7H3 can be used interchangeably with CD276. B7-H3 is a member of the immunoglobulin (Ig) superfamily. This gene is located on chromosome 15 in humans and chromosome 9 in mice and is highly conserved across different species. B7H3 most commonly exists as a transmembrane protein with a residual cytoplasmic domain and no known signaling motifs. As a result of alternative splicing, multiple isoforms of B7-H3 are produced. Isoform 1 (also known as 4Ig-B7-H3) is the most abundant in humans and most other mammals. It has four Ig subunits in its extracellular domain arranged in the pattern of Ig-V-1, Ig-C-1, Ig-V-2, Ig-C-2. Ig-V-1 and Ig-C-1 are more than 96% identical to Ig-V-2 and Ig-C-2, and the repeating pattern is thought to be due to exon duplication. The second most abundant isoform is isoform 2 (also known as 2Ig-B7-H3), a 2Ig membrane-bound protein composed of Ig-V-1-Ig-C-2. Soluble isoforms of B7H3 are also present in the tumor microenvironment and serum of cancer patients. There is also an artificial truncated isoform, T-B7-H3, which consists of an Ig-V-like type 2 subunit and an Ig-C-like type 2 subunit.

[0029] Antigen-binding molecule As used herein, the term "antigen-binding molecule" includes the whole antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An antigen-binding molecule includes a binding domain. The binding domain interacts with an antigen. For example, an antigen-binding molecule can include a binding domain that binds to B7H3.

[0030] An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. The disclosed antibodies are class-switchable. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The regions of VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).

[0031] CDRs are mainly involved in antigen binding. The CDRs of each chain are typically called CDR1, CDR2, and CDR3 (from the N-terminus to the C-terminus), and are typically identified by the chain on which a particular CDR is located. The light chain CDRs may also be called LCDR1, LCDR2, and LCDR3. The heavy chain CDRs may also be called HCDR1, HCDR2, and HCDR3. The CDR sequences typically run in the order of LCDR1, LCDR2, and LCDR3 in the N-terminus→C-terminus direction in the light chain variable domain, and HCDR1, HCDR2, and HCDR3 in the N-terminus→C-terminus direction in the heavy chain variable domain.

[0032] The sequences of the various light or heavy chain framework regions are relatively conserved within a species. The framework region of an antibody, which is the combination of the light and heavy chain framework regions that make up the construct, plays a role in the arrangement and alignment of the CDRs in three-dimensional space. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0033] The antibody can be a "monoclonal antibody". A monoclonal antibody is an immunoglobulin molecule that is identical to each other and has a single binding specificity and affinity for a specific epitope. These are produced by a single clone of B lymphocytes or by cells transfected with the genes of the light and heavy chains of a single antibody. Monoclonal antibodies (mAbs) can be generated by various techniques including conventional monoclonal antibody methods, such as those disclosed in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Hurrell (CRC Press, 1982).

[0034] The antibody may also be a "chimeric" antibody that contains the sequences of two different antibodies, usually from different species. For example, a chimeric antibody may contain the variable regions of the heavy and light chains from a first species, as well as the constant regions of the heavy and light chains from a second species. In other embodiments, the variable and constant regions of the light chain may be from a first species, while the variable region of the heavy chain is from the first species and the constant region of the heavy chain may be from a second species. In other embodiments, the variable and constant regions of the light chain may be from a first species, while the variable and constant regions of the heavy chain may be from a second species.

[0035] The term "fragment" of an antibody typically refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen, i.e., the "antigen-binding fragment" of the antibody. This antigen-binding molecule or antigen-binding fragment retains the ability to specifically bind to B7H3, preferably human B7H3. Examples of antigen-binding fragments include Fab, Fab’, F(ab)’2, Fd, Fv, single-chain Fab (scFab), single-chain Fv protein (scFv), tandem scFv protein, disulfide-stabilized Fv protein (dsFv), scFv-Fc protein, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, triabody, tetrabody, or any epitope-binding fragment of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). The antigen-binding fragments of the present invention include Fab, Fab’, F(ab)’2, Fd, Fv, single-chain Fab (scFab), single-chain Fv protein (scFv), tandem scFv protein, disulfide-stabilized Fv protein (dsFv), or scFv-Fc protein that specifically bind to human B7H3. These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art. For example, antigen-binding fragments can be prepared by modifying the whole antibody or can be synthesized de novo using recombinant DNA techniques. In one embodiment, the antigen-binding molecule of the present invention is preferably an scFv or scFv-Fc protein. The scFv protein is a fusion protein in which the variable region of the immunoglobulin light chain and the variable region of the immunoglobulin heavy chain are linked by a linker. In dsFv, the chains are mutated to introduce disulfide bonds to stabilize the binding of the chains. This term also includes genetically engineered forms such as chimeric antibodies and hetero-conjugate antibodies such as bispecific antibodies. Also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3 rdSee Ed., W.H. Freeman & Co., New York, 1997.

[0036] The term "binding affinity" refers to the tendency of an antibody molecule to bind or not bind to a target. Binding affinity can be quantified by determining the dissociation constant (Kd) for the antibody and its target. Similarly, the specificity of an antibody's binding to its target can be defined in terms of the comparative dissociation constant (Kd) of the antibody for its target compared to the dissociation constants for the antibody and another non-target molecule. Typically, the Kd of an antibody for its target is 2-fold, preferably 5-fold, more preferably 10-fold lower than the Kd for other non-target molecules. More preferably, the Kd is 50-fold lower, even more preferably 100-fold lower, even more preferably 200-fold lower. The value of this dissociation constant can be determined directly by well-known methods and can be calculated by computer, even for complex mixtures, by methods such as those described in Caceci et al. (Byte 9:340-362, 1984). Preferred methods for evaluating the binding affinity of the antibodies of the present invention for B7H3 include ELISA or Biacore (i.e., surface plasmon resonance).

[0037] The antigen-binding molecules of the present invention bind (e.g., specifically bind) to B7H3 (preferably human B7H3), i.e., preferably bind to B7H3 but do not bind or bind with lower affinity to other molecules. "Specifically bind" means that the antibody binds to B7H3 with a higher affinity than to other targets. Specific binding can be determined by methods known in the art. The antigen-binding molecules of the present invention can preferably bind to B7H3 with an affinity that is at least 2-fold, 10-fold, 50-fold, 100-fold, or more greater than the affinity for binding to another non-target molecule. Preferably, the antigen-binding molecules of the present invention have a binding affinity (i.e., K -9 d) for B7H3 of 1 × 10 D M or less. In some embodiments, the antigen-binding molecule is about 1 × 10 -9 M or less, about 1 × 10 -10M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less of K D binds specifically to B7H3. The antigen-binding molecule of the present invention may have a certain degree of binding affinity for B7H3 of other mammals, such as primates or murine B7H3, such as mouse or rat B7H3. The binding affinity of the antigen-binding molecule of the present invention for B7H3 of other species gradually weakens as the binding epitope is not conserved with the phylogenetic distance. The antigen-binding molecule of the present invention can bind (e.g., specifically bind) to any isoform of B7H3. In one embodiment, the antigen molecule specifically binds to human B7H3. In one embodiment, the antigen-binding molecule specifically binds to human B7H3 isoform 4IgB7-H3. In one embodiment, the antigen-binding molecule specifically binds to human B7H3 isoform 2IgB7-H3. In one embodiment, the antigen-binding molecule has specificity for both human B7H3 isoform 4IgB7-H3 and human B7H3 isoform 2IgB7-H3. In one embodiment, the antigen-binding molecule specifically binds to isoform T-B7-H3. In one embodiment, the antigen-binding molecule has specificity for T-B7-H3, human 4IgB7-H3, and human 2IgB7-H3. In one embodiment, the antigen-binding molecule specifically binds to human B7H3 isoform 4IgB7-H3 and does not bind or has a lower binding affinity for other targets. In one embodiment, the antigen-binding molecule specifically binds to human B7H3 isoform 2IgB7-H3 and does not bind or has a lower binding affinity for other targets. In one embodiment, the antigen-binding molecule has specificity for both human B7H3 isoform 4IgB7-H3 and human B7H3 isoform 2IgB7-H3 and does not bind or has a low binding affinity for other targets. In one embodiment, the antigen-binding molecule specifically binds to isoform T-B7-H3 and does not bind or has a lower binding affinity for other targets. In one embodiment, the antigen-binding molecule has specificity for T-B7-H3, human 4IgB7-H3, and human 2IgB7-H3 and does not bind or has a lower binding affinity for other targets.

[0038] The antigen-binding molecules of the present invention typically bind to the same epitope as an antigen-binding molecule having variable region sequences of the heavy and light chains of (i) SEQ ID NO: 2 and SEQ ID NO: 10, respectively, (ii) SEQ ID NO: 18 and SEQ ID NO: 26, respectively, (iii) SEQ ID NO: 34 and SEQ ID NO: 42, respectively, (iv) SEQ ID NO: 34 and SEQ ID NO: 42, respectively, (v) SEQ ID NO: 66 and SEQ ID NO: 74, respectively, (vi) SEQ ID NO: 82 and SEQ ID NO: 90, respectively, (vii) SEQ ID NO: 98 and SEQ ID NO: 106, respectively, (viii) SEQ ID NO: 114 and SEQ ID NO: 122, respectively, (ix) SEQ ID NO: 130 and SEQ ID NO: 138, respectively, (x) SEQ ID NO: 146 and SEQ ID NO: 154, respectively, (xi) SEQ ID NO: 162 and SEQ ID NO: 170, respectively, (xii) SEQ ID NO: 178 and SEQ ID NO: 186, respectively, (xiii) SEQ ID NO: 194 and SEQ ID NO: 202, respectively, (xiv) SEQ ID NO: 210 and SEQ ID NO: 218, respectively, (xv) SEQ ID NO: 226 and SEQ ID NO: 234, respectively, (xvi) SEQ ID NO: 242 and SEQ ID NO: 250, respectively, or (xvii) SEQ ID NO: 258 and SEQ ID NO: 266, respectively. For example, the antigen-binding molecules of the present invention can bind to the same epitope as an antigen-binding molecule having variable region sequences of the heavy and light chains of SEQ ID NO: 66 and SEQ ID NO: 74, respectively. As used herein, the term "epitope" generally refers to a site on a target antigen that is recognized by an antibody. The position of an epitope can be identified by routine methods. For example, the general position of an epitope can be determined by evaluating the ability of antibodies to bind to different fragments or variant B7H3 polypeptides, e.g., by measuring binding after mutagenesis of specific residues of B7H3. Additionally, an antibody and a target molecule can be combined, and the antibody / target complex can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the antibody and its target.The antigen-binding molecule of the present invention can cross-compete for binding to human B7H3 with another antigen-binding molecule of the present invention, preferably an antigen-binding molecule having the variable region sequences of the heavy and light chains of (i) SEQ ID NO: 2 and 10, respectively, (ii) SEQ ID NO: 18 and 26, respectively, (iii) SEQ ID NO: 34 and 42, respectively, (iv) SEQ ID NO: 34 and 42, respectively, (v) SEQ ID NO: 66 and 74, respectively, (vi) SEQ ID NO: 82 and 90, respectively, (vii) SEQ ID NO: 98 and 106, respectively, (viii) SEQ ID NO: 114 and 122, respectively, (ix) SEQ ID NO: 130 and 138, respectively, (x) SEQ ID NO: 146 and 154, respectively, (xi) SEQ ID NO: 162 and 170, respectively, (xii) SEQ ID NO: 178 and 186, respectively, (xiii) SEQ ID NO: 194 and 202, respectively, (xiv) SEQ ID NO: 210 and 218, respectively, (xv) SEQ ID NO: 226 and 234, respectively, (xvi) SEQ ID NO: 242 and 250, respectively, or (xvii) SEQ ID NO: 258 and 266, respectively. For example, the antigen-binding molecule of the present invention can cross-compete for binding to human B7H3 with another antigen-binding molecule of the present invention, preferably an antigen-binding molecule having the variable region sequences of the heavy and light chains of (i) SEQ ID NO: 2 and 10, respectively, (ii) SEQ ID NO: 18 and 26, respectively, (iii) SEQ ID NO: 34 and 42, respectively, (iv) SEQ ID NO: 34 and 42, respectively, (v) SEQ ID NO: 66 and 74, respectively. Such cross-competing antigen-binding molecules can be identified based on their ability to cross-compete with known antigen-binding molecules of the present invention in standard binding assays such as Biacore analysis, ELISA assays, and flow cytometry.

[0039] The CDRs of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, and 258, and SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, and 266 (i.e., the CDR sequences found within the respective heavy chain variable domain sequences and light chain variable domain sequences of the above SEQ ID NOs) can be identified using any suitable method known in the art, such as any suitable antibody numbering scheme. In some embodiments, the CDRs are identified using either the Kabat numbering scheme (Kabat et al, U.S. Department of Health and Human Services, 1991), the Chothia numbering scheme (Chothia C, Lesk A M. J Mol Biol. (1987) 196:901-17), or the IMGT numbering scheme (Giudicelli V, et al. Nucleic Acids Res. (1997) 25:206-11; Lefranc MP. Immunol Today (1997) 18:509). Those skilled in the art will understand that these different CDR labeling systems may give slightly different results, but in any case, those skilled in the art will be able to easily identify the CDRs. The CDR sequences set forth in SEQ ID NOs: 4, 6, and 8, and 12, 14, and 16 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 2 and 10, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 20, 22, and 24, and 28, 30, and 32 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 18 and 26, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 36, 38, and 40, and 44, 46, and 48 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 34 and 42, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 52, 54, and 56, and 60, 62, and 64 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 50 and 58, respectively, as defined using the Kabat numbering scheme.The CDR sequences described in SEQ ID NOs: 68, 70, and 72, and 76, 78, and 80 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 66 and 74 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 84, 86, and 88, and 92, 94, and 96 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 82 and 90 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 100, 102, and 104, and 108, 110, and 112 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 98 and 106 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 116, 118, and 120, and 124, 126, and 128 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 114 and 122 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 132, 134, and 136, and 140, 142, and 144 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 130 and 138 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 148, 150, and 152, and 156, 158, and 160 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 146 and 154 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 164, 166, and 168, and 172, 174, and 176 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 162 and 170 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 180, 182, and 184, and 188, 190, and 192 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 178 and 186 defined using the Kabat numbering scheme, respectively. The CDR sequences described in SEQ ID NOs: 196, 198, and 200, and 204, 206, and 208 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 194 and 202 defined using the Kabat numbering scheme, respectively.The CDR sequences described in SEQ ID NOs: 212, 214, and 216, and 220, 222, and 224 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 210 and 218 respectively, defined using the Kabat numbering scheme. The CDR sequences described in SEQ ID NOs: 228, 230, and 232, and 236, 238, and 240 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 226 and 234 respectively, defined using the Kabat numbering scheme. The CDR sequences described in SEQ ID NOs: 244, 246, and 248, and 252, 254, and 256 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 242 and 250 respectively, defined using the Kabat numbering scheme. The CDR sequences described in SEQ ID NOs: 260, 262, and 264, and 268, 270, and 272 are the sequences of HCDR1-3 and LCDR1-3 of SEQ ID NOs: 258 and 266 respectively, defined using the Kabat numbering scheme.

[0040] The present invention relates to an antigen-binding molecule comprising a binding domain that specifically binds to B7H3 (e.g., human B7H3). In some embodiments, the binding domain comprises a heavy chain variable domain and / or a light chain variable domain. In some embodiments, the binding domain comprises a heavy chain variable domain. In some embodiments, the binding domain comprises a light chain variable domain. In some embodiments, the binding domain comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 2 and the light chain variable domain sequence of SEQ ID NO: 10. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 2 and SEQ ID NO: 10, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 2 and 10 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, and LCDR3 comprises the sequence of SEQ ID NO: 16. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 4, HCDR2 consists of the sequence of SEQ ID NO: 6, HCDR3 consists of the sequence of SEQ ID NO: 8, LCDR1 consists of the sequence of SEQ ID NO: 12, LCDR2 consists of the sequence of SEQ ID NO: 14, and LCDR3 consists of the sequence of SEQ ID NO: 16.

[0041] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 18 and the light chain variable domain sequence of SEQ ID NO: 26. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 18 and SEQ ID NO: 26. Preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NOs: 18 and 26 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, and LCDR3 comprises the sequence of SEQ ID NO: 32. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 20, HCDR2 consists of the sequence of SEQ ID NO: 22, HCDR3 consists of the sequence of SEQ ID NO: 24, LCDR1 consists of the sequence of SEQ ID NO: 28, LCDR2 consists of the sequence of SEQ ID NO: 30, and LCDR3 consists of the sequence of SEQ ID NO: 32.

[0042] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 34 and the light chain variable domain sequence of SEQ ID NO: 42. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 34 and SEQ ID NO: 42, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 34 and 42 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, and LCDR3 comprises the sequence of SEQ ID NO: 48. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 36, HCDR2 consists of the sequence of SEQ ID NO: 38, HCDR3 consists of the sequence of SEQ ID NO: 40, LCDR1 consists of the sequence of SEQ ID NO: 44, LCDR2 consists of the sequence of SEQ ID NO: 46, and LCDR3 consists of the sequence of SEQ ID NO: 48.

[0043] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 50 and the light chain variable domain sequence of SEQ ID NO: 58. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 50 and SEQ ID NO: 58. Preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 50 and 58 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, and LCDR3 comprises the sequence of SEQ ID NO: 64. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 52, HCDR2 consists of the sequence of SEQ ID NO: 54, HCDR3 consists of the sequence of SEQ ID NO: 56, LCDR1 consists of the sequence of SEQ ID NO: 60, LCDR2 consists of the sequence of SEQ ID NO: 62, and LCDR3 consists of the sequence of SEQ ID NO: 64.

[0044] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 66 and the light chain variable domain sequence of SEQ ID NO: 74. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 66 and SEQ ID NO: 74, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 66 and 74 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, and LCDR3 comprises the sequence of SEQ ID NO: 80. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 68, HCDR2 consists of the sequence of SEQ ID NO: 70, HCDR3 consists of the sequence of SEQ ID NO: 72, LCDR1 consists of the sequence of SEQ ID NO: 76, LCDR2 consists of the sequence of SEQ ID NO: 78, and LCDR3 consists of the sequence of SEQ ID NO: 80.

[0045] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 82 and the light chain variable domain sequence of SEQ ID NO: 90. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 82 and SEQ ID NO: 90, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 82 and 90 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, and LCDR3 comprises the sequence of SEQ ID NO: 96. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 84, HCDR2 consists of the sequence of SEQ ID NO: 86, HCDR3 consists of the sequence of SEQ ID NO: 88, LCDR1 consists of the sequence of SEQ ID NO: 92, LCDR2 consists of the sequence of SEQ ID NO: 94, and LCDR3 consists of the sequence of SEQ ID NO: 96.

[0046] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 98 and the light chain variable domain sequence of SEQ ID NO: 106. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 98 and SEQ ID NO: 106, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 98 and 106 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 100, HCDR2 comprises the sequence of SEQ ID NO: 102, HCDR3 comprises the sequence of SEQ ID NO: 104, LCDR1 comprises the sequence of SEQ ID NO: 108, LCDR2 comprises the sequence of SEQ ID NO: 110, and LCDR3 comprises the sequence of SEQ ID NO: 112. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 100, HCDR2 consists of the sequence of SEQ ID NO: 102, HCDR3 consists of the sequence of SEQ ID NO: 104, LCDR1 consists of the sequence of SEQ ID NO: 108, LCDR2 consists of the sequence of SEQ ID NO: 110, and LCDR3 consists of the sequence of SEQ ID NO: 112.

[0047] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 114 and the light chain variable domain sequence of SEQ ID NO: 122. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 114 and SEQ ID NO: 122, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 114 and 122 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 116, HCDR2 comprises the sequence of SEQ ID NO: 118, HCDR3 comprises the sequence of SEQ ID NO: 120, LCDR1 comprises the sequence of SEQ ID NO: 124, LCDR2 comprises the sequence of SEQ ID NO: 126, and LCDR3 comprises the sequence of SEQ ID NO: 128. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 116, HCDR2 consists of the sequence of SEQ ID NO: 118, HCDR3 consists of the sequence of SEQ ID NO: 120, LCDR1 consists of the sequence of SEQ ID NO: 124, LCDR2 consists of the sequence of SEQ ID NO: 126, and LCDR3 consists of the sequence of SEQ ID NO: 128.

[0048] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 130 and the light chain variable domain sequence of SEQ ID NO: 138. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 130 and SEQ ID NO: 138, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 130 and 138 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be appreciated by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 132, HCDR2 comprises the sequence of SEQ ID NO: 134, HCDR3 comprises the sequence of SEQ ID NO: 136, LCDR1 comprises the sequence of SEQ ID NO: 140, LCDR2 comprises the sequence of SEQ ID NO: 142, and LCDR3 comprises the sequence of SEQ ID NO: 144. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 132, HCDR2 consists of the sequence of SEQ ID NO: 134, HCDR3 consists of the sequence of SEQ ID NO: 136, LCDR1 consists of the sequence of SEQ ID NO: 140, LCDR2 consists of the sequence of SEQ ID NO: 142, and LCDR3 consists of the sequence of SEQ ID NO: 144.

[0049] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 146 and the light chain variable domain sequence of SEQ ID NO: 154. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 146 and SEQ ID NO: 154, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 146 and 154 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 148, HCDR2 comprises the sequence of SEQ ID NO: 150, HCDR3 comprises the sequence of SEQ ID NO: 152, LCDR1 comprises the sequence of SEQ ID NO: 156, LCDR2 comprises the sequence of SEQ ID NO: 158, and LCDR3 comprises the sequence of SEQ ID NO: 160. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 148, HCDR2 consists of the sequence of SEQ ID NO: 150, HCDR3 consists of the sequence of SEQ ID NO: 152, LCDR1 consists of the sequence of SEQ ID NO: 156, LCDR2 consists of the sequence of SEQ ID NO: 158, and LCDR3 consists of the sequence of SEQ ID NO: 160.

[0050] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 162 and the light chain variable domain sequence of SEQ ID NO: 170. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 162 and SEQ ID NO: 170, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 162 and 170 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 164, HCDR2 comprises the sequence of SEQ ID NO: 166, HCDR3 comprises the sequence of SEQ ID NO: 168, LCDR1 comprises the sequence of SEQ ID NO: 172, LCDR2 comprises the sequence of SEQ ID NO: 174, and LCDR3 comprises the sequence of SEQ ID NO: 176. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 164, HCDR2 consists of the sequence of SEQ ID NO: 166, HCDR3 consists of the sequence of SEQ ID NO: 168, LCDR1 consists of the sequence of SEQ ID NO: 172, LCDR2 consists of the sequence of SEQ ID NO: 174, and LCDR3 consists of the sequence of SEQ ID NO: 176.

[0051] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 178 and the light chain variable domain sequence of SEQ ID NO: 186. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 178 and SEQ ID NO: 186. Preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 178 and 186 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 180, HCDR2 comprises the sequence of SEQ ID NO: 182, HCDR3 comprises the sequence of SEQ ID NO: 184, LCDR1 comprises the sequence of SEQ ID NO: 188, LCDR2 comprises the sequence of SEQ ID NO: 190, and LCDR3 comprises the sequence of SEQ ID NO: 192. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 180, HCDR2 consists of the sequence of SEQ ID NO: 182, HCDR3 consists of the sequence of SEQ ID NO: 184, LCDR1 consists of the sequence of SEQ ID NO: 188, LCDR2 consists of the sequence of SEQ ID NO: 190, and LCDR3 consists of the sequence of SEQ ID NO: 192.

[0052] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 194 and the light chain variable domain sequence of SEQ ID NO: 202. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 194 and SEQ ID NO: 202, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 194 and 202 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 196, HCDR2 comprises the sequence of SEQ ID NO: 198, HCDR3 comprises the sequence of SEQ ID NO: 200, LCDR1 comprises the sequence of SEQ ID NO: 204, LCDR2 comprises the sequence of SEQ ID NO: 206, and LCDR3 comprises the sequence of SEQ ID NO: 208. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 196, HCDR2 consists of the sequence of SEQ ID NO: 198, HCDR3 consists of the sequence of SEQ ID NO: 200, LCDR1 consists of the sequence of SEQ ID NO: 204, LCDR2 consists of the sequence of SEQ ID NO: 206, and LCDR3 consists of the sequence of SEQ ID NO: 208.

[0053] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 210 and the light chain variable domain sequence of SEQ ID NO: 218. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 210 and SEQ ID NO: 218, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 210 and 218 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 212, HCDR2 comprises the sequence of SEQ ID NO: 214, HCDR3 comprises the sequence of SEQ ID NO: 216, LCDR1 comprises the sequence of SEQ ID NO: 220, LCDR2 comprises the sequence of SEQ ID NO: 222, and LCDR3 comprises the sequence of SEQ ID NO: 224. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 212, HCDR2 consists of the sequence of SEQ ID NO: 214, HCDR3 consists of the sequence of SEQ ID NO: 216, LCDR1 consists of the sequence of SEQ ID NO: 220, LCDR2 consists of the sequence of SEQ ID NO: 222, and LCDR3 consists of the sequence of SEQ ID NO: 224.

[0054] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 226 and the light chain variable domain sequence of SEQ ID NO: 234. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 226 and SEQ ID NO: 234. Preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 226 and 234 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 228, HCDR2 comprises the sequence of SEQ ID NO: 230, HCDR3 comprises the sequence of SEQ ID NO: 232, LCDR1 comprises the sequence of SEQ ID NO: 236, LCDR2 comprises the sequence of SEQ ID NO: 238, and LCDR3 comprises the sequence of SEQ ID NO: 240. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 228, HCDR2 consists of the sequence of SEQ ID NO: 230, HCDR3 consists of the sequence of SEQ ID NO: 232, LCDR1 consists of the sequence of SEQ ID NO: 236, LCDR2 consists of the sequence of SEQ ID NO: 238, and LCDR3 consists of the sequence of SEQ ID NO: 240.

[0055] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 242 and the light chain variable domain sequence of SEQ ID NO: 250. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 242 and SEQ ID NO: 250, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus as in SEQ ID NO: 242 and 250 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 244, HCDR2 comprises the sequence of SEQ ID NO: 246, HCDR3 comprises the sequence of SEQ ID NO: 248, LCDR1 comprises the sequence of SEQ ID NO: 252, LCDR2 comprises the sequence of SEQ ID NO: 254, and LCDR3 comprises the sequence of SEQ ID NO: 256. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 244, HCDR2 consists of the sequence of SEQ ID NO: 246, HCDR3 consists of the sequence of SEQ ID NO: 248, LCDR1 consists of the sequence of SEQ ID NO: 252, LCDR2 consists of the sequence of SEQ ID NO: 254, and LCDR3 consists of the sequence of SEQ ID NO: 256.

[0056] In some other embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and the antigen-binding molecule comprises the CDR sequences of the heavy chain variable domain sequence of SEQ ID NO: 258 and the light chain variable domain sequence of SEQ ID NO: 266. Typically, the antigen-binding molecule comprises all six CDR sequences of SEQ ID NO: 258 and SEQ ID NO: 266, and preferably, the CDR sequences are arranged in the same order from the N-terminus to the C-terminus, similar to SEQ ID NO: 258 and 266 in the heavy and light chains of the antigen-binding molecule. These CDR sequences are defined using the Kabat numbering scheme. As will be understood by those skilled in the art, the exact CDR sequences may vary depending on the numbering scheme used (e.g., Kabat, Chothia, or IMGT). In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 260, HCDR2 comprises the sequence of SEQ ID NO: 262, HCDR3 comprises the sequence of SEQ ID NO: 264, LCDR1 comprises the sequence of SEQ ID NO: 268, LCDR2 comprises the sequence of SEQ ID NO: 270, and LCDR3 comprises the sequence of SEQ ID NO: 272. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1 consists of the sequence of SEQ ID NO: 260, HCDR2 consists of the sequence of SEQ ID NO: 262, HCDR3 consists of the sequence of SEQ ID NO: 264, LCDR1 consists of the sequence of SEQ ID NO: 268, LCDR2 consists of the sequence of SEQ ID NO: 270, and LCDR3 consists of the sequence of SEQ ID NO: 272.

[0057] The present invention relates to an antigen-binding molecule comprising a binding domain that specifically binds to B7H3 (e.g., human B7H3). In some embodiments, the binding domain comprises a heavy chain variable domain and / or a light chain variable domain. In some embodiments, the binding domain comprises a heavy chain variable domain. In some embodiments, the binding domain comprises a light chain variable domain. In some embodiments, the binding domain comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, and LCDR3 comprises the sequence of SEQ ID NO: 16. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 4, HCDR2 consists of the sequence of SEQ ID NO: 6, HCDR3 consists of the sequence of SEQ ID NO: 8, LCDR1 consists of the sequence of SEQ ID NO: 12, LCDR2 consists of the sequence of SEQ ID NO: 14, and LCDR3 consists of the sequence of SEQ ID NO: 16.

[0058] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, and LCDR3 comprises the sequence of SEQ ID NO: 32. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 20, HCDR2 consists of the sequence of SEQ ID NO: 22, HCDR3 consists of the sequence of SEQ ID NO: 24, LCDR1 consists of the sequence of SEQ ID NO: 28, LCDR2 consists of the sequence of SEQ ID NO: 30, and LCDR3 consists of the sequence of SEQ ID NO: 32.

[0059] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, and LCDR3 comprises the sequence of SEQ ID NO: 48. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 36, HCDR2 consists of the sequence of SEQ ID NO: 38, HCDR3 consists of the sequence of SEQ ID NO: 40, LCDR1 consists of the sequence of SEQ ID NO: 44, LCDR2 consists of the sequence of SEQ ID NO: 46, and LCDR3 consists of the sequence of SEQ ID NO: 48.

[0060] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, and LCDR3 comprises the sequence of SEQ ID NO: 64. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 52, HCDR2 consists of the sequence of SEQ ID NO: 54, HCDR3 consists of the sequence of SEQ ID NO: 56, LCDR1 consists of the sequence of SEQ ID NO: 60, LCDR2 consists of the sequence of SEQ ID NO: 62, and LCDR3 consists of the sequence of SEQ ID NO: 64.

[0061] In some preferred embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, and LCDR3 comprises the sequence of SEQ ID NO: 80. In some preferred embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 68, HCDR2 consists of the sequence of SEQ ID NO: 70, HCDR3 consists of the sequence of SEQ ID NO: 72, LCDR1 consists of the sequence of SEQ ID NO: 76, LCDR2 consists of the sequence of SEQ ID NO: 78, and LCDR3 consists of the sequence of SEQ ID NO: 80.

[0062] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, and LCDR3 comprises the sequence of SEQ ID NO: 96. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 84, HCDR2 consists of the sequence of SEQ ID NO: 86, HCDR3 consists of the sequence of SEQ ID NO: 88, LCDR1 consists of the sequence of SEQ ID NO: 92, LCDR2 consists of the sequence of SEQ ID NO: 94, and LCDR3 consists of the sequence of SEQ ID NO: 96.

[0063] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 100, HCDR2 comprises the sequence of SEQ ID NO: 102, HCDR3 comprises the sequence of SEQ ID NO: 104, LCDR1 comprises the sequence of SEQ ID NO: 108, LCDR2 comprises the sequence of SEQ ID NO: 110, and LCDR3 comprises the sequence of SEQ ID NO: 112. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 100, HCDR2 consists of the sequence of SEQ ID NO: 102, HCDR3 consists of the sequence of SEQ ID NO: 104, LCDR1 consists of the sequence of SEQ ID NO: 108, LCDR2 consists of the sequence of SEQ ID NO: 110, and LCDR3 consists of the sequence of SEQ ID NO: 112.

[0064] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 116, HCDR2 comprises the sequence of SEQ ID NO: 118, HCDR3 comprises the sequence of SEQ ID NO: 120, LCDR1 comprises the sequence of SEQ ID NO: 124, LCDR2 comprises the sequence of SEQ ID NO: 126, and LCDR3 comprises the sequence of SEQ ID NO: 128. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 116, HCDR2 consists of the sequence of SEQ ID NO: 118, HCDR3 consists of the sequence of SEQ ID NO: 120, LCDR1 consists of the sequence of SEQ ID NO: 124, LCDR2 consists of the sequence of SEQ ID NO: 126, and LCDR3 consists of the sequence of SEQ ID NO: 128.

[0065] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 132, HCDR2 comprises the sequence of SEQ ID NO: 134, HCDR3 comprises the sequence of SEQ ID NO: 136, LCDR1 comprises the sequence of SEQ ID NO: 140, LCDR2 comprises the sequence of SEQ ID NO: 142, and LCDR3 comprises the sequence of SEQ ID NO: 144. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 132, HCDR2 consists of the sequence of SEQ ID NO: 134, HCDR3 consists of the sequence of SEQ ID NO: 136, LCDR1 consists of the sequence of SEQ ID NO: 140, LCDR2 consists of the sequence of SEQ ID NO: 142, and LCDR3 consists of the sequence of SEQ ID NO: 144.

[0066] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 148, HCDR2 comprises the sequence of SEQ ID NO: 150, HCDR3 comprises the sequence of SEQ ID NO: 152, LCDR1 comprises the sequence of SEQ ID NO: 156, LCDR2 comprises the sequence of SEQ ID NO: 158, and LCDR3 comprises the sequence of SEQ ID NO: 160. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 148, HCDR2 consists of the sequence of SEQ ID NO: 150, HCDR3 consists of the sequence of SEQ ID NO: 152, LCDR1 consists of the sequence of SEQ ID NO: 156, LCDR2 consists of the sequence of SEQ ID NO: 158, and LCDR3 consists of the sequence of SEQ ID NO: 160.

[0067] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 164, HCDR2 comprises the sequence of SEQ ID NO: 166, HCDR3 comprises the sequence of SEQ ID NO: 168, LCDR1 comprises the sequence of SEQ ID NO: 172, LCDR2 comprises the sequence of SEQ ID NO: 174, and LCDR3 comprises the sequence of SEQ ID NO: 176. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 164, HCDR2 consists of the sequence of SEQ ID NO: 166, HCDR3 consists of the sequence of SEQ ID NO: 168, LCDR1 consists of the sequence of SEQ ID NO: 172, LCDR2 consists of the sequence of SEQ ID NO: 174, and LCDR3 consists of the sequence of SEQ ID NO: 176.

[0068] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 180, HCDR2 comprises the sequence of SEQ ID NO: 182, HCDR3 comprises the sequence of SEQ ID NO: 184, LCDR1 comprises the sequence of SEQ ID NO: 188, LCDR2 comprises the sequence of SEQ ID NO: 190, and LCDR3 comprises the sequence of SEQ ID NO: 192. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 180, HCDR2 consists of the sequence of SEQ ID NO: 182, HCDR3 consists of the sequence of SEQ ID NO: 184, LCDR1 consists of the sequence of SEQ ID NO: 188, LCDR2 consists of the sequence of SEQ ID NO: 190, and LCDR3 consists of the sequence of SEQ ID NO: 192.

[0069] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 196, HCDR2 comprises the sequence of SEQ ID NO: 198, HCDR3 comprises the sequence of SEQ ID NO: 200, LCDR1 comprises the sequence of SEQ ID NO: 204, LCDR2 comprises the sequence of SEQ ID NO: 206, and LCDR3 comprises the sequence of SEQ ID NO: 208. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 196, HCDR2 consists of the sequence of SEQ ID NO: 198, HCDR3 consists of the sequence of SEQ ID NO: 200, LCDR1 consists of the sequence of SEQ ID NO: 204, LCDR2 consists of the sequence of SEQ ID NO: 206, and LCDR3 consists of the sequence of SEQ ID NO: 208.

[0070] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 212, HCDR2 comprises the sequence of SEQ ID NO: 214, HCDR3 comprises the sequence of SEQ ID NO: 216, LCDR1 comprises the sequence of SEQ ID NO: 220, LCDR2 comprises the sequence of SEQ ID NO: 222, and LCDR3 comprises the sequence of SEQ ID NO: 224. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 212, HCDR2 consists of the sequence of SEQ ID NO: 214, HCDR3 consists of the sequence of SEQ ID NO: 216, LCDR1 consists of the sequence of SEQ ID NO: 220, LCDR2 consists of the sequence of SEQ ID NO: 222, and LCDR3 consists of the sequence of SEQ ID NO: 224.

[0071] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 228, HCDR2 comprises the sequence of SEQ ID NO: 230, HCDR3 comprises the sequence of SEQ ID NO: 232, LCDR1 comprises the sequence of SEQ ID NO: 236, LCDR2 comprises the sequence of SEQ ID NO: 238, and LCDR3 comprises the sequence of SEQ ID NO: 240. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 228, HCDR2 consists of the sequence of SEQ ID NO: 230, HCDR3 consists of the sequence of SEQ ID NO: 232, LCDR1 consists of the sequence of SEQ ID NO: 236, LCDR2 consists of the sequence of SEQ ID NO: 238, and LCDR3 consists of the sequence of SEQ ID NO: 240.

[0072] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 244, HCDR2 comprises the sequence of SEQ ID NO: 246, HCDR3 comprises the sequence of SEQ ID NO: 248, LCDR1 comprises the sequence of SEQ ID NO: 252, LCDR2 comprises the sequence of SEQ ID NO: 254, and LCDR3 comprises the sequence of SEQ ID NO: 256. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 244, HCDR2 consists of the sequence of SEQ ID NO: 246, HCDR3 consists of the sequence of SEQ ID NO: 248, LCDR1 consists of the sequence of SEQ ID NO: 252, LCDR2 consists of the sequence of SEQ ID NO: 254, and LCDR3 consists of the sequence of SEQ ID NO: 256.

[0073] In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 260, HCDR2 comprises the sequence of SEQ ID NO: 262, HCDR3 comprises the sequence of SEQ ID NO: 264, LCDR1 comprises the sequence of SEQ ID NO: 268, LCDR2 comprises the sequence of SEQ ID NO: 270, and LCDR3 comprises the sequence of SEQ ID NO: 272. In some embodiments, the binding domain comprises a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and a light chain variable domain comprising LCDR1, LCDR2, and LCDR3, HCDR1 consists of the sequence of SEQ ID NO: 260, HCDR2 consists of the sequence of SEQ ID NO: 262, HCDR3 consists of the sequence of SEQ ID NO: 264, LCDR1 consists of the sequence of SEQ ID NO: 268, LCDR2 consists of the sequence of SEQ ID NO: 270, and LCDR3 consists of the sequence of SEQ ID NO: 272.

[0074] The present invention relates to an antigen-binding molecule comprising a binding domain that specifically binds to B7H3 (e.g., human B7H3). In some embodiments, the binding domain comprises a heavy chain variable domain and / or a light chain variable domain. In some embodiments, the binding domain comprises a heavy chain variable domain. In some embodiments, the binding domain comprises a light chain variable domain. In some embodiments, the binding domain comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 2, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 2, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 2, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 2, and the light chain variable domain comprises the sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 2, and the light chain variable domain consists of the sequence of SEQ ID NO: 10.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, LCDR3 comprises the sequence of SEQ ID NO: 16, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, LCDR3 comprises the sequence of SEQ ID NO: 16, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 10.In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, LCDR3 comprises the sequence of SEQ ID NO: 16, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 2, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 10. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 2. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 2 and the light chain variable domain comprises the sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 2 and the light chain variable domain consists of the sequence of SEQ ID NO: 10.

[0075] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 18, and the light chain variable domain comprises the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 18, and the light chain variable domain consists of the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, LCDR3 comprises the sequence of SEQ ID NO: 32, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 18.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, LCDR3 comprises the sequence of SEQ ID NO: 32, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 26. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, LCDR3 comprises the sequence of SEQ ID NO: 32, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 26. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 18. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 18 and the light chain variable domain comprises the sequence of SEQ ID NO: 26. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 18 and the light chain variable domain consists of the sequence of SEQ ID NO: 26.

[0076] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 34, and the light chain variable domain comprises the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 34, and the light chain variable domain consists of the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, LCDR3 comprises the sequence of SEQ ID NO: 48, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 34.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, LCDR3 comprises the sequence of SEQ ID NO: 48, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 42. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, LCDR3 comprises the sequence of SEQ ID NO: 48, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 42. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 34. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 34 and the light chain variable domain comprises the sequence of SEQ ID NO: 42. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 34 and the light chain variable domain consists of the sequence of SEQ ID NO: 42.

[0077] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 50, and the light chain variable domain comprises the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 50, and the light chain variable domain consists of the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, LCDR3 comprises the sequence of SEQ ID NO: 64, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 50.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, LCDR3 comprises the sequence of SEQ ID NO: 64, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 58. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, LCDR3 comprises the sequence of SEQ ID NO: 64, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 58. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 50. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 50 and the light chain variable domain comprises the sequence of SEQ ID NO: 58. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 50 and the light chain variable domain consists of the sequence of SEQ ID NO: 58.

[0078] In some preferred embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 74. In some preferred embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 74. In some preferred embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 74. In some preferred embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 66, and the light chain variable domain comprises the sequence of SEQ ID NO: 74. In some preferred embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 66, and the light chain variable domain consists of the sequence of SEQ ID NO: 74. In some preferred embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, LCDR3 comprises the sequence of SEQ ID NO: 80, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 66.In some preferred embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, LCDR3 comprises the sequence of SEQ ID NO: 80, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 74. In some preferred examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, LCDR3 comprises the sequence of SEQ ID NO: 80, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 74. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 66. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 74. In some preferred embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 66 and the light chain variable domain comprises the sequence of SEQ ID NO: 74.In some preferred embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 66, and the light chain variable domain consists of the sequence of SEQ ID NO: 74.

[0079] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 82, and the light chain variable domain comprises the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 82, and the light chain variable domain consists of the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, LCDR3 comprises the sequence of SEQ ID NO: 96, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 82.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, LCDR3 comprises the sequence of SEQ ID NO: 96, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 90. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, LCDR3 comprises the sequence of SEQ ID NO: 96, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 90. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 82. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 82 and the light chain variable domain comprises the sequence of SEQ ID NO: 90. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 82 and the light chain variable domain consists of the sequence of SEQ ID NO: 90.

[0080] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 106. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 106. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 106. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 98, and the light chain variable domain comprises the sequence of SEQ ID NO: 106. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 98, and the light chain variable domain consists of the sequence of SEQ ID NO: 106. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 100, HCDR2 comprises the sequence of SEQ ID NO: 102, HCDR3 comprises the sequence of SEQ ID NO: 104, LCDR1 comprises the sequence of SEQ ID NO: 108, LCDR2 comprises the sequence of SEQ ID NO: 110, LCDR3 comprises the sequence of SEQ ID NO: 112, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 98.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 100, HCDR2 comprises the sequence of SEQ ID NO: 102, HCDR3 comprises the sequence of SEQ ID NO: 104, LCDR1 comprises the sequence of SEQ ID NO: 108, LCDR2 comprises the sequence of SEQ ID NO: 110, LCDR3 comprises the sequence of SEQ ID NO: 112, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 106. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 100, HCDR2 comprises the sequence of SEQ ID NO: 102, HCDR3 comprises the sequence of SEQ ID NO: 104, LCDR1 comprises the sequence of SEQ ID NO: 108, LCDR2 comprises the sequence of SEQ ID NO: 110, LCDR3 comprises the sequence of SEQ ID NO: 112, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 106. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 98. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 106. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 98 and the light chain variable domain comprises the sequence of SEQ ID NO: 106.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 98, and the light chain variable domain consists of the sequence of SEQ ID NO: 106.

[0081] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 122. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 122. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 122. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 114, and the light chain variable domain comprises the sequence of SEQ ID NO: 122. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 114, and the light chain variable domain consists of the sequence of SEQ ID NO: 122. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 116, HCDR2 comprises the sequence of SEQ ID NO: 118, HCDR3 comprises the sequence of SEQ ID NO: 120, LCDR1 comprises the sequence of SEQ ID NO: 124, LCDR2 comprises the sequence of SEQ ID NO: 126, LCDR3 comprises the sequence of SEQ ID NO: 128, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 114.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 116, HCDR2 comprises the sequence of SEQ ID NO: 118, HCDR3 comprises the sequence of SEQ ID NO: 120, LCDR1 comprises the sequence of SEQ ID NO: 124, LCDR2 comprises the sequence of SEQ ID NO: 126, LCDR3 comprises the sequence of SEQ ID NO: 128, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 122. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 116, HCDR2 comprises the sequence of SEQ ID NO: 118, HCDR3 comprises the sequence of SEQ ID NO: 120, LCDR1 comprises the sequence of SEQ ID NO: 124, LCDR2 comprises the sequence of SEQ ID NO: 126, LCDR3 comprises the sequence of SEQ ID NO: 128, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 122. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 114. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 122. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 114 and the light chain variable domain comprises the sequence of SEQ ID NO: 122.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 114, and the light chain variable domain consists of the sequence of SEQ ID NO: 122.

[0082] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 138. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 138. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 138. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 130, and the light chain variable domain comprises the sequence of SEQ ID NO: 138. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 130, and the light chain variable domain consists of the sequence of SEQ ID NO: 138. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence of SEQ ID NO: 132, HCDR2 comprises the sequence of SEQ ID NO: 134, HCDR3 comprises the sequence of SEQ ID NO: 136, LCDR1 comprises the sequence of SEQ ID NO: 140, LCDR2 comprises the sequence of SEQ ID NO: 142, LCDR3 comprises the sequence of SEQ ID NO: 144, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 130.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 132, HCDR2 comprises the sequence of SEQ ID NO: 134, HCDR3 comprises the sequence of SEQ ID NO: 136, LCDR1 comprises the sequence of SEQ ID NO: 140, LCDR2 comprises the sequence of SEQ ID NO: 142, LCDR3 comprises the sequence of SEQ ID NO: 144, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 138. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 132, HCDR2 comprises the sequence of SEQ ID NO: 134, HCDR3 comprises the sequence of SEQ ID NO: 136, LCDR1 comprises the sequence of SEQ ID NO: 140, LCDR2 comprises the sequence of SEQ ID NO: 142, LCDR3 comprises the sequence of SEQ ID NO: 144, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 138. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 130. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 138. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 130 and the light chain variable domain comprises the sequence of SEQ ID NO: 138.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 130, and the light chain variable domain consists of the sequence of SEQ ID NO: 138.

[0083] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 154. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 154. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 154. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 146, and the light chain variable domain comprises the sequence of SEQ ID NO: 154. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 146, and the light chain variable domain consists of the sequence of SEQ ID NO: 154. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 148, HCDR2 comprises the sequence of SEQ ID NO: 150, HCDR3 comprises the sequence of SEQ ID NO: 152, LCDR1 comprises the sequence of SEQ ID NO: 156, LCDR2 comprises the sequence of SEQ ID NO: 158, LCDR3 comprises the sequence of SEQ ID NO: 160, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 146.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 148, HCDR2 comprises the sequence of SEQ ID NO: 150, HCDR3 comprises the sequence of SEQ ID NO: 152, LCDR1 comprises the sequence of SEQ ID NO: 156, LCDR2 comprises the sequence of SEQ ID NO: 158, LCDR3 comprises the sequence of SEQ ID NO: 160, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 154. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 148, HCDR2 comprises the sequence of SEQ ID NO: 150, HCDR3 comprises the sequence of SEQ ID NO: 152, LCDR1 comprises the sequence of SEQ ID NO: 156, LCDR2 comprises the sequence of SEQ ID NO: 158, LCDR3 comprises the sequence of SEQ ID NO: 160, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 154. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 146. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 154. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 146 and the light chain variable domain comprises the sequence of SEQ ID NO: 154.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 146, and the light chain variable domain consists of the sequence of SEQ ID NO: 154.

[0084] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 170. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 170. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 170. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 162, and the light chain variable domain comprises the sequence of SEQ ID NO: 170. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 162, and the light chain variable domain consists of the sequence of SEQ ID NO: 170. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 164, HCDR2 comprises the sequence of SEQ ID NO: 166, HCDR3 comprises the sequence of SEQ ID NO: 168, LCDR1 comprises the sequence of SEQ ID NO: 172, LCDR2 comprises the sequence of SEQ ID NO: 174, LCDR3 comprises the sequence of SEQ ID NO: 176, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 162.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 164, HCDR2 comprises the sequence of SEQ ID NO: 166, HCDR3 comprises the sequence of SEQ ID NO: 168, LCDR1 comprises the sequence of SEQ ID NO: 172, LCDR2 comprises the sequence of SEQ ID NO: 174, LCDR3 comprises the sequence of SEQ ID NO: 176, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 170. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 164, HCDR2 comprises the sequence of SEQ ID NO: 166, HCDR3 comprises the sequence of SEQ ID NO: 168, LCDR1 comprises the sequence of SEQ ID NO: 172, LCDR2 comprises the sequence of SEQ ID NO: 174, LCDR3 comprises the sequence of SEQ ID NO: 176, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 170. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 162. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 170. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 162 and the light chain variable domain comprises the sequence of SEQ ID NO: 170.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 162, and the light chain variable domain consists of the sequence of SEQ ID NO: 170.

[0085] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 186. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 186. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 186. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 178, and the light chain variable domain comprises the sequence of SEQ ID NO: 186. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 178, and the light chain variable domain consists of the sequence of SEQ ID NO: 186. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 180, HCDR2 comprises the sequence of SEQ ID NO: 182, HCDR3 comprises the sequence of SEQ ID NO: 184, LCDR1 comprises the sequence of SEQ ID NO: 188, LCDR2 comprises the sequence of SEQ ID NO: 190, LCDR3 comprises the sequence of SEQ ID NO: 192, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 178.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 180, HCDR2 comprises the sequence of SEQ ID NO: 182, HCDR3 comprises the sequence of SEQ ID NO: 184, LCDR1 comprises the sequence of SEQ ID NO: 188, LCDR2 comprises the sequence of SEQ ID NO: 190, LCDR3 comprises the sequence of SEQ ID NO: 192, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 186. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 180, HCDR2 comprises the sequence of SEQ ID NO: 182, HCDR3 comprises the sequence of SEQ ID NO: 184, LCDR1 comprises the sequence of SEQ ID NO: 188, LCDR2 comprises the sequence of SEQ ID NO: 190, LCDR3 comprises the sequence of SEQ ID NO: 192, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 186. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 178. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 186. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 178 and the light chain variable domain comprises the sequence of SEQ ID NO: 186.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 178, and the light chain variable domain consists of the sequence of SEQ ID NO: 186.

[0086] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 202. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 202. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 202. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 194, and the light chain variable domain comprises the sequence of SEQ ID NO: 202. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 194, and the light chain variable domain consists of the sequence of SEQ ID NO: 202. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 196, HCDR2 comprises the sequence of SEQ ID NO: 198, HCDR3 comprises the sequence of SEQ ID NO: 200, LCDR1 comprises the sequence of SEQ ID NO: 204, LCDR2 comprises the sequence of SEQ ID NO: 206, LCDR3 comprises the sequence of SEQ ID NO: 208, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 194.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 196, HCDR2 comprises the sequence of SEQ ID NO: 198, HCDR3 comprises the sequence of SEQ ID NO: 200, LCDR1 comprises the sequence of SEQ ID NO: 204, LCDR2 comprises the sequence of SEQ ID NO: 206, LCDR3 comprises the sequence of SEQ ID NO: 208, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 202. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 196, HCDR2 comprises the sequence of SEQ ID NO: 198, HCDR3 comprises the sequence of SEQ ID NO: 200, LCDR1 comprises the sequence of SEQ ID NO: 204, LCDR2 comprises the sequence of SEQ ID NO: 206, LCDR3 comprises the sequence of SEQ ID NO: 208, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 202. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 194. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 202. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 194 and the light chain variable domain comprises the sequence of SEQ ID NO: 202.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 194, and the light chain variable domain consists of the sequence of SEQ ID NO: 202.

[0087] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 218. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 218. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 218. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 210, and the light chain variable domain comprises the sequence of SEQ ID NO: 218. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 210, and the light chain variable domain consists of the sequence of SEQ ID NO: 218. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 212, HCDR2 comprises the sequence of SEQ ID NO: 214, HCDR3 comprises the sequence of SEQ ID NO: 216, LCDR1 comprises the sequence of SEQ ID NO: 220, LCDR2 comprises the sequence of SEQ ID NO: 222, LCDR3 comprises the sequence of SEQ ID NO: 224, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 210.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 212, HCDR2 comprises the sequence of SEQ ID NO: 214, HCDR3 comprises the sequence of SEQ ID NO: 216, LCDR1 comprises the sequence of SEQ ID NO: 220, LCDR2 comprises the sequence of SEQ ID NO: 222, LCDR3 comprises the sequence of SEQ ID NO: 224, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 218. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 212, HCDR2 comprises the sequence of SEQ ID NO: 214, HCDR3 comprises the sequence of SEQ ID NO: 216, LCDR1 comprises the sequence of SEQ ID NO: 220, LCDR2 comprises the sequence of SEQ ID NO: 222, LCDR3 comprises the sequence of SEQ ID NO: 224, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 218. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 210. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 218. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 210 and the light chain variable domain comprises the sequence of SEQ ID NO: 218.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 210, and the light chain variable domain consists of the sequence of SEQ ID NO: 218.

[0088] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 234. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 234. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 234. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 226, and the light chain variable domain comprises the sequence of SEQ ID NO: 234. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 226, and the light chain variable domain consists of the sequence of SEQ ID NO: 234. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 228, HCDR2 comprises the sequence of SEQ ID NO: 230, HCDR3 comprises the sequence of SEQ ID NO: 232, LCDR1 comprises the sequence of SEQ ID NO: 236, LCDR2 comprises the sequence of SEQ ID NO: 238, LCDR3 comprises the sequence of SEQ ID NO: 240, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 226.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 228, HCDR2 comprises the sequence of SEQ ID NO: 230, HCDR3 comprises the sequence of SEQ ID NO: 232, LCDR1 comprises the sequence of SEQ ID NO: 236, LCDR2 comprises the sequence of SEQ ID NO: 238, LCDR3 comprises the sequence of SEQ ID NO: 240, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 234. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 228, HCDR2 comprises the sequence of SEQ ID NO: 230, HCDR3 comprises the sequence of SEQ ID NO: 232, LCDR1 comprises the sequence of SEQ ID NO: 236, LCDR2 comprises the sequence of SEQ ID NO: 238, LCDR3 comprises the sequence of SEQ ID NO: 240, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 234. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 226. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 234. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 226 and the light chain variable domain comprises the sequence of SEQ ID NO: 234.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 226, and the light chain variable domain consists of the sequence of SEQ ID NO: 234.

[0089] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 250. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 250. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 250. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 242, and the light chain variable domain comprises the sequence of SEQ ID NO: 250. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 242, and the light chain variable domain consists of the sequence of SEQ ID NO: 250. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 244, HCDR2 comprises the sequence of SEQ ID NO: 246, HCDR3 comprises the sequence of SEQ ID NO: 248, LCDR1 comprises the sequence of SEQ ID NO: 252, LCDR2 comprises the sequence of SEQ ID NO: 254, LCDR3 comprises the sequence of SEQ ID NO: 256, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 242.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 244, HCDR2 comprises the sequence of SEQ ID NO: 246, HCDR3 comprises the sequence of SEQ ID NO: 248, LCDR1 comprises the sequence of SEQ ID NO: 252, LCDR2 comprises the sequence of SEQ ID NO: 254, LCDR3 comprises the sequence of SEQ ID NO: 256, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 250. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 244, HCDR2 comprises the sequence of SEQ ID NO: 246, HCDR3 comprises the sequence of SEQ ID NO: 248, LCDR1 comprises the sequence of SEQ ID NO: 252, LCDR2 comprises the sequence of SEQ ID NO: 254, LCDR3 comprises the sequence of SEQ ID NO: 256, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 250. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 242. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 250. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 242 and the light chain variable domain comprises the sequence of SEQ ID NO: 250.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 242, and the light chain variable domain consists of the sequence of SEQ ID NO: 250.

[0090] In some embodiments, the heavy chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 258, and the light chain variable domain comprises the sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 258, and the light chain variable domain consists of the sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 260, HCDR2 comprises the sequence of SEQ ID NO: 262, HCDR3 comprises the sequence of SEQ ID NO: 264, LCDR1 comprises the sequence of SEQ ID NO: 268, LCDR2 comprises the sequence of SEQ ID NO: 270, LCDR3 comprises the sequence of SEQ ID NO: 272, and the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 258.In some embodiments, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 260, HCDR2 comprises the sequence of SEQ ID NO: 262, HCDR3 comprises the sequence of SEQ ID NO: 264, LCDR1 comprises the sequence of SEQ ID NO: 268, LCDR2 comprises the sequence of SEQ ID NO: 270, LCDR3 comprises the sequence of SEQ ID NO: 272, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 266. In some examples, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, HCDR1 comprises the sequence of SEQ ID NO: 260, HCDR2 comprises the sequence of SEQ ID NO: 262, HCDR3 comprises the sequence of SEQ ID NO: 264, LCDR1 comprises the sequence of SEQ ID NO: 268, LCDR2 comprises the sequence of SEQ ID NO: 270, LCDR3 comprises the sequence of SEQ ID NO: 272, the heavy chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 95%, at least 98%, at least 99%, at least 99.5% sequence identity to the sequence of SEQ ID NO: 266. The heavy chain variable domain may comprise or consist of the sequence of SEQ ID NO: 258. The light chain variable domain may comprise or consist of the sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 258 and the light chain variable domain comprises the sequence of SEQ ID NO: 266.In some embodiments, the heavy chain variable domain consists of the sequence of SEQ ID NO: 258, and the light chain variable domain consists of the sequence of SEQ ID NO: 266.

[0091] Sequence identity, including determination of sequence complementarity of nucleic acid or polynucleotide sequences, can be determined by sequence comparison and alignment algorithms known in the art. To determine the percentage of identity between two nucleic acid sequences (or polynucleotide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the first sequence or the second sequence for optimal alignment). Next, the nucleotides at corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), and optionally, a penalty is set for the number of gaps introduced and / or the score for the length of the gaps introduced.

[0092] Comparison of sequences and determination of the percentage of identity between two sequences can be performed using mathematical algorithms. In one embodiment, the alignment is performed over a particular portion of the aligned sequences that have sufficient identity but not over portions with low degrees of identity (i.e., local alignment). A preferred non-limiting example of a local alignment algorithm utilized for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such algorithms are incorporated into the BLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0093] In another embodiment, alignment is optimized by introducing appropriate gaps and the percent identity is determined over the length of the aligned sequences (i.e., gapped alignment). To obtain a gapped alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, alignment is optimized by introducing appropriate gaps and the percent identity is determined over the entire length of the aligned sequences (i.e., global alignment). A preferred non-limiting example of a mathematical algorithm utilized for global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.

[0094] "Polypeptide" is used herein in its broadest sense to refer to compounds of two or more subunit amino acids, amino acid analogs, or other peptidomimetic compounds. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to both natural and / or unnatural, i.e., synthetic, amino acids including glycine and both D- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0095] Alternatively, the antigen-binding molecule of the present invention may contain one or more variants of a designated sequence. A "variant" may be any substitution, deletion, or addition variant of the above amino acid sequence. A variant may contain one, two, three, four, five, up to ten, up to twenty, up to thirty, or more amino acid substitutions and / or deletions from the specific sequences and fragments described above while maintaining the activity of the antigen-binding molecule described herein. A "deletion" variant may contain, for example, deletions of 1, 2, 3, 4, or 5 individual amino acids. A "substitution" variant preferably includes substituting one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid may be substituted with another amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of several of the 20 major amino acids that can be used to select suitable substituents are as follows:

[0096] JPEG2025522826000002.jpg80154

[0097] The substituent can also be selected from the amino acids selenocysteine and pyrrolidine.

[0098] Preferred "derivatives" or "variants" include those in which the amino acids present in the sequence are structural analogs of the naturally occurring amino acids. Also, the amino acids used in the sequence may be derivatized or modified, for example, labeled, as long as the function of the antigen-binding molecule is not significantly adversely affected. The above derivatives and variants can be prepared during the synthesis of the antigen-binding molecule, or by post-generation modification, or, when the antigen-binding molecule is in recombinant form, by known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids. For example, the antigen-binding molecule can be labeled with a radiolabeled amino acid. Examples of radiolabels include the following radioisotopes or radiolabeled nucleotides:3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 Examples include, but are not limited to, I. The radiolabel can be used for both diagnostic and therapeutic purposes.

[0099] The antigen-binding molecules described herein may be derivatized or linked to another molecule (such as another peptide or protein, etc.). Generally, the antigen-binding molecules are derivatized such that their binding to B7H3 is not adversely affected by the derivatization or labeling. For example, the antigen-binding molecules can be functionally linked to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide (such as a streptavidin core region or a polyhistidine tag, etc.) that can mediate the association of an antibody or antibody portion with another molecule, by, for example, chemical coupling, gene fusion, non-covalent binding, or other methods. The antigen-binding domain that specifically binds to B7H3 can be labeled with a detectable moiety or marker as described herein.

[0100] Means for detecting the label are well known to those skilled in the art. Thus, for example, a radiolabel can be detected using a photographic film or a scintillation counter, and a fluorescent marker can be detected using a light detector that detects the emitted illumination. An enzyme label is usually detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and a colorimetric label is detected simply by visualizing the colored label.

[0101] The antigen-binding molecules can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be useful for improving the biological characteristics of the antigen-binding domain, such as extending the serum half-life or increasing tissue binding.

[0102] Other features of the antigen-binding molecule of the present invention The antigen-binding molecule may be human or may be humanized. A "humanized" antigen-binding molecule comprises a human framework region and one or more CDRs derived from a non-human antigen-binding molecule such as an antibody (e.g., monkey, mouse, rat, or synthetic antibody). The non-human antigen-binding molecule that provides the CDRs is the "donor", and the human antigen-binding molecule that provides the framework is the "acceptor". Preferably, all six CDR sequences in the humanized antigen-binding molecule are derived from the antigen-binding molecule. The humanized antigen-binding molecule may not contain a constant region. When a constant region is present in the humanized antigen-binding molecule, it is usually substantially identical to the constant region of a human antigen-binding molecule, e.g., having at least 85%, at least 90%, at least 95%, at least 98%, or about 100% sequence identity with the human constant region; preferably having at least 90%, or most preferably at least 95% sequence identity with the human constant region. Thus, in a preferred embodiment, all portions of the humanized antigen-binding molecule except the CDRs are substantially identical to the corresponding portions of a native human antigen-binding molecule sequence (i.e., having at least 90%, preferably at least 95% sequence identity). A "humanized antigen-binding molecule" may comprise a humanized light chain and a humanized heavy chain. The humanized antigen-binding molecule binds to the same antigen as the donor antigen-binding molecule that provides the CDRs. The acceptor framework of the humanized antigen-binding molecule may have a limited number of substitutions by amino acids taken from the donor framework (typically about 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 substitutions, preferably 1-20, most preferably 1-10 substitutions). The humanized antigen-binding molecule or other monoclonal antibody can have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. The humanized immunoglobulin can be constructed using genetic engineering (see, e.g., U.S. Patent No. 5,585,089). In some embodiments, the binding domain of the antigen-binding molecule of the invention may be human or may be humanized. In some embodiments, the heavy chain variable domain and / or the light chain variable domain may be human or may be humanized.In some embodiments, the antigen-binding domain is a humanized antigen-binding domain and includes one or more human framework regions.

[0103] In some embodiments, the antigen-binding molecule may be an antibody fragment or a single-chain antibody, and optionally, the fragment may be a Fab fragment, a Fab’ fragment, an F(ab)’2 fragment, a single-chain Fab (scFab) fragment, a single-chain Fv protein (scFv), a tandem scFv protein, or a disulfide-stabilized Fv protein (dsFv), an scFv-Fc protein, a bivalent, trivalent, or tetravalent antibody, a Bis-scFv, a diabody, a triabody, a tetrabody, or an epitope-binding fragment of any of the above (e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for making and producing antibody fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). In some preferred embodiments, the antigen-binding molecule is an scFv or an scFv-Fc. The scFv protein is a fusion protein in which the light-chain variable region (LCVR) and the heavy-chain variable region (HCVR) of an immunoglobulin are linked by a linker. In some embodiments, the scFv has a linker sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 273). In some embodiments, the scFv contains the linker of SEQ ID NO: 273, and the heavy-chain variable domain is connected to the light-chain variable domain via the linker. In some embodiments, the components of the scFv are arranged in the order of 5’-HCVR-linker-LCVR-3’. In some embodiments, the scFv has a heavy-chain variable domain containing the sequence of SEQ ID NO: 2 linked to a light-chain variable domain containing the sequence of SEQ ID NO: 10 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy-chain variable domain containing the sequence of SEQ ID NO: 18 linked to a light-chain variable domain containing the sequence of SEQ ID NO: 26 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy-chain variable domain containing the sequence of SEQ ID NO: 34 linked to a light-chain variable domain containing the sequence of SEQ ID NO: 42 via a linker having the sequence of SEQ ID NO: 273.In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 50 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 58 via a linker having the sequence of SEQ ID NO: 273. In some preferred embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 66 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 74 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 82 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 90 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 98 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 106 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 114 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 122 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 130 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 138 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 146 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 154 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 162 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 170 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 178 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 186 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 194 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 202 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 210 linked to a light chain variable domain comprising the sequence of SEQ ID NO: 218 via a linker having the sequence of SEQ ID NO: 273.In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 226 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 234 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 242 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 250 via a linker having the sequence of SEQ ID NO: 273. In some embodiments, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 258 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 266 via a linker having the sequence of SEQ ID NO: 273. In a preferred embodiment, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 50 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 58 via a linker having the sequence of SEQ ID NO: 273. In a more preferred embodiment, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 258 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 266 via a linker having the sequence of SEQ ID NO: 273. In a more preferred embodiment, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 66 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 74 via a linker having the sequence of SEQ ID NO: 273. Optionally, the components of the scFv may be arranged in the order of 5’-LCVR-linker-HCVR-3’.

[0104] In some embodiments, the antigen-binding molecule is a multispecific molecule. In some embodiments, the antigen-binding molecule is, for example, a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain corresponds to the antigen-binding molecule of the present invention and specifically binds to B7H3, and the second antigen-binding domain specifically binds to a different target antigen. In some embodiments, the antigen-binding molecule may be, for example, a bivalent molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain corresponds to the antigen-binding molecule of the present invention, the second antigen-binding domain is derived from a second different antigen-binding molecule of the present invention, and the first antigen-binding domain and the second antigen-binding domain specifically bind to B7H3 and recognize distinct, non-overlapping epitopes. In some embodiments, the antigen-binding molecule is, for example, a trispecific molecule comprising first, second, and third antigen-binding domains, wherein the first antigen-binding domain corresponds to the antigen-binding molecule of the present invention and specifically binds to B7H3, and the second (and third) antigen-binding domains specifically bind to different target antigens.

[0105] In some embodiments, the antigen-binding molecule is a bispecific T cell engager (BiTE). In some embodiments, the BiTE comprises first and second binding domains, wherein the first antigen-binding domain corresponds to the antigen-binding molecule of the present invention and specifically binds to B7H3, and the second antigen-binding domain specifically binds to CD3 on the surface of T cells. For example, the second antigen-binding domain specifically binds to the CD3 subunit of the T cell receptor (TCR). Antigen-binding molecules containing such binding domains are well known to those skilled in the art. For example, anti-CD3 monoclonal antibodies contain such binding domains and are readily available. In some embodiments, the second antigen-binding domain specifically binds to other T cell-specific surface molecules.

[0106] In one aspect, the antigen-binding molecule of the present invention is a bispecific molecule or BiTE molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to B7H3, the first antigen-binding domain comprises a heavy-chain variable domain and a light-chain variable domain, the heavy-chain variable domain comprises HCDR1 comprising the sequence of SEQ ID NO: 68, HCDR2 comprising the sequence of SEQ ID NO: 70, and HCDR3 comprising the sequence of SEQ ID NO: 72, the light-chain variable domain comprises LCDR1 comprising the sequence of SEQ ID NO: 76, LCDR2 comprising the sequence of SEQ ID NO: 78, and LCDR3 comprising the sequence of SEQ ID NO: 80, preferably, the heavy-chain variable domain comprises the sequence of SEQ ID NO: 66, and the light-chain variable domain comprises the sequence of SEQ ID NO: 74.

[0107] Bispecific molecules, trispecific molecules, or multispecific molecules can be generated by crosslinking two or more antigen-binding domains. Suitable crosslinking agents include those that are heterobifunctional (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate) having two distinct reactive groups separated by an appropriate spacer. In some aspects, the bispecific (e.g., BiTE) or biparatopic molecules described herein comprise two scFvs. For example, in the case of a BiTE molecule, the first antigen-binding domain may be an scFv of the present invention that specifically binds to B7H3, and the second CD3 antigen-binding domain may also be an scFv. The two scFv antibodies can be covalently linked, for example, using a short peptide linker of 5-20 amino acids.

[0108] In some embodiments, the antigen-binding molecule may be linked to an effector molecule. For example, an antigen-binding molecule that specifically binds to B7H3 may be covalently linked to an effector molecule or a toxin. The linkage may be by chemical means or recombinant means (e.g., a peptide linker). When the linkage is chemical, a reaction may occur that results in a covalent bond linking the antibody or a fragment thereof to the effector molecule. The linkage may include, for example, a peptide linker comprising 1 to 50, 1 to 40, 1 to 30, 1 to 20, or preferably 1 to 10 amino acids. Optionally, the antigen-binding molecule that is bound to the effector molecule may further be linked to a lipid, protein, polypeptide, or carbohydrate that prolongs, preferably shortens, its half-life in the body. The effector molecule may be selected from the group consisting of an anti-cancer agent, a cytotoxic agent, a cell division inhibitor, a drug, a radioisotope, a detectable label, an enzyme, a fluorophore, a fluorescent protein, a chemiluminescent agent, a radioactive label, a heavy metal, a tracer molecule, or other detectable compounds known to those skilled in the art. In a preferred embodiment, the antibody may be conjugated to an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor. In some embodiments, the antigen-binding molecule may be conjugated to a pyrrolobenzodiazepine (PBD) or monomethyl auristatin E (MMAE). MMAE is a synthetic anti-cancer agent having the following structure:

[0109]

Chemical Structure

[0110] PBD has the following general structure:

[0111]

Chemical Structure

[0112] PBDs may differ in the number, type, and position of substituents in the aromatic A-ring and pyrrolo C-ring, as well as the degree of saturation of the C-ring. In the B-ring, an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) is present at the N10-C11 position. PBDs typically have an (S) configuration at the chiral C11a position and are right-handed when viewed from the C-ring towards the A-ring. Many naturally occurring PBDs have been identified and more than 10 synthetic routes to various analogs have been developed (see, for example, Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. and Thurston, D. E., Chem. Rev. 2011 111(4), 2815-2864). The term "PBD" may be understood to include PBD dimers.

[0113] Chimeric antigen receptors (CARs), chimeric costimulatory receptors (CCRs), and cells comprising them The present invention provides a CAR comprising an antigen-binding molecule described herein that specifically binds to B7H3. A CAR is an artificial receptor that can be transplanted into immune effector cells with any specificity. In classical CARs, the specificity of an antibody is transplanted into T cells. The nucleic acid encoding the CAR can be introduced into T cells using, for example, a retroviral vector. In this way, a large number of cancer-specific T cells for adoptive cell transfer can be produced.

[0114] CARs are modular and typically include an extracellular target antigen-binding domain, a hinge region (or spacer), a transmembrane domain, and one or more intracellular signaling domains (or intracellular domains, or endodomains). The antigen-binding ability of a CAR is defined by the extracellular target antigen-binding domain. Typically, the extracellular target antigen-binding domain is an scFv. The transmembrane domain anchors the CAR to the cell membrane and the intracellular signaling domain transmits an activation signal. The intracellular signaling domain may include one or more costimulatory domains. In some embodiments, the CAR further includes a hinge region, a transmembrane domain, and an intracellular signaling domain.

[0115] The extracellular target antigen-binding domain of a CAR is generally fused to an intracellular signaling domain (or endodomain) via a spacer (or hinge) and a transmembrane domain, and this domain contains or associates with an intracellular T cell signaling domain. When the CAR binds to the target antigen, an activation signal is transmitted to T cells expressing it, etc. The hinge provides flexibility for the CAR (e.g., scFv) to access the target antigen. A longer hinge increases flexibility and improves access to membrane-proximal epitopes, while a shorter hinge results in more effective binding to membrane-distal epitopes. Those skilled in the art will understand that any suitable hinge or spacer sequence can be used. The hinge may be an IgG-based hinge derived from IgG1, IgG2, or IgG4. The hinge may be derived from native CD28 or CD8. The hinge or spacer sequence includes, for example, a short flexible linker, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk, or combinations thereof. Alternatively, the linker may include an alternative linker sequence having the same length and / or domain-spacing characteristics as an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk. In some embodiments, the hinge region of the CAR of the present invention is derived from CD8.

[0116] The CAR may also include a transmembrane domain that spans the membrane. It may include a hydrophobic α-helix. The transmembrane domain may be derived from, for example, CD4, CD8α (sometimes referred to herein as CD8), or CD28. The transmembrane domain may be the ICOS transmembrane domain. Among the intracellular signaling domains, the transmembrane domain of the most membrane-proximal component is often used, but different transmembrane domains can also be used. In some embodiments, the transmembrane domain of the CAR of the present invention is derived from CD8 (sometimes referred to herein as CD8α) or CD28.

[0117] The CAR may include an intracellular signaling domain (or endodomain), which is the part of the CAR involved in signal transduction. The intracellular signaling domain may include or be associated with an intracellular T cell signaling domain. After antigen recognition, the receptor clusters and activation signals are transmitted to the cell. The most commonly used T cell signaling component is that of CD3-zeta (or CD3ζ) containing three ITAMs (immunoreceptor tyrosine-based activation motifs). This transmits activation signals to the T cell after antigen binding. In some embodiments, the intracellular signaling domain includes CD3-zeta. CD3-zeta alone may not be able to provide a sufficiently potent activation signal and additional co-stimulatory signaling may be required. One or more co-stimulatory molecules can also be used. The co-stimulatory molecule may be from the CD28 family (including CD28 and ICOS) or the tumor necrosis factor receptor family (including 4-1BB, OX40, or CD27). Those skilled in the art will understand that any suitable co-stimulatory domain can be used. Depending on the number of co-stimulatory domains, the CAR can be classified into first-generation (CD3ζ only), second-generation (one co-stimulatory domain + CD3ζ), and third-generation (more than one co-stimulatory domain + CD3ζ) CARs. In some embodiments, the intracellular signaling domain includes a co-stimulatory domain. In some embodiments, the intracellular signaling domain includes a co-stimulatory domain derived from CD28 or 4-1BB.

[0118] The present invention further provides a chimeric co-stimulatory receptor (CCR) comprising an antigen-binding molecule described herein that specifically binds to B7H3. The CCR is similar to a CAR and typically comprises an extracellular target antigen-binding domain, a hinge region (or spacer), a transmembrane domain, and one or more intracellular signaling domains (or intracellular domains, or endodomains). The antigen-binding ability of the CCR is defined by the extracellular target antigen-binding domain. Typically, the extracellular target antigen-binding domain is a scFv. The transmembrane domain anchors the CCR to the cell membrane, and the intracellular signaling domain transmits an activation signal. However, in contrast to a CAR, the intracellular signaling domain of the CCR is unable to transduce T cell signals. That is, the intracellular signaling domain of the CCR does not confer signal 1 (the signal generated after the interaction of the endogenous TCR with its ligand, i.e., the T cell activation signal). Rather, the intracellular signaling domain of the CCR confers signal 2 (the signal generated by the interaction of a co-stimulatory molecule on an antigen-presenting cell with its cognate receptor on a T cell). Thus, the CCR provides signal 2 but not signal 1. The CCR provides co-stimulation but does not provide TCR signaling. Accordingly, the intracellular signaling domain of the CCR lacks an intracellular T cell signaling domain and does not associate with an intracellular T cell signaling domain. For example, the intracellular signaling domain of the CCR lacks CD3-zeta and cannot associate with CD3-zeta. The intracellular signaling domain of the CCR may comprise or consist of one or more co-stimulatory domains. The one or more co-stimulatory domains can be any co-stimulatory domain known in the art. For example, the intracellular signaling domain of the CCR may comprise or consist of (i) a co-stimulatory domain derived from CD28 and / or (ii) a co-stimulatory domain derived from 4-1BB. That is, the intracellular signaling domain of the CCR may comprise or consist of a CD28 co-stimulatory domain and / or a 4-1BB co-stimulatory domain derived from the 4-1BB co-stimulatory domain.

[0119] The extracellular target antigen-binding domain of the CCR is generally fused to an intracellular signaling domain (or endodomain) via a spacer (or hinge) and a transmembrane domain. When the CCR binds to the target antigen, a co-stimulatory signal is transmitted to T cells and the like expressing it. The hinge provides flexibility for the CCR (e.g., scFv) to access the target antigen. A longer hinge increases flexibility and improves access to membrane-proximal epitopes, while a shorter hinge makes binding to membrane-distal epitopes more effective. Those skilled in the art will understand that any suitable hinge or spacer sequence can be used. The hinge may be an IgG-based hinge derived from IgG1, IgG2, or IgG4. The hinge may be derived from native CD28 or CD8. The hinge or spacer sequence may include, for example, a short flexible linker, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk, or combinations thereof. Alternatively, the linker may include an alternative linker sequence having the same length and / or domain-spacing characteristics as the IgG1 Fc region, IgG1 hinge, or CD8 stalk. In some embodiments, the hinge region of the CAR of the present invention is derived from CD8.

[0120] The CCR may also include a transmembrane domain spanning the membrane. It may include a hydrophobic α-helix. The transmembrane domain may be derived from, for example, CD4, CD8α (sometimes referred to as CD8 herein), or CD28. The transmembrane domain may be the ICOS transmembrane domain. Among the intracellular signaling domains, the transmembrane domain of the component closest to the membrane is often used, but different transmembrane domains can also be used. In some embodiments, the transmembrane domain of the CCR of the present invention is derived from CD8 (sometimes referred to as CD8α herein) or CD28.

[0121] The CAR or CCR of the present invention described herein may comprise an antigen-binding molecule described herein that specifically binds to B7H3. In some embodiments, the CAR or CCR of the present invention comprises an extracellular target antigen-binding domain that is an antigen-binding molecule described herein. All descriptions of the antigen-binding molecules of the present invention can be directly applied to the CARs and CCRs described herein. Any feature of the antigen-binding molecules of the present invention described herein applies to the CARs and CCRs described herein. Specifically, any of the antigen-binding molecules described herein can be incorporated into the CARs or CCRs described herein. The CAR or CCR comprises an extracellular target antigen-binding domain that is an antigen-binding molecule of the present invention.

[0122] The CAR or CCR of the present invention described herein may comprise a signal peptide such that when the CAR or CCR is expressed intracellularly in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface where it is expressed.

[0123] For example, the CAR or CCR of the present invention may preferably comprise an antigen-binding molecule of the present invention that comprises a binding domain that specifically binds to B7H3, the binding domain comprising a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising HCDR1 comprising the sequence of SEQ ID NO: 68, HCDR2 comprising the sequence of SEQ ID NO: 70, and HCDR3 comprising the sequence of SEQ ID NO: 72, the light chain variable domain comprising LCDR1 comprising the sequence of SEQ ID NO: 76, LCDR2 comprising the sequence of SEQ ID NO: 78, and LCDR3 comprising the sequence of SEQ ID NO: 80, more preferably, the heavy chain variable domain comprises the sequence of SEQ ID NO: 66 and the light chain variable domain comprises the sequence of SEQ ID NO: 74. Preferably, the CAR of the present invention may comprise an scFv described herein having a heavy chain variable domain comprising the sequence of SEQ ID NO: 66 connected to a light chain variable domain comprising the sequence of SEQ ID NO: 74 via a linker having the sequence of SEQ ID NO: 273. Preferably, the CAR of the present invention may comprise a hinge and transmembrane domain derived from CD8, an intracellular signaling domain comprising CD3-zeta, and further a co-stimulatory domain derived from CD28.

[0124] The present invention also provides a cell comprising the CAR described herein, preferably a T cell (i.e., a CAR-T cell). In some embodiments, the T cell is an alpha-beta T cell. In some embodiments, the T cell is a gamma-delta T cell. In some embodiments, the cell can be an NK cell or an iPS cell. The present invention further provides a cell comprising the CCR described herein, preferably a T cell. In some embodiments, the T cell is an alpha-beta T cell. In some embodiments, the T cell is a gamma-delta T cell. In some embodiments, the cell can be an NK cell or an iPS cell.

[0125] The T cell can be a T cell or T lymphocyte, a type of lymphocyte that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on their cell surface. There are various types of T cells, such as helper T cells, cytotoxic T cells, memory T cells, and regulatory T cells. Any type of T cell can be used to produce CAR-T cells or T cells expressing CCR.

[0126] Helper T cells (TH cells) assist other white blood cells in immunological processes including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when a peptide antigen is presented by MHC class II molecules on the surface of an antigen-presenting cell (APC). These cells can differentiate into one of several subtypes including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0127] Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection reactions. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated and rendered anergic, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0128] Memory T cells are a subset of antigen-specific T cells that persist for long periods after recovery from an infection. They rapidly amplify into large numbers of effector T cells upon re-exposure to the same antigen, thereby giving the immune system "memory" of past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0129] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are extremely important for maintaining immune tolerance. Their main role is to halt T cell-mediated immunity as the immune response nears its end and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. Two major classifications of CD4+ Treg cells, naturally occurring Treg cells and adaptive Treg cells, have been reported. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) are generated in the thymus and are associated with the interaction of developing T cells with dendritic cells of the bone marrow (CD11c+) and plasmacytoid cells (CD123+) activated by TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene prevent the development of regulatory T cells and cause IPEX, a lethal autoimmune disease. Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can occur during a normal immune response.

[0130] The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide a rapid response to natural signals from virus-infected cells without depending on MHC. NK cells (which belong to a group of natural lymphocytes) are defined as large granular lymphocytes (LGL) and constitute a third type of cell that differentiates from lymphoid common progenitor cells that give rise to B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and then enter the circulation. NK cells can form CAR-NK cells using a CAR construct designed for CAR-T cells. NK cells can form NK cells expressing CCR using a CCR construct designed for T cells.

[0131] The T cells (or NK cells) comprising the CAR or CCR of the present invention (i) Isolate a sample containing T cells (or NK cells) from a subject or from other sources listed below, (ii) Transducing or transfecting the nucleic acid sequence(s) encoding the CAR or CCR of the present invention into T cells (or NK cells). This can be prepared by.

[0132] The T cells (or NK cells) containing or expressing the CAR or CCR according to the present invention can be prepared ex vivo from the patient's own peripheral blood (autologous treatment), or in the context of hematopoietic stem cell transplantation from a donor's peripheral blood (allogeneic treatment), or from the peripheral blood of an unrelated donor (allogeneic treatment). The T cells (or NK cells) expressing the CAR or CCR according to the present invention can also be induced from the ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into T cells (or NK cells). Immortalized T cell lines that retain lytic function and can act as therapeutic agents may also be used.

[0133] White blood cells in a blood sample can be isolated, for example, using a cell separator (leukapheresis). Peripheral blood mononuclear cells (PBMCs) can be separated and recovered from the sample. T cells (or NK cells) in PBMCs may be activated and / or amplified by treatment with, for example, anti-CD3 monoclonal antibody or IL-2 before being transduced or transfected with the nucleic acid encoding the CAR or CCR according to the present invention.

[0134] The cells containing the CAR or CCR of the present invention can be prepared by introducing the DNA or RNA encoding the CAR or CCR by one of many means including transduction with a viral vector or transfection with DNA or RNA. For example, after purifying the expanded T cells, they are transduced or transfected with the nucleic acid sequence encoding the CAR or CCR of the present invention via, for example, a retroviral vector (e.g., an integrative gammaretroviral (RV) vector or a lentiviral (LV) vector) or through the use of the CRISPR / Cas9 system.

[0135] Thereafter, T cells (or NK cells) may be purified and selected, for example, based on the expression of CAR or CCR.

[0136] Nucleic acid molecules, vectors, and host cells There is provided a nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding molecule or CAR according to the present invention. There is also provided a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable domain or the light chain variable domain described herein.

[0137] The nucleic acid molecule (or polynucleotide) may encode all or part of the antigen-binding molecule of the present invention. Thus, the nucleic acid molecule of the present invention may encode "all", i.e., the full length, of any antigen-binding molecule, variant, or fragment described herein. "Part" of an antigen-binding molecule usually means a heavy chain or a light chain or a region thereof, such as a variable region. For example, "part" of an antigen-binding molecule may mean a heavy chain variable domain or a light chain variable domain. The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, which can be either deoxyribonucleotides or ribonucleotides or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotide of the present invention may be provided in an isolated or purified form.

[0138] A polynucleotide sequence that "encodes" a selected polynucleotide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. For the purposes of the present invention, such polynucleotide sequences can include, but are not limited to, cDNA derived from viruses, prokaryotic or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. Transcription termination sequences may be located on the 3' side of the coding sequence.

[0139] In one aspect, the nucleic acid molecule of the present invention comprises a nucleotide sequence encoding the sequence of the heavy chain variable domain or the light chain variable domain described herein. Such nucleic acid molecules may consist of or may contain the nucleotide sequences of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, or 257 corresponding to VH and VL, respectively, or SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, or 265. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 1 and 9. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 17 and 25. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 33 and 41. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 49 and 57. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 65 and 73. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 81 and 89. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 97 and 105. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 113 and 121. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 129 and 137. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 145 and 153. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 161 and 169. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 177 and 185. The nucleic acid molecule of the present invention may consist of or may contain both of the nucleotide sequences of SEQ ID NO: 193 and 201.The nucleic acid molecule of the present invention may comprise or consist of both of the nucleotide sequences of SEQ ID NO: 209 and 217. The nucleic acid molecule of the present invention may comprise or consist of both of the nucleotide sequences of SEQ ID NO: 225 and 233. The nucleic acid molecule of the present invention may comprise or consist of both of the nucleotide sequences of SEQ ID NO: 241 and 249. The nucleic acid molecule of the present invention may comprise or consist of both of the nucleotide sequences of SEQ ID NO: 257 and 265. The nucleic acid molecule of the present invention preferably encodes an antigen-binding molecule comprising the heavy chain variable domain amino acid sequence of SEQ ID NO: 66 and the light chain variable domain amino acid sequence of SEQ ID NO: 74, or variants or fragments thereof such as those described above. The nucleic acid molecule of the present invention may contain a nucleotide sequence encoding a CAR according to the present invention.

[0140] To generate an scFv, the sequences of the heavy chain variable domain and the light chain variable domain are operably linked to a DNA fragment encoding the heavy chain variable domain and the light chain variable domain, which can be expressed as a continuous single-chain protein in which the heavy chain variable domain and the light chain variable domain are joined by a flexible linker, to another fragment encoding a flexible linker, for example, the amino acid sequence (Gly4-Ser)3. Optionally, a cleavage site such as a furin cleavage site may be included in the linker. The nucleic acid encoding VH and / or VL optionally encodes an Fc domain (immunoadhesin). The Fc domain can be an Fc domain of IgA, IgM, or IgG. The Fc domain can be an optimized Fc domain. In one example, the immunoadhesin is IgG1 Fc.

[0141] Alternatively, a suitable polynucleotide sequence may be a variant of one of these specific polynucleotide sequences. For example, the variant may be a substitution, deletion, or addition variant of any of the above nucleic acid sequences. The variant polynucleotide may contain one, two, three, four, five, up to ten, up to twenty, up to thirty, up to forty, up to fifty, up to sixty, up to seventy, up to eighty, up to ninety, or up to one hundred or more nucleic acid substitutions and / or deletions from the sequences provided in the sequence listing.

[0142] Suitable variants may be at least 70% homologous to any one of the polynucleotides of the nucleic acid sequences disclosed herein, preferably at least 80% or 90%, more preferably at least 95%, 97%, or 99% homologous. Preferably, this level of homology and identity exists at least with respect to the coding region of the polynucleotide. Methods for measuring homology are well known in the art, and in the present context, it will be understood by those skilled in the art that homology is calculated based on nucleic acid identity. The calculation of homology is also described in the previous section.

[0143] Variant sequences may differ from the specific sequences given in the sequence listing due to the redundancy of the genetic code. The DNA code has four main nucleic acid residues (A, T, C, and G), which are used to "spell out" three-letter codons that represent the amino acids of the proteins encoded by an organism's genes. The linear sequence of codons along a DNA molecule is translated into the linear sequence of amino acids of the protein(s) encoded by that gene. This code is highly degenerate, with 61 codons encoding 20 natural amino acids and three codons representing "stop" signals. Thus, most amino acids are encoded by more than one codon, and in fact, some are encoded by four or more different codons. Accordingly, the variant polynucleotides of the present invention may encode the same polypeptide sequence as another polynucleotide of the present invention, but may have different nucleic acid sequences because they use different codons to encode the same amino acids.

[0144] A polynucleotide "fragment" according to the present invention can be produced, for example, by cleavage such as removing one or more nucleotides from one or both ends of a polynucleotide. One or more amino acids of 10 or less, 20 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, or 100 or less, or more can be removed in this way from the 3' end and / or 5' end of the polynucleotide. Fragments can also arise from one or more internal deletions. Such fragments may be derived from the sequences described herein or from the variant polynucleotides described herein. Preferably, such fragments are 90 to 1000 residues in length, for example, 90 to 300 residues, 90 to 500 residues, 100 to 800 residues, 200 to 900 residues, or 300 to 100 residues. Alternatively, a fragment of the present invention may be a longer sequence that includes, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the full-length polynucleotide of the present invention.

[0145] Accordingly, the antigen-binding molecules of the present invention may be produced from polynucleotides that encode them and are capable of expressing them, or may be delivered in the form of such polynucleotides. When the antibody contains two or more chains, the polynucleotide of the present invention may encode one or more antibody chains. For example, the polynucleotide of the present invention may encode an antibody light chain variable domain, an antibody heavy chain variable domain, or both. It is also possible to provide two polynucleotides, one encoding an antibody light chain variable domain and the other encoding the corresponding antibody heavy chain variable domain. Such polynucleotides or polynucleotide pairs may be co-expressed so that the antigen-binding molecules of the present invention are produced.

[0146] The polynucleotides of the present invention can be synthesized according to methods well known in the art, for example, as described in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0147] The nucleic acid molecules of the present invention may be provided in the form of an expression cassette that is operably linked to the inserted sequence and thereby capable of expressing the antigen-binding molecules of the present invention in vivo. These expression cassettes are then typically provided within a vector (e.g., a plasmid or recombinant viral vector). Such expression cassettes may be administered directly to the host subject. Alternatively, a vector containing the polynucleotide of the present invention may be administered to the host subject. Preferably, gene vectors are used to prepare and / or administer the polynucleotide. Suitable vectors may be any vector that carries a sufficient amount of genetic information and is capable of expressing the polypeptide of the present invention.

[0148] Accordingly, the present invention also provides an expression vector comprising the nucleic acid molecule described herein. Such expression vectors are routinely constructed in the art of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals that may be required and arranged in the correct orientation, to express the polypeptide of the present invention. Other suitable vectors will be apparent to those skilled in the art. See, for example, Sambrook et al. for further examples in this regard.

[0149] The present invention also provides a host cell comprising the nucleic acid molecule or vector described herein. The cell is modified to express the antigen-binding molecule of the present invention. Such cells include transient or preferably stable higher eukaryotic cell lines such as mammalian cells or insect cells, lower eukaryotic cells such as yeast, or prokaryotic cells such as bacterial cells. Specific examples of cells that can be modified by insertion of a vector or expression cassette encoding the antigen-binding molecule of the present invention include mammalian HEK293T, CHO, HeLa, NS0, and COS cells. Cell lines available as hosts for expressing antigen-binding molecules are well known in the art. Preferably, the cell line selected is not only stable but also allows for mature glycosylation. Such cell lines of the present invention may be cultured using conventional methods for producing the antigen-binding molecule of the present invention.

[0150] Antibody-drug conjugate (ADC) The present invention provides an antibody-drug conjugate (ADC) comprising the antigen-binding molecule described herein linked to a drug. In some embodiments, the drug is an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor. In some embodiments, the drug is selected from pyrrolobenzodiazepine (PBD) and monomethyl auristatin E (MMAE). In some embodiments, the drug is a fluorophore or a tracer molecule.

[0151] An antibody-drug conjugate of formula (I) is also described herein: Ab-(L-D)p (I) (wherein Ab is an antigen-binding molecule of the present invention described herein, L is a linker that connects Ab to D, D is an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor, and p is preferably from 1 to 8).

[0152] In some embodiments, L may be absent and may simply be a covalent bond between the antibody (Ab) and the drug (D).

[0153] Antibody-drug conjugates (i.e., immunoconjugates) enable the targeted delivery of a cytotoxic agent or a cell division inhibitor (i.e., a drug that kills cells or inhibits growth and division, such as a drug useful in the treatment of cancer) to cells such as cancer cells. The antigen-binding molecule portion enables selective binding to the target tumor. The ADC then internalizes into the target cells, typically cancer cells, resulting in the accumulation of the drug inside the cells. The ADC may include a linker that connects the antibody and the drug payload. When the ADC internalizes into the target cells, the linker may be cleaved, thereby releasing the payload into the cytoplasm. Systemic administration of the unconjugated drug usually results in unacceptable levels of toxicity to normal or non-target cells. Accordingly, the present invention provides an antibody-drug conjugate that targets the delivery of a drug to B7H3-positive cells. The B7H3-positive cells may be cancer cells. Accordingly, the present invention provides an antibody-drug conjugate comprising an antigen-binding molecule of the present invention that specifically binds to B7H3 as defined above and a drug, wherein the drug is an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor.

[0154] The antibody-drug conjugates described herein can have several advantageous features. For example, after administration to a subject in need thereof, the antibody-drug conjugate can be rapidly cleared from the subject's system to minimize residual toxicity. The Ab may be selected, modified, or genetically engineered to have a short half-life in the subject of interest. The antibody-drug conjugates described herein are highly specific and targeted, potent agents.

[0155] Antibody (Ab) In the antibody-drug conjugates described herein, the Ab can be any antigen-binding molecule of the invention described herein. All descriptions of the antigen-binding molecules of the invention apply mutatis mutandis to the Ab within the antibody-drug conjugates described herein. Any feature of the antigen-binding molecules of the invention described herein applies to the Ab of the antibody-drug conjugates described herein. Specifically, any of the antigen-binding molecules described herein can be incorporated into the antibody-drug conjugates described herein. The Ab of the antibody-drug conjugates of the invention is an antigen-binding molecule of the invention.

[0156] For example, the Ab can be an antigen-binding molecule of the invention that includes a binding domain that specifically binds to B7H3, wherein the binding domain includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain includes HCDR1 comprising the sequence of SEQ ID NO: 68, HCDR2 comprising the sequence of SEQ ID NO: 70, and HCDR3 comprising the sequence of SEQ ID NO: 72, the light chain variable domain includes LCDR1 comprising the sequence of SEQ ID NO: 76, LCDR2 comprising the sequence of SEQ ID NO: 78, and LCDR3 comprising the sequence of SEQ ID NO: 80, and preferably, the heavy chain variable domain comprises the sequence of SEQ ID NO: 66 and the light chain variable domain comprises the sequence of SEQ ID NO: 74.

[0157] Drug unit An antibody-drug conjugate can comprise any antibody (Ab) described herein that is conjugated (i.e., linked or fused) to an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor (D). Thus, D is an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor. An anti-cancer agent (also referred to as an anti-tumor agent) is any agent, small molecule, or biologic that is effective in the treatment of cancer. A cytotoxic agent is any agent that results in cell death, preferably cancer cell death and tumor shrinkage. A cell division inhibitor is any agent that inhibits, reduces, or blocks the growth or division of cells, preferably cancer cells, and inhibits tumor growth. D may be a known anti-cancer therapeutic agent that has demonstrated anti-cancer, cytotoxic, or cell division inhibitory properties.

[0158] Drug loading is the average number of drug units (D) per antibody (Ab) and is represented by p. The average number of drugs per antibody in a preparation of antibody-drug conjugate obtained from a conjugation reaction can be characterized by conventional means such as UV, reverse-phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis. In some cases, the separation, purification, and characterization of homogeneous antibody-drug conjugates with a specific value of p from antibody-drug conjugates with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis. The drug loading (p) is usually limited by the number of binding sites for the drug and linker on the antibody. The binding site can be understood to generally mean the site on the antibody to which the drug unit binds via a linker. For example, an antibody can have 1, 2, 3, 4, 5, 6, 7, or 8 binding sites to which a drug linker can bind. In some embodiments, the antibody has 1 to 8, 1 to 6, 1 to 4, or 1 to 2 such binding sites, preferably 1 to 8, most preferably 1 to 4 such binding sites. Usually, the drug moiety conjugated to the antibody during the conjugation reaction is less than the theoretical maximum number. The loading (drug / antibody ratio) of the ADC can be controlled in several different ways including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent with respect to the antibody and (ii) limiting the time or temperature of the conjugation reaction. Antibody-drug conjugate compositions are described herein that include a mixture of antibody-drug conjugates, where one or more drug units are bound to the antibody and the drug units can bind to the antibody at various different sites such as different amino acid residues. The drug unit usually binds to the antibody via a linker. Suitable linkers are further described herein. Suitable means for binding or conjugating the antibody to the linker are also further described herein.

[0159] In some embodiments, the average number (D) of drug units per antibody (Ab) in the antibody-drug conjugate of the present invention ranges from 1 to 8. In some embodiments, the range is selected from 1 to 4, 2 to 4, 1 to 3, 2 to 3, or 1 to 2, preferably 1 to 4. In some embodiments, the antibody-drug conjugate of the present invention has 1 or 2 drug units (D) per antibody (Ab). In some embodiments, p is from 1 to 8, preferably from 1 to 4. In some embodiments, p is about 2.

[0160] In some embodiments, each D is independently selected from the group consisting of an anti-cancer agent, a cytotoxic agent, a cell division inhibitor, a drug, a radioisotope, a detectable label, an enzyme, a fluorophore, a fluorescent protein, a chemiluminescent agent, a radioactive label, a heavy metal, or any other detectable compound known to those skilled in the art. In preferred embodiments, D is an anti-cancer agent, a cytotoxic agent, or a cell division inhibitor. In more preferred embodiments, D is pyrrolobenzodiazepine (PBD) or monomethyl auristatin E (MMAE).

[0161] Linker L is a linker that connects antibody Ab to drug D. L can be any linker suitable for connecting, covalently bonding, or conjugating antibody Ab to drug D. The linker L may be cleavable or non-cleavable. The linker L is preferably stable extracellularly. Thus, prior to intracellular transport or delivery, the antibody-drug conjugate of the present invention is preferably stable and remains intact, i.e., the antibody Ab remains linked to the drug D. In some embodiments, the linker L is stable outside the target cell (i.e., in the extracellular environment), but is cleaved intracellularly (i.e., in the intracellular environment) to release the drug D from the antibody Ab. Thus, the antibody Ab targets target cells expressing B7H3 with an anti-cancer drug, a cytotoxic drug, and / or a cell division inhibitor drug D. Usually, the cleavage of the linker occurs at a rate fast enough for the drug to have an anti-cancer, cytotoxic, or cell division inhibitory effect on the target cell. The linker can be cleaved at any time after the antibody-drug conjugate has internalized into the target cell. In some embodiments, the linker can be preferentially cleaved in a specific intracellular compartment within the target cell. For example, the linker L can be preferentially cleaved within lysosomes. An effective linker (i) maintains the specific binding properties of the antibody; (ii) enables intracellular delivery of the conjugate and / or the drug; (iii) remains stable and intact, i.e., is not cleaved, until the conjugate and / or the drug is delivered or transported to its target site; and (iv) maintains the cytotoxic, anti-cancer, cell-killing, and / or cell division inhibitory effects of the drug D. The stability of the antibody-drug conjugate can be measured by standard analytical techniques such as mass spectrometry, HPLC, and LC / MS, which is a separation / analysis technique.

[0162] The linker may be a non-cleavable linker, i.e., a linker that is not readily cleaved by enzymatic activity such as protease activity or under specific conditions such as acidic conditions. In some preferred embodiments, L is a cleavable linker, i.e., a linker that is readily cleaved in the presence of a suitable cleavage moiety or under specific conditions. L may be selected from the group consisting of an acid-cleavable linker, a protease-cleavable linker, a disulfide linker, an enzyme-cleavable linker, a pH-sensitive linker, a thiol-sensitive linker, or a reactive oxygen species-sensitive linker. L can be any suitable linker that enables targeted delivery of the drug unit to B7H3-positive cells. Suitable linkers are described, for example, in Yang et al. Med Res Rev. 2020;1-32, and one of ordinary skill in the art will be able to select a suitable linker.

[0163] Conjugates of antibodies and cytotoxic agents can be prepared using any suitable method as disclosed in the art, such as, for example, "Bioconjugate Techniques", G.T. Hermanson, 3rd Ed., Elsevier Inc., 2013. The linker can be conjugated to the antibody (Ab) using, for example, cleavable disulfide or non-cleavable thioether linker chemistries. The linker can bind to a lysine residue of the antibody, and the lysine can be native or genetically engineered. The linker can also bind to a cysteine residue of the antibody. The cysteine can be native, such as one of the cysteines in the interchain disulfide bridges of the antibody, or the cysteine can be genetically engineered, i.e., site-specifically inserted into the antibody sequence at the desired conjugation site. The linker can bind to non-natural amino acids (such as acetyl-phenylalanine, p-acetyl-L-phenylalanine (pAcF), selenocysteine, or para-azidomethyl-L-phenylalanine, etc.) in the antibody, for example, by an oxime bond. The antibody can be genetically engineered to contain non-natural amino acids at the desired conjugation site. Chemoselective site-specific conjugation can also be used. For example, an azide group can be formed at an asparagine residue in the antibody constant region and linked to the drug unit using a copper-mediated click reaction or the like. The azide group can be formed by a selective hydrolysis reaction mediated by the chemoselective enzyme Endo-β-N-acetylglucosaminidase (EndoS). Other strategies for site-specific conjugation of the linker and the drug unit bound thereto are known in the art and are extensively covered in G.T. Hermanson "Bioconjugate Techniques", 2013, Elsevier Inc.

[0164] Pharmaceutical Compositions, Methods, and Uses The present invention also provides a pharmaceutical composition comprising an antigen-binding molecule, CAR, cell (such as a T cell), or ADC described herein. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0165] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for parenteral administration, for example, intravenous, intramuscular, or subcutaneous administration (e.g., by injection or infusion). Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995 describes compositions and formulations suitable for the pharmaceutical delivery of the antigen-binding molecules, CARs, cells comprising CARs, or ADCs described herein.

[0166] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water, and physiological saline. Examples of other carriers include aqueous dextrose, glycerol, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, by using coating materials such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants, appropriate fluidity can be maintained. In many cases, it is preferred to include in the composition an isotonic agent, for example, a polyalcohol such as sugar, mannitol, sorbitol, or sodium chloride.

[0167] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the active agent (e.g., an antibody) in a suitable solvent with one or a combination of the ingredients enumerated above, as required, followed by sterile filtration through a fine filter. The pharmaceutical compositions of the present invention may contain additional active ingredients in addition to the antigen-binding molecules, CARs, cells comprising a CAR, or ADCs of the present invention. For example, the pharmaceutical composition may further contain an additional therapeutic or prophylactic agent.

[0168] The present invention further provides a method of treating cancer, comprising administering to a subject in need thereof an antigen-binding molecule, CAR, cell comprising a CAR, ADC, or pharmaceutical composition described herein. An effective amount of an antigen-binding molecule, CAR, cell comprising a CAR, ADC, or pharmaceutical composition described herein can be administered. "Effective amount" refers to an amount effective at the required dosage and for the required period to achieve the desired therapeutic result. The present invention also provides an antigen-binding molecule, CAR, cell comprising a CAR, ADC, or pharmaceutical composition described herein for use in a method of treating cancer.

[0169] In some examples, the cancer is selected from the group consisting of solid tumors, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma, rhabdomyosarcoma, hematological malignancies, acute myeloid leukemia, desmoplastic small round cell tumor (DSRCT), melanoma, breast cancer, prostate cancer, colon cancer, lung cancer, kidney cancer, or pancreatic cancer, or oral squamous cell carcinoma (SCC).

[0170] When a disease or disorder (e.g., cancer such as solid tumors) is "treated" as discussed herein (e.g., in the methods or uses of the present invention), this means that one or more symptoms of the disease or disorder (e.g., cancer such as solid tumors) are alleviated. It does not mean that the symptoms of the disease or disorder (e.g., cancer such as solid tumors) are completely improved such that there are no longer any symptoms in the patient, although in some methods this may be the case. Thus, in all instances, the terms "treat" or "treating" can be replaced with the terms "alleviate" or "alleviating", respectively. The methods or uses of the present invention (such as methods of treating or treating) can reduce the severity of one or more of the symptoms of a disease or disorder (e.g., cancer such as solid tumors) compared to before treatment.

[0171] The antigen-binding molecules, CARs, cells comprising CARs, or ADCs of the present invention, or pharmaceutical compositions comprising the antigen-binding molecules, CARs, cells comprising CARs, or ADCs of the present invention can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route of administration and / or method of administration will vary depending on the desired result. Preferably, the antigen-binding molecules, CARs, cells comprising CARs, or ADCs, or pharmaceutical compositions of the present invention can be administered by parenteral administration. As used herein, the expression "parenteral administration" means a route of administration other than enteral administration and topical administration, and is usually by injection. Preferred routes of administration of the antigen-binding molecules, CARs, cells comprising CARs, ADCs, or pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes of administration, e.g., injection or infusion. Alternatively, the antigen-binding molecules, CARs, cells comprising CARs, ADCs, or pharmaceutical compositions of the present invention may be administered via a parenteral route, e.g., a topical, epidermal, or mucosal route of administration. Local administration, including administration around, near, within the tumor, within, around the lesion, intra-cavitary injection, intravesical administration, and inhalation, is also possible.

[0172] A suitable dosage of the antigen-binding molecule, CAR, cell comprising a CAR, or ADC of the present invention can be determined by those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied so that it is not toxic to the patient and an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration is obtained. The dosage level selected will depend on various pharmacokinetic factors including the activity of the particular antibody being used, the route of administration, the time of administration, the rate of excretion of the antibody, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition being used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0173] A suitable dosage of the antigen-binding molecule, CAR, cell comprising a CAR, or ADC of the present invention can be, for example, in the range of about 100 ng / kg (body weight of the patient being treated) to about 25 mg / kg per day. For example, a suitable dosage can be about 1 μg / kg (body weight) to about 10 mg / kg per week, about 100 μg / kg (body weight) to about 10 mg / kg per week, or about 10 μg / kg (body weight) to about 5 mg / kg per week. A suitable dosage can be about 1 μg / kg (body weight) to about 10 mg / kg per day, about 100 μg / kg (body weight) to about 10 mg / kg per day, or about 10 μg / kg (body weight) to about 5 mg / kg per day. In some embodiments, 1×10 6 or 5×10 6 CAR T cells per kg can be administered.

[0174] The dosing regimen may be adjusted to provide an optimal desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time, and the dose may be proportionally decreased or increased as indicated by the requirements of the treatment situation. It is particularly advantageous to formulate a composition for parenteral administration in unit dosage form to facilitate administration and to make the dosage uniform. As used herein, unit dosage form refers to physically discrete units suitable as a single dosage for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic or conditioning effect in association with the required pharmaceutical carrier.

[0175] The antigen-binding molecules, CARs, cells comprising a CAR, or ADCs described herein may be administered as a single dose or in multiple doses. In multiple dosing, administration may be at the same or different locations via the same or different routes. Alternatively, the antigen-binding molecule may be administered as a sustained-release formulation, in which case the required dosing frequency will be less. The dosage and frequency may vary depending on the half-life of the antigen-binding molecule in the patient and the desired treatment period.

[0176] The pharmaceutical composition may comprise any of the antigen-binding molecules, CARs, cells comprising a CAR, or ADCs of the invention described herein. In some embodiments, the pharmaceutical composition may comprise a single antigen-binding molecule, CAR, cell comprising a CAR, or ADC of the invention. In some embodiments, the pharmaceutical composition may comprise two or more different species of the antigen-binding molecules, CARs, cells comprising a CAR, or ADCs of the invention within the same composition. The invention also provides for the co-administration of two different pharmaceutical compositions each comprising a single but different species of the antigen-binding molecules, CARs, cells comprising a CAR, or ADCs of the invention.

[0177] Method for detecting cancer The invention also provides a method for detecting cancer in a subject, comprising Contacting a biological sample from a subject with an antigen-binding molecule described herein and detecting the antigen-binding molecule bound to the sample, wherein binding of the antigen-binding molecule to the sample indicates that the subject has cancer. In some examples, the cancer is selected from the group consisting of solid tumor, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma, rhabdomyosarcoma, hematological malignancy, acute myeloid leukemia, desmoplastic small round cell tumor (DSRCT), melanoma, breast cancer, prostate cancer, colon cancer, lung cancer, kidney cancer, or pancreatic cancer, or oral squamous cell carcinoma (SCC). In some examples, the antigen-binding molecule specifically binds to human B7H3, and binding of the antigen-binding molecule indicates that the subject has cancer.

[0178] The present invention is illustrated by the following examples:

Examples

[0179] Example 1 - Materials and Methods Cells and Culture Conditions The following cell lines were used in this study: Jurkat, 293T, CHO, MEXi 293E, 293F, LAN-1, Kelly, SupT1, K562. CHO was cultured in CHO culture medium (Gibco) + 8 mM GlutaMAX (Gibco) + 0.4 mM hypoxanthine + 0.32 mM thymidine (Gibco). MEXi 293E cells were cultured in MEXi culture medium (IBA) + 50 mg / L gentamicin and 8 mM GlutaMAX. 293F cells were cultured in Freestyle 293 expression medium (Thermofisher). All three of these cell types were cultured at 37°C, 5% CO2, in an orbital shaker. A temperature of 32°C was used for protein production. 293T cells were cultured in IMDM (Sigma) supplemented with 10% FCS (Gibco) and 100 U penicillin / 0.1 mg streptomycin / L. They were grown at 37°C, 5% CO2. The remaining cells were cultured at 37°C, 5% CO2 in RPMI (Sigma) + 10% FCS 100 U penicillin / 0.1 mg streptomycin / L.

[0180] γ-Retroviral transduction 293T cells were plated at 1.5×10 24 hours prior to transfection. The GOI (gene of interest) expression cassette and helper plasmids env (RD114), gagpol (PegPam-env) were transduced into the cells using GeneJuice (Merck). The supernatant containing retrovirus was collected 48 hours and 72 hours later. For stable transduction, target cells were plated on a 24-well plate coated with RetroNectin (Takara) and incubated with the retroviral supernatant for 72 hours. 6 The supernatant containing retrovirus was collected 48 hours and 72 hours later. For stable transduction, target cells were plated on a 24-well plate coated with RetroNectin (Takara) and incubated with the retroviral supernatant for 72 hours.

[0181] Construction of the B7-H3 phage display library Jurkat cells were stably transduced using γ-retroviral transduction to produce recombinant B7-H3-mouse Fc fusion protein. The protein was produced in a bioreactor and purified using a protein A column. Three BALB / cJ mice were injected with the recombinant protein (Figure 1A). Seroconversion was confirmed by flow cytometry from serial extraction of serum. Spleen mRNA was extracted using the RNeasy Mini Kit (QIAGEN). After reverse transcription of the mRNA (Superscript III Reverse Transcriptase, Invitrogen), it was amplified by PCR (Amplitaq Polymerase, Applied Biosystems). Using further PCR reactions, the heavy and light chain DNAs were connected with a serine-glycine linker. The amplified DNA was first cloned into the intermediate pSP73 vector and then into the pHEN vector. Escherichia coli was transduced using the electroporation method (Figure 1).

[0182] Panning of the B7-H3 library The 4Ig-B7-H3 cDNA was purchased (Sinobiological) and cloned into two types of vectors to generate the B7-H3-His tag and the B7-H3-Strep tag. These constructs were transiently transfected into CHO cells and MEXi293E cells respectively. The cells were cultured until the survival rate decreased, and the proteins were filtered from the cell supernatant using HiTrap MabSelect Protein-A Columns (Cytiva) or Strep-Tactin XT:Twin Strep tag purification columns (IBA).

[0183] E. coli was inoculated into 2TY medium. The bacteria were cultured until the OD reached 0.5. The bacteria were infected with M13KO7 helper phage (New England Biolabs) and cultured overnight. The bacteria were removed by centrifugation, and the phage particles in the supernatant were precipitated with PEG600 / 2.5M NaCl. After washing, the phage was resuspended in sterile water.

[0184] The immunotubes were incubated with the B7-H3-His tag, and the MagStrep “type3” XT beads (IBA) were incubated with the B7-H3-Strep tag at 4°C overnight to coat the tubes / beads. The tubes / beads were washed three times with PBS and blocked by incubating with Marvel Milk at room temperature for 2 hours. The precipitated phage was blocked separately with Marvel Milk. The tubes / beads were washed three times with PBS. The blocked phage was applied to the coated and blocked tubes and incubated at room temperature for 2 hours. The tubes / beads were washed, and the bound phage was eluted with 1 mL of 100 μM triethylamine. The eluted phage was incubated with TG1 E. coli with an OD of 0.5 for 40 minutes. The bacteria were pelleted and plated on agar plates (Figure 1B).

[0185] Selection of anti-B7-H3 scFv Colonies selected from the panned library were grown in 96-well plates. Positive binders were identified using ELISA against immobilized recombinant B7-H3. Bound scFv-myc was detected with anti-myc (Sigma), followed by anti-rabbit HRP (Sigma). The selected binders were cloned into the scFv-Fc format in a pcDNA3.1 expression vector. 293F cells were transiently transfected with PEI (Sigma). Cells were cultured until the viability decreased, and the supernatant was collected. The protein was purified using a HiTrap MabSelect Protein-A column (Cytiva). The purified and diluted protein was used to stain B7-H3 bound to cells on Jurkats, and binding was analyzed using flow cytometry.

[0186] Generation of B7-H3-positive Jurkat cells The truncated B7-H3 (T-B7-H3) in the SFG γ-retroviral expression cassette was used. The 4Ig-B7-H3 isoform of B7-H3 was purchased (Sinobiological) and cloned into the γ-retroviral expression cassette. 4Ig-B7-H3 was digested to generate 2Ig-B7-H3. These three isoforms of B7-H3 were stably transduced into Jurkat cells by retroviral transduction, respectively.

[0187] Isolation of PBMC and T cells Leukocyte cone apheresis was obtained from NHS Blood and Transplant. PBMC were separated by Ficoll centrifugation using Lymphoprep (Stemcell Technologies). PBMC were washed and residual red blood cells were lysed with ACK lysis buffer (Thermofisher). NK cells were depleted using magnetic CD56 depletion beads (Miltenyi Biotec) and LD depletion columns (Miltenyi Biotec).

[0188] Generation of CAR-T construct The gene blocks of each anti-B7-H3 CAR T cell were designed and cloned into the expression vector scFv-CH2-CH3-CD28-CD3zγ-retroviral CAR expression cassette (Philip et al., 2014) with the previously reported RQR8 marker gene for selection / exclusion using the restriction sites at the 3' prime and 5' prime ends of the CAR (Thermofisher). These gene blocks included TE9-CD8H / Tm-CD28-CD3z, TC6-CD8H / Tm-CD28-CD3z, TF9-CD8H / Tm-CD28-CD3z, BF9-CD8H / Tm-CD28-CD3z, BH6-CD8H / Tm-CD28-CD3z, TE9-CD8H / Tm-4-1BB-CD3z, TE9-CD28H / Tm-CD28-CD3z, TE9-CD8H / Tm-CD28-ILR2-CD3z.

[0189] CAR T cell transduction PBMCs were suspended in RPMI containing FCS and L-glutamine at a concentration of 1×10 6 cells / mL. It was activated with 0.5 μg / mL of anti-CD3 antibody (Miltenyi Biotec) and anti-CD28 antibody (Miltenyi Biotec). Recombinant human IL-2 (Proleukin, Novartis) at 100 IU / mL was added 48 hours before transduction and on the day of transduction. T cells were transduced using γ-retroviral transduction.

[0190] T cell function assay In the co-culture assay with CAR-T cells, the targets were LAN-1, Kelly, K562, or AML target cells (MV411, NOMO1, THP1) or without antigen stimulation in a 48-well plate at an effector:target ratio of 2:1. In most experiments, the control target cells were Jurkat cells or SupT1 cells stably transduced to express the target antigen 4×Ig human B7H3. For the 18-hour co-culture assay, the CAR T cells were co-cultured with the targets. After 18 hours, the supernatant was removed for ELISA, and the cells were incubated with monensin (BioLegend). The activation markers CD69 and CD25 and the degranulation marker CD107a were detected by flow cytometry. For the 7-day co-culture, the CAR T cells were labeled with CSFE or cell trace violet and co-cultured with LAN-1, Kelly, K562, or AML target cells (MV411, NOMO1, THP1) or without antigen target at an effector:target ratio of 2:1 in a 24-well plate for 6 days. On day 6, the plates were centrifuged to pellet the cells, 1 mL of the medium was removed, and 1 mL containing fresh target cells was added. After an additional 24 hours, the supernatant was removed for ELISA, the cells were pelleted, and flow cytometry was used to examine the levels of the exhaustion markers Tim3, Lag-3, and PD-1 and the proliferation as measured by CSFE dilution. To evaluate the proliferative capacity of the IL-2Rβ-modified CAR construct, the CAR T cells were labeled with CellTrace Violet (ThermoFisher) and co-cultured in a 48-well plate at an effector:target ratio of 1:1 with wild-type Jurkats, B7-H3-expressing Jurkats or without target cells for 6 days. The cells were plated with either no cytokine, 70 ng / mL of IL-15 (PeproTech), or 100 IU / mL of IL-2 (Proleukin, Novartis), and fresh target cells were supplied on days 2 and 4 of the co-culture. The cell proliferation and fold expansion were evaluated on day 6 by flow cytometry analysis using Precision Count Beads (BioLegend).In the 28-day co-culture assay, CAR T cells were co-cultured with irradiated LAN-1 and Kelly or without target cells at an effector:target ratio of 2:1 in 24-well plates. The cell medium was replenished every 2 - 3 days. CAR T cells were challenged with irradiated target cells every 6 days and further cultured for 24 hours and analyzed. The cells were pelleted and the supernatant was removed weekly for ELISA. The proliferation of CAR-T cells was measured weekly by flow cytometry using Precision Count Beads (BioLegend). The levels of cytokines IL-2 and IFN-γ were quantified using ELISA MAX Deluxe Set Human IL-2 and ELISA MAX Deluxe Set Human IFN-γ (BioLegend). Cytotoxicity was tested using the Cr. 51 release cytotoxicity assay. Target cells were incubated with Cr 51 for 1 hour, washed, and plated in 96-well plates. CAR T cells or non-transduced cells were plated at effector:target ratios of 10:1, 5:1, 2.5:1, and 1.25:1. The plates were incubated at 37°C for 4 hours, the supernatant was removed, and the plates were incubated with scintillation fluid (Perkin Elmer) overnight at room temperature. The Cr 51 released into the supernatant was measured using a 1450 MicroBeta TriLux (Perkin Elmer). The activity of CAR T cells against decreasing concentrations of B7-H3 protein was measured using a plate-based assay. ELISA plates were coated with decreasing concentrations of recombinant B7-H3 and incubated overnight at 4°C. The plates were washed and CAR T cells or non-transformed cells were added. The plates were incubated overnight at 37°C, the cells were pelleted, and the supernatant was removed for use in ELISA.

[0191] Hematopoietic colony assay The clonogenic assay [also known as the colony-forming cell (CFC) assay, colony-forming unit (CFU) assay, and methylcellulose assay] is an in vitro assay used in the study of hematopoietic stem cells. This assay is based on the ability of individual hematopoietic progenitor cells, called colony-forming units (CFU), to proliferate and differentiate into colonies in a semi-solid medium in response to cytokine stimulation. The colonies formed can be enumerated and characterized according to their unique morphology. This assay was used to examine colony formation from cord blood (CB) and NOMO-1 leukemia cells after treatment with TE9-CD8-28ζCAR T cells. Non-transduced T cells were used as a control.

[0192] H4434 Classic Methocult medium (STEMCELL Technologies) was used in this assay as the semi-solid matrix: it contains rh SCF (stem cell factor), rh GM-CSF (granulocyte macrophage colony-stimulating factor), rh IL-3, rh EPO and enables the growth of CFU-E (erythroid progenitor cells), BFU-E (burst-forming cells of erythroblasts), CFU-GM (granulocyte and / or macrophage progenitor cells), and CFU-GEMM (pluripotent progenitor cells) as well as leukemia colonies in CB.

[0193] Prior to the experiment, Methocult medium was aliquoted at 2 mL / aliquot into Sterilin (trademark) 7 mL Bijou (Thermo Scientific) and stored at -20 °C. Briefly, effector cells (non-transduced T cells and TE9-CD8-28ζCAR T) and target cells (CB and NOMO-1) were co-cultured at an E:T ratio of 5:1 in a tissue culture-treated 48-well plate at 37 °C for 18 hours.

[0194] After incubation, cells under each co-culture condition were separately harvested and washed with Iscove's MDM supplemented with 2% FBS (STEMCELL Technologies), which is the recommended medium for sample preparation and washing for the CFU assay. Thereafter, if no lysis occurred, the cells were resuspended in Iscove's medium at the desired concentrations such that there were 20,000 CB cells in 40 μL of the cell suspension and 2,000 NOMO-1 cells in 40 μL of the cell suspension.

[0195] Next, 40 μL of the cell suspension was transferred into one 2 mL Methocult aliquot and shaken vigorously to evenly distribute the cells in the Methocult. Then, 1 mL of Methocult containing the desired number of cells (10,000 for CB and 1,000 for NOMO-1) was plated into the wells of a 6-well plate that had been tissue culture-treated using a 16 Gauge Blunt-End Needle (STEMCELL Technologies).

[0196] These seeding densities were estimated based on the assumption that only 1% of the cells from CB are colony-forming units of hematopoietic progenitors and were selected to make the conditions of NOMO-1 and CB as equivalent as possible. Other densities were also tested, but the wells were too crowded or not enough colonies were generated to be appropriately quantified (data not shown).

[0197] To maintain high humidity and prevent drying of the Methocult, the plate was surrounded with PBS and incubated at 37 °C for 14 days. On day 14, the number and morphology of the colonies were evaluated microscopically, and then the colonies were stained dark purple with p-iodonitrotetrazolium violet (Sigma) so that the colonies appeared in the photograph.

[0198] Antibodies and Flow Cytometry Analysis The following antibodies were used in this assay: anti-B7-H3 (FM276, Miltenyi Biotech), anti-GD2 (14.G2a, BD Biosciences), human Ig (polyclonal, Thermofisher), anti-mouse IgG (polyclonal, R&D), anti-CD3 (UCHT1, BioLegend), anti-HisTag (J095G45, BioLegend), anti-CD34 (QBEnd10, R&D), anti-αβ-TCR (IP26, BioLegend), anti-CD107a (H4A3, BioLegend), anti-cD25 (BC96, BioLegend), anti-CD69 (FN50, BioLegend), anti-Tim3 (F38-2E2, BioLegend), anti-Lag3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-mouse CD45 (30-F11, BioLegend), anti-human CD45 (HI30, BioLegend), Ghost Red™ 780 (Tonbo Biosciences), Zombie Yellow Viability Dye (BioLegend), propidium iodide (Gibco), Cell Trace Violet (ThermoFisher), Precision Count Beads (BioLegend).

[0199] Cross-reactivity of TC6, TE9, and BH6 full antibodies TC6, TF9, and BH6 were generated as chimeric antibodies with a human IgG1 Fc domain. The antibodies were purified on a protein A column (Cytiva), tested by ELISA against the antigen bound to the plate, and detected using goat anti-human IgG (H+L) (SeraCare). Cross-reactivity against mouse B7-H3 was tested using flow cytometry against the mouse cell line 3T3 / NA1.

[0200] In vivo models of LAN-1 neuroblastoma and Med8A medulloblastoma Animal protocols were approved by the local institutional research committee and were in accordance with the guidelines of the UK Home Office. Male NSG mice, 6 - 8 weeks old, were provided by UCL. All experiments were carried out under UK Home Office project license number 15981 / 01 and personal license number 12972. For the neuroblastoma LAN-1 experiment, 1×10 6 individual LAN-1-BFP / Luc were subcutaneously injected into the flanks of NSG mice. On day 10, 1×10 6 individual CAR T cells were intravenously injected into the tail vein. Tumor size was monitored twice a week using a digital caliper. 200 μL of luciferin was administered into the neck muscle of the mice and imaged weekly using a PhotonIMAGER™ optical imaging system (Biospace Lab). Mice were sacrificed when the tumors reached the threshold size, and samples of blood, spleen, and tumor were collected. Cells were dissociated using a cell strainer and residual red blood cells were removed using ACK lysis buffer (ThermoFisher). Cells were stained and markers were analyzed using flow cytometry. For the Med8A medulloblastoma experiment, 1×10 6 individual Med8A medulloblastoma cells stably transduced with luciferase were stereotactically implanted into the hemisphere in a volume of 3 - 5 microliters. 48 hours later, 5×10 6 individual CAR-T cells or non-transduced controls were injected into the lateral ventricle. Tumor growth was evaluated by bioluminescence imaging.

[0201] Amplification of gamma delta T cells PBMCs were isolated from purchased whole blood leukocyte cones by density gradient centrifugation using Lymphoprep (Stemcell) according to the manufacturer's instructions. PBMCs were cryopreserved in 90% FBS 10% DMSO or resuspended in complete T cell culture medium for further processing. Complete T cell culture medium consists of serum-free and xenogeneic component-free CTS-OpTmizer (Thermo Fisher) containing 10% synthetic serum substitute (Thermo Fisher) and GlutaMAX (Thermo Fisher), all of which are available from Thermo Fisher in research grade and GMP grade with the following product catalog numbers: research grade CTS-OpTmizer (A1048501) and GMP grade OpTmizer-CTS (A3705003) which is a GMP compliant substitute, synthetic immunocyte serum substitute (A2596101) compliant with both manufacturing standards, and GlutaMAX (35050061) also compliant with both standards. When starting from cryopreserved material, to avoid excessive stress to the lymphocytes and to enhance the quality of depletion, PBMCs were thawed and rested overnight at 10×10 6 cells / mL in complete pre-warmed medium. Subsequently, PBMCs at a density of 2 - 4×10 6 cells / mL were immediately stimulated in standard cell culture plates or first depleted of αβ T cells using TCRα / β Product Line (Miltenyi Biotec) according to the manufacturer's instructions and simultaneously depleted of CD56 positive cells using CD56 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, cells were first labeled with anti-TCRα / β-biotin, then labeled with a mixture of anti-biotin microbeads and anti-CD56 beads, and then depleted using a MACS Cell Separation LD column (Miltenyi Biotec). When culturing in G-Rex vessels (Wilson Wolf), 2 - 4×10 6 cells / cm 2Started with depleted PBMC. The thus prepared PBMC were stimulated with 1 μg / mL of OKT-3 (Miltenyi Biotec Cat#130-093-387, RRID:AB_1036144) or 1 μg / mL of PHA (Merck) and combinations of various cytokines: (i) 100 IU / mL Aldesleukin (Proleukin; Novartis), (ii) 70 ng / mL IL-15 (Peprotech), (iii) 20 ng / mL rhIL-7 (Peprotech), or (iv) a cytokine cocktail consisting of a first culture in 100 ng / mL rIL-4, 70 ng / mL rIFN-γ, 7 ng / mL rIL-21, and 15 ng / mL rIL-1β followed by a second culture in 70 ng / mL rIL-15 and 30 ng / mL IFN-γ (all from Peprotech), the "DOT protocol". When comparing the complete "DOT protocol" to the test amplification protocol, the positive selection step using OKT-3 after alpha-beta TCR depletion was omitted, but the methodology described by Almeida et al. (2016) was used. Briefly, depleted PBMC were stimulated for a first cytokine culture with 70 ng / mL OKT-3 followed by a second cytokine culture with 1 μg / mL OKT-3. Live cells before and during amplification were counted using trypan blue exclusion, an automated cell counter (Invitrogen), and flow cytometry-based Precision Count Beads (Biolegend).

[0202] Three stages of amplification: Vδ2 γδ T cells were depleted from PBMC at one of three stages: before start, during split, or at collection. 0.5 μg / 10 6Depletion of all individual PBMCs was performed using anti-TCR / Vδ2 mAb clone B6 (BioLegend Cat#331404, RRID:AB_1089228). When depleting at the start, Vδ2 cell initiation was incorporated into the αβTCR / CD56 depletion process. This was done as follows: PBMCs were coinubated with αβTCR-biotin mAb and Vδ2 (clone: B6)-biotin mAb, washed, then coinubated with anti-biotin and anti-CD56 microbeads according to the manufacturer's protocol, then washed, as above, and depleted using Miltenyi LD magnetic column separation according to the manufacturer's protocol. When depleting during splitting or at final collection, the expanding cells were collected, washed, labeled with 0.5 μg of clone B6 / 10 6 individual PBMCs, incubated for 20 minutes, washed, incubated, and depleted using Miltenyi anti-biotin microbeads and LD column.

[0203] Viral transduction of gamma delta T cells by CAR-T 293T cells (ATCC Cat#CRL-3216, RRID:CVCL_0063) were seeded at 1.5×10 2 per 10 cm 6Cells were plated. When they reached 70% confluence, 293T cells were transfected with GeneJuice (Merck) according to the manufacturer's protocol. Triple plasmid transient transfection was performed using an SFG-gamma retroviral vector (RRID:Addgene_22493) containing B7H3-CAR, gag+pol (RRID:Addgene_8449), and RD114 envelope (RRID:Addgene_17576) plasmids in equimolar ratios. Retroviral supernatants were collected 48 and 72 hours after transfection and immediately used for T cell transduction. Briefly, uncoated 24-well plates (Costar) were coated with RetroNectin (Takara) in PBS (final concentration 1 mg / mL) and incubated at 4°C for 24 hours. RetroNectin was removed, and 1.5 mL of retroviral supernatant was added to each well coated with RetroNectin. Thereafter, 3×10 5 stimulated T cells in 500 μL were added, and the plates were centrifuged at 1000×g for 40 minutes at room temperature, followed by incubation in complete T cell culture medium at 37°C with IL-15 added to a final concentration of 70 ng / mL (~140 IU / mL). Transduced T cells were collected 3 days later, washed, and resuspended in complete T cell culture medium with the designated cytokines added for expansion. Transduction efficiency was evaluated by flow cytometry detection of the CD34 marker gene.

[0204] Statistical analysis All statistical analyses were performed using GraphPad Prism v8. Unless otherwise specified, data are presented as mean ± range. Statistical analysis of in vitro assays was performed using two-way ANOVA with Cr 51 one-way ANOVA with Tukey's multiple comparisons, except for the cytotoxicity assay. In in vivo analysis, tumor size and ROI were compared using the Kruskal-Wallis test, and survival was analyzed using the log-rank (Mantle-Cox) test. *** p < 0.0001, ** p < 0.001, and * p < 0.01.

[0205] Example 2 - Development of a novel anti-B7-H3 antibody in single-stranded format Targeting of the B7-H3 cancer antigen by T cells genetically engineered to express a CAR is promising in preclinical models and is moving into clinical trials. To date, most studies have used scFvs from lipapus antibodies derived from existing monoclonal antibodies. To generate potentially more finely tuned novel B7-H3 binders for CAR-T applications, mice were immunized with a recombinant B7H3-Fc fusion protein, and spleen RNA from the immunized mice was used as a substrate for generating an ScFv library in bacteriophage (Figure 1A). Individual scFvs were obtained from the phage library by panning with human B7-H3 and screened as CAR-T binding elements by direct cloning into the CAR-T format to experimentally compare CAR-T effector functions (Figure 1B). Seventeen binders were identified by ELISA screening (Figure 2). Based on ELISA and genetic heterogeneity of the clones, 10 scFvs were selected for generation in the scFv-Fc format. Of the 10 scFv-Fc fusion proteins, five (TE9, TC6, BH6, TF9, and BF9) were selected for further evaluation in the CAR-T format based on the strength and specificity of their binding to B7-H3 isoforms (isoform 1, isoform 2, or the artificially cleaved isoform T-B7-H3 used as an immunogen) by flow cytometry (Figure 2). Four of the anti-B7-H3 binders (TE9, TC6, TF9, and BF9) bound to both human B7-H3 isoforms, while BH6 showed specificity for 4Ig-B7-H3 (Figure 2D). The other five binders (TB8, BG4, BD9, BC10, BB5) showed overall weak binding and no reactivity to the physiological target isoforms 4Ig B7H3 or 2Ig B7H3 (Figure 3). Sequencing of the entire library of binders selected in the original ELISA and assessment of their sequence similarity to each other revealed a high degree of diversity among the selected binders (Figure 4). The binders TE9, TC6, and BH6 were generated in the full antibody format.These antibodies showed specific binding to B7-H3 in an ELISA assay but not to other members of the human B7 family. BH6 bound to B7-H3 in both human and mouse, while TE9 and TC6 were specific for human and cynomolgus monkey (Figure 5) and showed antigen specificity similar to that of commercially available anti-B7-H3 monoclonal antibodies against neuroblastoma and synthetic cell lines (Figure 6).

[0206] Example 3 - B7-H3 binders in the CAR-T format exhibit various antigen-specific effector functions By evaluating cytotoxicity and cytokine secretion, five candidate scFv sequences were evaluated for their ability to confer antigen-specific T cell function in a second-generation CD8 hinge and transmembrane (H / Tm)-CD28-CD3ζ (28ζ) CAR format containing the CD28 and CD3ζ signaling domains (Figure 7). CARs were evaluated for effector function by culturing with neuroblastoma cells that naturally express B7-H3 (Figure 7C). All five CAR-T constructs showed similar transduction efficiency in human T cells (data not shown). Two binders (TE9, TC6) showed significant cytotoxicity specific for B7-H3-expressing target cells in a 4-hour killing assay (Figure 7B) and also showed the highest degree of cytokine response against neuroblastoma targets. However, the binder BH6 showed less B7-H3-specific cytokine production in a 24-hour assay (Figure 7D).

[0207] To determine how CAR behaves during longer-term co-culture, CAR was evaluated as CAR-T in a repeated antigen challenge assay in which CAR T cells received stimulation from irradiated tumor cells four times over a four-week period. Three of the anti-B7-H3 binders were compared to an FMC63 anti-CD19 CAR-T construct. From these experiments, it was shown that TE9-28ζ and CD19-28ζ CAR-T cells maintained the ability to produce IL-2 in response to the fourth rechallenge by B7-H3 positive leukemia cells (Figure 7E). Therefore, the TE9 binder was selected to further optimize the function of CAR-T.

[0208] Example 4 - CD28 costimulation and CD8 hinge / transmembrane region confer optimal long-term persistence to TE9 CAR-T cells Next, the endodomains of CD28 and 4-1BB in combination with the CD8 hinge and transmembrane region (H / Tm: Figure 8A) were compared by assaying effector function against neuroblastoma cells expressing B7-H3. Similar levels of transduction efficiency were observed for these two constructs (Figure 8B). Cytotoxic degranulation as determined by CD107a and upregulation of the CD25 and CD69 activation markers after target addition were higher, although not significantly, for the CD28ζ construct than for the 4-1BBζ construct (Figure 8C). In short-term co-culture, TE9-28ζ produced more interferon-γ (IFN-γ) and significantly more IL-2 than its 4-1BB counterpart (Figure 8D). The 4-1BB endodomain in CAR-T cells has been well reported to confer longer-term effector function against antigen rechallenge. Therefore, cytokine production was evaluated after rechallenge with neuroblastoma cells seven days after the first antigen challenge. Here, TE9-4-1BBζ CAR induced significantly lower levels of IL-2 and IFN-γ after repeated challenge with neuroblastoma (Figure 8E). Greater activation by the CD28 endodomain was reflected in a significantly higher upregulation of activation / exhaustion markers (data not shown).

[0209] Previous studies have shown that CD28 H / Tm confers significantly higher sensitivity to target antigens than CD8 H / Tm (Majzner et al., 2020 & Muller et al., 2021). Therefore, when TE9-28z was compared using two different H / Tm arrangements, both were expressed at similar levels in T cells (Figures 9A, 9C). When each CAR-T construct was stimulated with a decreasing concentration of recombinant B7-H3, only a slight enhancement of the IFN-γ and IL-2 responses of the CD28 H / Tm construct was shown, which was most prominent at the IFN-γ and lowest antigen concentrations (Figure 9B). After co-culturing with neuroblastoma cells LAN-1 and Kelly with different expression levels of B7-H3 and 562 cells with low B7-H3 expression for 18 hours, cytokine production was evaluated. After antigen re-challenge after 7 days of incubation, cytokine production and proliferation were evaluated, and cytokine analysis was p...

Claims

**Claim 1** An antigen-binding molecule comprising a binding domain that specifically binds to B7H3, wherein the binding domain comprises a heavy chain variable domain and / or a light chain variable domain, the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3, the light chain variable domain comprises light chain complementarity-determining regions (LCDRs) 1, 2, and 3, and the antigen-binding molecule is (a) a heavy chain variable domain sequence of SEQ ID NO: 66 and a light chain variable domain sequence of SEQ ID NO: 74; or (b) a heavy chain variable domain sequence of SEQ ID NO: 2 and a light chain variable domain sequence of SEQ ID NO: 10; or (c) a heavy chain variable domain sequence of SEQ ID NO: 18 and a light chain variable domain sequence of SEQ ID NO: 26; or (d) a heavy chain variable domain sequence of SEQ ID NO: 34 and a light chain variable domain sequence of SEQ ID NO: 42; or (e) a heavy chain variable domain sequence of SEQ ID NO: 50 and a light chain variable domain sequence of SEQ ID NO: 58; or (f) a heavy chain variable domain sequence of SEQ ID NO: 82 and a light chain variable domain sequence of SEQ ID NO: 90; or (g) a heavy chain variable domain sequence of SEQ ID NO: 98 and a light chain variable domain sequence of SEQ ID NO: 106; or (h) a heavy chain variable domain sequence of SEQ ID NO: 114 and a light chain variable domain sequence of SEQ ID NO: 122; or (i) a heavy chain variable domain sequence of SEQ ID NO: 130 and a light chain variable domain sequence of SEQ ID NO: 138; or (j) a heavy chain variable domain sequence of SEQ ID NO: 146 and a light chain variable domain sequence of SEQ ID NO: 154; or (k) a heavy chain variable domain sequence of SEQ ID NO: 162 and a light chain variable domain sequence of SEQ ID NO: 170; or (l) a heavy chain variable domain sequence of SEQ ID NO: 178 and a light chain variable domain sequence of SEQ ID NO: 186; or (m) a heavy chain variable domain sequence of SEQ ID NO: 194 and a light chain variable domain sequence of SEQ ID NO: 202; or (n) a heavy chain variable domain sequence of SEQ ID NO: 210 and a light chain variable domain sequence of SEQ ID NO: 218; or (o) a heavy chain variable domain sequence of SEQ ID NO: 226 and a light chain variable domain sequence of SEQ ID NO: 234; or (p) a heavy chain variable domain sequence of SEQ ID NO: 242 and a light chain variable domain sequence of SEQ ID NO: 250; or (q) a heavy chain variable domain sequence of SEQ ID NO: 258 and a light chain variable domain sequence of SEQ ID NO: 266 and an antigen-binding molecule comprising the complementarity-determining region (CDR) sequences thereof. **Claim 2** An antigen-binding molecule comprising a binding domain that specifically binds to B7H3, wherein the binding domain comprises a heavy-chain variable domain and / or a light-chain variable domain, the heavy-chain variable domain comprises heavy-chain complementarity-determining regions (HCDRs) 1, 2, and 3, and the light-chain variable domain comprises light-chain complementarity-determining regions (LCDRs) 1, 2, and 3, (a) HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, and LCDR3 comprises the sequence of SEQ ID NO: 80; or (b) HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 6, HCDR3 comprises the sequence of SEQ ID NO: 8, LCDR1 comprises the sequence of SEQ ID NO: 12, LCDR2 comprises the sequence of SEQ ID NO: 14, and LCDR3 comprises the sequence of SEQ ID NO: 16; or (c) HCDR1 comprises the sequence of SEQ ID NO: 20, HCDR2 comprises the sequence of SEQ ID NO: 22, HCDR3 comprises the sequence of SEQ ID NO: 24, LCDR1 comprises the sequence of SEQ ID NO: 28, LCDR2 comprises the sequence of SEQ ID NO: 30, and LCDR3 comprises the sequence of SEQ ID NO: 32; or (d) HCDR1 comprises the sequence of SEQ ID NO: 36, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 44, LCDR2 comprises the sequence of SEQ ID NO: 46, and LCDR3 comprises the sequence of SEQ ID NO: 48; or (e) HCDR1 comprises the sequence of SEQ ID NO: 52, HCDR2 comprises the sequence of SEQ ID NO: 54, HCDR3 comprises the sequence of SEQ ID NO: 56, LCDR1 comprises the sequence of SEQ ID NO: 60, LCDR2 comprises the sequence of SEQ ID NO: 62, and LCDR3 comprises the sequence of SEQ ID NO: 64; or (f) HCDR1 comprises the sequence of SEQ ID NO: 84, HCDR2 comprises the sequence of SEQ ID NO: 86, HCDR3 comprises the sequence of SEQ ID NO: 88, LCDR1 comprises the sequence of SEQ ID NO: 92, LCDR2 comprises the sequence of SEQ ID NO: 94, and LCDR3 comprises the sequence of SEQ ID NO: 96; or (g) the HCDR1 comprises the sequence of SEQ ID NO: 100, the HCDR2 comprises the sequence of SEQ ID NO: 102, the HCDR3 comprises the sequence of SEQ ID NO: 104, the LCDR1 comprises the sequence of SEQ ID NO: 108, the LCDR2 comprises the sequence of SEQ ID NO: 110, and the LCDR3 comprises the sequence of SEQ ID NO: 112; or (h) the HCDR1 comprises the sequence of SEQ ID NO: 116, the HCDR2 comprises the sequence of SEQ ID NO: 118, the HCDR3 comprises the sequence of SEQ ID NO: 120, the LCDR1 comprises the sequence of SEQ ID NO: 124, the LCDR2 comprises the sequence of SEQ ID NO: 126, and the LCDR3 comprises the sequence of SEQ ID NO: 128; or (i) the HCDR1 comprises the sequence of SEQ ID NO: 132, the HCDR2 comprises the sequence of SEQ ID NO: 134, the HCDR3 comprises the sequence of SEQ ID NO: 136, the LCDR1 comprises the sequence of SEQ ID NO: 140, the LCDR2 comprises the sequence of SEQ ID NO: 142, and the LCDR3 comprises the sequence of SEQ ID NO: 144; or (j) the HCDR1 comprises the sequence of SEQ ID NO: 148, the HCDR2 comprises the sequence of SEQ ID NO: 150, the HCDR3 comprises the sequence of SEQ ID NO: 152, the LCDR1 comprises the sequence of SEQ ID NO: 156, the LCDR2 comprises the sequence of SEQ ID NO: 158, and the LCDR3 comprises the sequence of SEQ ID NO: 160; or (k) the HCDR1 comprises the sequence of SEQ ID NO: 164, the HCDR2 comprises the sequence of SEQ ID NO: 166, the HCDR3 comprises the sequence of SEQ ID NO: 168, the LCDR1 comprises the sequence of SEQ ID NO: 172, the LCDR2 comprises the sequence of SEQ ID NO: 174, and the LCDR3 comprises the sequence of SEQ ID NO: 176; or (l) the HCDR1 comprises the sequence of SEQ ID NO: 180, the HCDR2 comprises the sequence of SEQ ID NO: 182, the HCDR3 comprises the sequence of SEQ ID NO: 184, the LCDR1 comprises the sequence of SEQ ID NO: 188, the LCDR2 comprises the sequence of SEQ ID NO: 190, and the LCDR3 comprises the sequence of SEQ ID NO: 192; or (m) the HCDR1 comprises the sequence of SEQ ID NO: 196, the HCDR2 comprises the sequence of SEQ ID NO: 198, the HCDR3 comprises the sequence of SEQ ID NO: 200, the LCDR1 comprises the sequence of SEQ ID NO: 204, the LCDR2 comprises the sequence of SEQ ID NO: 206, and the LCDR3 comprises the sequence of SEQ ID NO: 208; or (n) the HCDR1 comprises the sequence of SEQ ID NO: 212, the HCDR2 comprises the sequence of SEQ ID NO: 214, the HCDR3 comprises the sequence of SEQ ID NO: 216, the LCDR1 comprises the sequence of SEQ ID NO: 220, the LCDR2 comprises the sequence of SEQ ID NO: 222, and the LCDR3 comprises the sequence of SEQ ID NO: 224; or (o) the HCDR1 comprises the sequence of SEQ ID NO: 228, the HCDR2 comprises the sequence of SEQ ID NO: 230, the HCDR3 comprises the sequence of SEQ ID NO: 232, the LCDR1 comprises the sequence of SEQ ID NO: 236, the LCDR2 comprises the sequence of SEQ ID NO: 238, and the LCDR3 comprises the sequence of SEQ ID NO: 240; or (p) the HCDR1 comprises the sequence of SEQ ID NO: 244, the HCDR2 comprises the sequence of SEQ ID NO: 246, the HCDR3 comprises the sequence of SEQ ID NO: 248, the LCDR1 comprises the sequence of SEQ ID NO: 252, the LCDR2 comprises the sequence of SEQ ID NO: 254, and the LCDR3 comprises the sequence of SEQ ID NO: 256; or (q) the HCDR1 comprises the sequence of SEQ ID NO: 260, the HCDR2 comprises the sequence of SEQ ID NO: 262, the HCDR3 comprises the sequence of SEQ ID NO: 264, the LCDR1 comprises the sequence of SEQ ID NO: 268, the LCDR2 comprises the sequence of SEQ ID NO: 270, and the LCDR3 comprises the sequence of SEQ ID NO: 272, an antigen-binding molecule. (Claim 3) An antigen-binding molecule comprising a binding domain that specifically binds to B7H3, wherein the binding domain comprises a heavy-chain variable domain and / or a light-chain variable domain, (a) the heavy-chain variable domain comprises the sequence of SEQ ID NO: 66 or a sequence having at least 90% identity thereto, and the light-chain variable domain comprises the sequence of SEQ ID NO: 74 or a sequence having at least 90% identity thereto; or (b) the heavy-chain variable domain comprises the sequence of SEQ ID NO: 2 or a sequence having at least 90% identity thereto, and the light-chain variable domain comprises the sequence of SEQ ID NO: 10 or a sequence having at least 90% identity thereto; or (c) the heavy-chain variable domain comprises the sequence of SEQ ID NO: 18 or a sequence having at least 90% identity thereto, and the light-chain variable domain comprises the sequence of SEQ ID NO: 26 or a sequence having at least 90% identity thereto; or (d) the heavy chain variable domain comprises the sequence of SEQ ID NO: 34 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 42 or a sequence having at least 90% identity thereto; or (e) the heavy chain variable domain comprises the sequence of SEQ ID NO: 50 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 58 or a sequence having at least 90% identity thereto; or (f) the heavy chain variable domain comprises the sequence of SEQ ID NO: 82 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 90 or a sequence having at least 90% identity thereto; or (g) the heavy chain variable domain comprises the sequence of SEQ ID NO: 98 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 106 or a sequence having at least 90% identity thereto; or (h) the heavy chain variable domain comprises the sequence of SEQ ID NO: 114 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 122 or a sequence having at least 90% identity thereto; or (i) the heavy chain variable domain comprises the sequence of SEQ ID NO: 130 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 138 or a sequence having at least 90% identity thereto; or (j) the heavy chain variable domain comprises the sequence of SEQ ID NO: 146 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 154 or a sequence having at least 90% identity thereto; or (k) the heavy chain variable domain comprises the sequence of SEQ ID NO: 162 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 170 or a sequence having at least 90% identity thereto; or (l) the heavy chain variable domain comprises the sequence of SEQ ID NO: 178 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 186 or a sequence having at least 90% identity thereto; or (m) the heavy chain variable domain comprises the sequence of SEQ ID NO: 194 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 202 or a sequence having at least 90% identity thereto; or (n) the heavy chain variable domain comprises the sequence of SEQ ID NO: 210 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 218 or a sequence having at least 90% identity thereto; or (o) the heavy chain variable domain comprises the sequence of SEQ ID NO: 226 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 234 or a sequence having at least 90% identity thereto; or (p) the heavy chain variable domain comprises the sequence of SEQ ID NO: 242 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 250 or a sequence having at least 90% identity thereto; or (q) the heavy chain variable domain comprises the sequence of SEQ ID NO: 258 or a sequence having at least 90% identity thereto, and the light chain variable domain comprises the sequence of SEQ ID NO: 266 or a sequence having at least 90% identity thereto, an antigen-binding molecule.

4. the heavy chain variable domain comprises heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3, and the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, (a) HCDR1 comprises the sequence of SEQ ID NO: 68, HCDR2 comprises the sequence of SEQ ID NO: 70, HCDR3 comprises the sequence of SEQ ID NO: 72, LCDR1 comprises the sequence of SEQ ID NO: 76, LCDR2 comprises the sequence of SEQ ID NO: 78, and LCDR3 comprises the sequence of SEQ ID NO: 80; or (b) HCDR1 contains the sequence of SEQ ID NO: 4, HCDR2 contains the sequence of SEQ ID NO: 6, HCDR3 contains the sequence of SEQ ID NO: 8, LCDR1 contains the sequence of SEQ ID NO: 12, LCDR2 contains the sequence of SEQ ID NO: 14, and LCDR3 contains the sequence of SEQ ID NO: 16; or (c) HCDR1 contains the sequence of SEQ ID NO: 20, HCDR2 contains the sequence of SEQ ID NO: 22, HCDR3 contains the sequence of SEQ ID NO: 24, LCDR1 contains the sequence of SEQ ID NO: 28, LCDR2 contains the sequence of SEQ ID NO: 30, and LCDR3 contains the sequence of SEQ ID NO: 32; or (d) HCDR1 contains the sequence of SEQ ID NO: 36, HCDR2 contains the sequence of SEQ ID NO: 38, HCDR3 contains the sequence of SEQ ID NO: 40, LCDR1 contains the sequence of SEQ ID NO: 44, LCDR2 contains the sequence of SEQ ID NO: 46, and LCDR3 contains the sequence of SEQ ID NO: 48; or (e) HCDR1 contains the sequence of SEQ ID NO: 52, HCDR2 contains the sequence of SEQ ID NO: 54, HCDR3 contains the sequence of SEQ ID NO: 56, LCDR1 contains the sequence of SEQ ID NO: 60, LCDR2 contains the sequence of SEQ ID NO: 62, and LCDR3 contains the sequence of SEQ ID NO: 64; or (f) HCDR1 contains the sequence of SEQ ID NO: 84, HCDR2 contains the sequence of SEQ ID NO: 86, HCDR3 contains the sequence of SEQ ID NO: 88, LCDR1 contains the sequence of SEQ ID NO: 92, LCDR2 contains the sequence of SEQ ID NO: 94, and LCDR3 contains the sequence of SEQ ID NO: 96; or (g) HCDR1 contains the sequence of SEQ ID NO: 100, HCDR2 contains the sequence of SEQ ID NO: 102, HCDR3 contains the sequence of SEQ ID NO: 104, LCDR1 contains the sequence of SEQ ID NO: 108, LCDR2 contains the sequence of SEQ ID NO: 110, and LCDR3 contains the sequence of SEQ ID NO: 112; or (h) HCDR1 contains the sequence of SEQ ID NO: 116, HCDR2 contains the sequence of SEQ ID NO: 118, HCDR3 contains the sequence of SEQ ID NO: 120, LCDR1 contains the sequence of SEQ ID NO: 124, LCDR2 contains the sequence of SEQ ID NO: 126, and LCDR3 contains the sequence of SEQ ID NO: 128; or (i) HCDR1 contains the sequence of SEQ ID NO: 132, HCDR2 contains the sequence of SEQ ID NO: 134, HCDR3 contains the sequence of SEQ ID NO: 136, LCDR1 contains the sequence of SEQ ID NO: 140, LCDR2 contains the sequence of SEQ ID NO: 142, and LCDR3 contains the sequence of SEQ ID NO: 144; or (j) HCDR1 contains the sequence of SEQ ID NO: 148, HCDR2 contains the sequence of SEQ ID NO: 150, HCDR3 contains the sequence of SEQ ID NO: 152, LCDR1 contains the sequence of SEQ ID NO: 156, LCDR2 contains the sequence of SEQ ID NO: 158, and LCDR3 contains the sequence of SEQ ID NO: 160; or (k) HCDR1 contains the sequence of SEQ ID NO: 164, HCDR2 contains the sequence of SEQ ID NO: 166, HCDR3 contains the sequence of SEQ ID NO: 168, LCDR1 contains the sequence of SEQ ID NO: 172, LCDR2 contains the sequence of SEQ ID NO: 174, and LCDR3 contains the sequence of SEQ ID NO: 176; or (l) HCDR1 contains the sequence of SEQ ID NO: 180, HCDR2 contains the sequence of SEQ ID NO: 182, HCDR3 contains the sequence of SEQ ID NO: 184, LCDR1 contains the sequence of SEQ ID NO: 188, LCDR2 contains the sequence of SEQ ID NO: 190, and LCDR3 contains the sequence of SEQ ID NO: 192; or (m) HCDR1 contains the sequence of SEQ ID NO: 196, HCDR2 contains the sequence of SEQ ID NO: 198, HCDR3 contains the sequence of SEQ ID NO: 200, LCDR1 contains the sequence of SEQ ID NO: 204, LCDR2 contains the sequence of SEQ ID NO: 206, and LCDR3 contains the sequence of SEQ ID NO: 208; or (n) HCDR1 contains the sequence of SEQ ID NO: 212, HCDR2 contains the sequence of SEQ ID NO: 214, HCDR3 contains the sequence of SEQ ID NO: 216, LCDR1 contains the sequence of SEQ ID NO: 220, LCDR2 contains the sequence of SEQ ID NO: 222, and LCDR3 contains the sequence of SEQ ID NO: 224; or (o) HCDR1 contains the sequence of SEQ ID NO: 228, HCDR2 contains the sequence of SEQ ID NO: 230, HCDR3 contains the sequence of SEQ ID NO: 232, LCDR1 contains the sequence of SEQ ID NO: 236, LCDR2 contains the sequence of SEQ ID NO: 238, and LCDR3 contains the sequence of SEQ ID NO: 240; or (p) the HCDR1 comprises the sequence of SEQ ID NO: 244, the HCDR2 comprises the sequence of SEQ ID NO: 246, the HCDR3 comprises the sequence of SEQ ID NO: 248, the LCDR1 comprises the sequence of SEQ ID NO: 252, the LCDR2 comprises the sequence of SEQ ID NO: 254, and the LCDR3 comprises the sequence of SEQ ID NO: 256; or (q) the HCDR1 comprises the sequence of SEQ ID NO: 260, the HCDR2 comprises the sequence of SEQ ID NO: 262, the HCDR3 comprises the sequence of SEQ ID NO: 264, the LCDR1 comprises the sequence of SEQ ID NO: 268, the LCDR2 comprises the sequence of SEQ ID NO: 270, and the LCDR3 comprises the sequence of SEQ ID NO: 272, the antigen-binding molecule according to claim 1 or 3. [

5. ] The antigen-binding molecule according to any one of claims 1 to 4, which specifically binds to human B7H3. [

6. ] The antigen-binding molecule according to claim 5, which specifically binds to human B7H3 isoform 4IgB7-H3 or 2IgB7-H3. [

7. ] (i) specifically binds to isoform T-B7-H3; or (ii) has specificity for isoforms T-B7-H3, 4IgB7-H3, and 2IgB7-H3, the antigen-binding molecule according to any one of claims 1 to 4. [

8. ] (i) the binding domain is human or humanized; and / or (ii) the heavy chain variable domain and / or the light chain variable domain is human or humanized; and / or (iii) the antigen-binding molecule is a single domain fragment, Fab fragment, Fab' fragment, F(ab)'2 fragment, single-chain Fab (scFab) fragment, single-chain Fv protein (scFv), tandem scFv protein, disulfide-stabilized Fv protein (dsFv), or scFv-Fc protein, the antigen-binding molecule according to any one of claims 1 to 7. [

9. ] The antigen-binding molecule is scFv, optionally, the scFv further comprises a linker having the sequence of SEQ ID NO: 273, and the heavy chain variable domain is connected to the light chain variable domain via the linker, (a) the heavy chain variable domain comprises the sequence of SEQ ID NO: 66, and the light chain variable domain comprises the sequence of SEQ ID NO: 74; or (b) the heavy chain variable domain comprises the sequence of SEQ ID NO: 2, and the light chain variable domain comprises the sequence of SEQ ID NO: 10; or (c) the heavy chain variable domain sequence comprises the sequence of SEQ ID NO: 18, and the light chain variable domain sequence comprises the sequence of SEQ ID NO: 26; or (d) the heavy chain variable domain comprises the sequence of SEQ ID NO: 34, and the light chain variable domain comprises the sequence of SEQ ID NO: 42; or (e) the heavy chain variable domain comprises the sequence of SEQ ID NO: 50, and the light chain variable domain comprises the sequence of SEQ ID NO: 58; or (f) the heavy chain variable domain sequence comprises the sequence of SEQ ID NO: 82, and the light chain variable domain sequence comprises the sequence of SEQ ID NO: 90; or (g) the heavy chain variable domain comprises the sequence of SEQ ID NO: 98, and the light chain variable domain comprises the sequence of SEQ ID NO: 106; or (h) the heavy chain variable domain comprises the sequence of SEQ ID NO: 114, and the light chain variable domain comprises the sequence of SEQ ID NO: 122; or (i) the heavy chain variable domain sequence comprises the sequence of SEQ ID NO: 130, and the light chain variable domain sequence comprises the sequence of SEQ ID NO: 138; or (j) the heavy chain variable domain comprises the sequence of SEQ ID NO: 146, and the light chain variable domain comprises the sequence of SEQ ID NO: 154; or (k) the heavy chain variable domain comprises the sequence of SEQ ID NO: 162, and the light chain variable domain comprises the sequence of SEQ ID NO: 170; or (l) the heavy chain variable domain sequence comprises the sequence of SEQ ID NO: 178, and the light chain variable domain sequence comprises the sequence of SEQ ID NO: 186; or (m) the heavy chain variable domain comprises the sequence of SEQ ID NO: 194, and the light chain variable domain comprises the sequence of SEQ ID NO: 202; or (n) the heavy chain variable domain comprises the sequence of SEQ ID NO: 210, and the light chain variable domain comprises the sequence of SEQ ID NO: 218; or (o) the heavy chain variable domain sequence comprises the sequence of SEQ ID NO: 226, and the light chain variable domain sequence comprises the sequence of SEQ ID NO: 234; or (p) the heavy chain variable domain comprises the sequence of SEQ ID NO: 242, and the light chain variable domain comprises the sequence of SEQ ID NO: 250; or (q) the heavy chain variable domain comprises the sequence of SEQ ID NO: 258, and the light chain variable domain comprises the sequence of SEQ ID NO: 266, the antigen-binding molecule according to claim 8(iii).

10. The antigen-binding molecule according to any one of claims 1 to 9, which is a multispecific molecule, optionally a bispecific molecule or a trispecific molecule.

11. An antigen-binding molecule according to claim 10, comprising a first binding domain that specifically binds to B7H3 and a further binding domain that specifically binds to a second antigen, optionally wherein said further binding domain specifically binds to CD3 on the surface of T cells.

12. The antigen-binding molecule according to claim 11, comprising two scFvs.

13. The antigen-binding molecule according to claim 11 or 12, which is a bispecific T cell engager (BiTE).

14. A chimeric antigen receptor (CAR) or chimeric costimulatory receptor (CCR) comprising the antigen-binding molecule according to any one of claims 1 to 13 that specifically binds to B7H3.

15. (i) said CAR or CCR further comprises a hinge region, a transmembrane domain, and an intracellular signaling domain; and / or (ii) said hinge region is derived from CD8; and / or (iii) said transmembrane domain is derived from CD8 or CD28, the CAR or CCR according to claim 14.

16. (a) The intracellular signaling domain of said CAR comprises a costimulatory domain, optionally wherein said costimulatory domain is derived from CD28 or 4-1BB, and further optionally wherein said intracellular signaling domain comprises CD3-zeta; or (b) The intracellular signaling domain of said CCR comprises a costimulatory domain, optionally wherein said costimulatory domain is derived from CD28 or 4-1BB, the CAR or CCR according to claim 15.

17. A cell comprising the CAR or CCR according to claim 15 or 16, which is a T cell, optionally wherein said T cell is an alpha-beta T cell or a gamma-delta T cell.

18. (i) The antigen-binding molecule according to any one of claims 1 to 13 or the CAR or CCR according to any one of claims 14 to 16, or (ii) A nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable domain or the light chain variable domain according to any one of claims 1 to 13.

19. An expression vector comprising the nucleic acid molecule according to claim 18.

20. A host cell comprising the nucleic acid molecule according to claim 18 or the vector according to claim 19.

21. An antibody-drug conjugate (ADC) comprising an antigen-binding molecule according to any one of claims 1 to 13 linked to a drug, optionally wherein the drug is an anti-cancer agent, a cytotoxic agent, a cell division inhibitor, and optionally wherein the drug is selected from pyrrolobenzodiazepine (PBD) and monomethyl auristatin E (MMAE).

22. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 13, a CAR or CCR according to any one of claims 14 to 16, a cell according to claim 17, or an ADC according to claim 20 or 21, and optionally a pharmaceutically acceptable carrier.

23. An antigen-binding molecule according to any one of claims 1 to 13, a CAR or CCR according to any one of claims 14 to 16, a cell according to claim 17, an ADC according to claim 20 or 21, or a pharmaceutical composition according to claim 22 for use in a method of treating cancer.

24. The antigen-binding molecule for use as described in claim 23, wherein the cancer is selected from the group consisting of solid tumors, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma, rhabdomyosarcoma, hematological malignancies, acute myeloid leukemia, desmoplastic small round cell tumor (DSRCT), melanoma, breast cancer, prostate cancer, colon cancer, lung cancer, kidney cancer, or pancreatic cancer, or squamous cell carcinoma (SCC) of the oral cavity.

25. A method for detecting cancer in a subject, comprising contacting a biological sample from the subject with an antigen-binding molecule according to any one of claims 1 to 13 and detecting the antigen-binding molecule bound to the sample, wherein binding of the antigen-binding molecule to the sample indicates that the subject has cancer, and optionally wherein the cancer is selected from the group consisting of solid tumors, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma, rhabdomyosarcoma, hematological malignancies, acute myeloid leukemia, desmoplastic small round cell tumor (DSRCT), melanoma, breast cancer, prostate cancer, colon cancer, lung cancer, kidney cancer, or pancreatic cancer, or squamous cell carcinoma (SCC) of the oral cavity, and further optionally wherein the antigen-binding molecule specifically binds to human B7H3 and binding of the antigen-binding molecule indicates that the subject has cancer.

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