B7H3 binding agent

Abstract

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

Description

Technical Field

[0001] The present invention relates to a new antigen binding molecule that specifically binds to B7H3. The present invention also relates to a chimeric antigen receptor (CAR) and an antibody-drug conjugate (ADC) comprising an antigen binding molecule. Also provided are the uses of antigen binding molecules, CAR and ADC, and pharmaceutical compositions comprising antigen binding molecules, CAR and ADC. Background Art

[0002] Immunotherapy in the form of CAR T cell technology has produced complete clinical responses and long-term cures in many patients with otherwise refractory B-cell malignancies. Despite this progress, similar success has not been replicated in solid tumors for a variety of reasons, including the relative lack of suitable antigenic targets and challenges with penetrance and persistence in the solid tumor setting. Pediatric solid tumors present additional challenges due to the rarity of neoantigens and their immunologically "cold", hostile microenvironment.

[0003] B7-H3 (CD276) has emerged as a potential target for cancer immunotherapy in solid and liquid malignancies arising in adults and children. B7-H3 is a member of the immunoglobulin superfamily and the B7 family, closely related to PD-L1. It is present in the majority of pediatric solid cancers, with a tendency for increased expression in high-grade tumors, but B7-H3 is relatively deficient in healthy cells.

[0004] B7-H3 exists in alternatively spliced ​​isoforms. In mice, a single isoform exists that contains two immunoglobulin domains (2xIg), C1 and V1. In contrast, human cells can express 2xIg C1 / V1, but also a larger C1 / V1 / C2 / V2 (4xIg), which is an almost exact replica of 2xIg. 4xIg is the predominant isoform in human cells, including cancer cells.

[0005] When initially identified, B7-H3 was thought to be involved in T cell activation, but over time, a growing body of evidence indicates that its primary role is as a suppressor of the innate and adaptive immune systems. The mechanism of action of B7-H3 remains unclear, and although several receptors have been implicated, no studies have conclusively identified the receptor or receptors through which B7-H3 signals. Furthermore, B7-H3 is thought to have non-immunological roles in cancer development, and its elevated expression is associated with increased invasion, metastasis, resistance to chemotherapy, and poor prognosis.

[0006] Two previous anti-B7-H3 CAR T cell products have been reported and translated into clinical trials. Both products incorporate single-chain Fv fragments (scFvs) modified from the following monoclonal antibodies: MGA271 and 376.96, respectively. Preclinical studies of anti-B7-H3 CAR-T using these two scFvs have demonstrated cytotoxicity against a range of solid tumors in vitro and in animal models.

[0007] There is therefore a need for improved treatments for cancers such as solid tumors. Summary of the Invention

[0008] The present inventors have identified new B7-H3 binding agents with favorable properties that can be used for cancer treatment, particularly cancer treatment of many solid tumors. They constructed a scFv library and then screened the binding agents in the library by ELISA. The scFv library was constructed by mice immunized with two domains of the 4xIg isoform of human B7-H3 (the most proximal end fused to the most distal end). A portion of the scFv identified in the initial screening was cloned into the scFv-Fc protein format and demonstrated specific binding to human 4xIg B7-H3 expressed on the surface of human cells. In addition, a portion of these scFvs was produced in the form of chimeric antibodies and showed specific binding to B7-H3 in ELISA, and showed binding to B7-H3 naturally expressed on neuroblastoma cell lines. The selected anti-human B7-H3 scFv was cloned into, for example, CAR-T and evaluated for anti-tumor reactivity in cytotoxicity, cytokine and proliferation assays. In addition, further studies have been conducted using the antigen-binding molecules (e.g., scFv) of the present invention in antibody-drug conjugates (ADCs) or multispecific antigen-binding molecules (e.g., bispecific antibodies, bispecific T cell engagers (BiTEs), etc.). Antigen-binding molecules (e.g., scFv) of the present invention have shown excellent antigen-specific cytotoxicity and cytokine secretion levels.

[0009] Therefore, in a first 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, wherein the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, and the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3, wherein the antigen-binding molecule comprises the following complementarity determining region (CDR) sequences:

[0010] (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

[0011] (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

[0012] (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

[0013] (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

[0014] (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

[0015] (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

[0016] (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

[0017] (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

[0018] (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

[0019] (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

[0020] (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

[0021] (1) the heavy chain variable domain sequence of SEQ ID NO: 178 and the light chain variable domain sequence of SEQ ID NO: 186; or

[0022] (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

[0023] (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

[0024] (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

[0025] (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

[0026] (q) The heavy chain variable domain sequence of SEQ ID NO: 258 and the light chain variable domain sequence of SEQ ID NO: 266.

[0027] In a preferred embodiment, the antigen binding molecule may comprise 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.

[0028] 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, wherein the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, and the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3, and wherein:

[0029] (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

[0030] (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

[0031] (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

[0032] (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

[0033] (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

[0034] (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

[0035] (g) 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; or

[0036] (h) 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; or

[0037] (i) 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; or

[0038] (j) 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; or

[0039] (k) 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; or

[0040] (1) 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; or

[0041] (m) 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; or

[0042] (n) 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; or

[0043] (o) 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; or

[0044] (p) 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; or

[0045] (q) 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.

[0046] In a third 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:

[0047] (a) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 2, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 10, or a sequence with at least 90% identity thereof; or

[0048] (b) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 18, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 26, or a sequence with at least 90% identity thereof; or

[0049] (c) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 34, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 42, or a sequence with at least 90% identity thereof; or

[0050] (d) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 50, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 58, or a sequence with at least 90% identity thereof; or

[0051] (e) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 66, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 74, or a sequence with at least 90% identity thereof; or

[0052] (f) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 82, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 90, or a sequence with at least 90% identity thereof; or

[0053] (g) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 98, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 106, or a sequence with at least 90% identity thereof; or

[0054] (h) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 114, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 122, or a sequence with at least 90% identity thereof; or

[0055] (i) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 130, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 138, or a sequence with at least 90% identity thereof; or

[0056] (j) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 146, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 154, or a sequence with at least 90% identity thereof; or

[0057] (k) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 162, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 170, or a sequence with at least 90% identity thereof; or

[0058] (1) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 178, or a sequence at least 90% identical thereto, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 186, or a sequence at least 90% identical thereto; or

[0059] (m) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 194, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 202, or a sequence with at least 90% identity thereof; or

[0060] (n) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 210, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 218, or a sequence with at least 90% identity thereof; or

[0061] (o) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 226, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 234, or a sequence with at least 90% identity thereof; or

[0062] (p) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 242, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 250, or a sequence with at least 90% identity thereof; or

[0063] (q) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 258, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 266, or a sequence with at least 90% identity thereof.

[0064] In a preferred embodiment, the binding domain may comprise 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.

[0065] In a fourth aspect, the present invention provides a chimeric antigen receptor (CAR) or a chimeric co-stimulatory receptor (CCR), which CAR or CCR comprises an antigen binding molecule as described herein that specifically binds to B7H3.

[0066] In a fifth aspect, the present invention provides a cell comprising a CAR as described herein, wherein the cell is a T cell.

[0067] In a sixth aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antigen binding molecule or CAR as described herein.

[0068] In a seventh aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable domain or a light chain variable domain as described herein.

[0069] In an eighth aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described herein.

[0070] In a ninth aspect, the present invention provides a host cell comprising a nucleic acid molecule or a vector as described herein.

[0071] In a tenth aspect, the present invention provides an antibody-drug conjugate (ADC), the ADC comprising an antigen binding molecule as described herein linked to a drug.

[0072] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising an antigen binding molecule, a CAR, a cell comprising a CAR, or an ADC as described herein, and optionally a pharmaceutically acceptable carrier.

[0073] In a twelfth aspect, the present invention provides a method for treating cancer, wherein the method comprises administering to a subject in need thereof an antigen binding molecule, CAR, cell comprising CAR, ADC or pharmaceutical composition as described herein.

[0074] In a thirteenth aspect, the present invention provides an antigen binding molecule, a CAR, a cell comprising a CAR, an ADC or a pharmaceutical composition as described herein for use in a method for treating cancer.

[0075] In a fourteenth aspect, the present invention provides a method for detecting cancer in a subject, the method comprising: contacting a biological sample from the subject with an antigen binding molecule as 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, optionally wherein the cancer is selected from a solid tumor, 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). BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Schematic diagram illustrating the strategy used to generate and pan the anti-B7-H3 library. A. Generation of the phage display library. Mice were immunized with B7-H3-mouse Fc fusion protein. Spleens were harvested from the immunized mice, and mRNA was extracted and reverse transcribed into the corresponding VH and VL cDNAs. PCR was used to add the linker and myc tag before cloning the scFv-myc into the pHEN phagemid. Figure generated using Biorender. B. The phage display library was panned against human 4Ig-B7-H3 immobilized on immunotubes or magnetic beads.

[0077] Figure 2- Anti-B7-H3 scFvs were identified that showed binding to plate-bound and cell-bound human 4xIg B7-H3. A. Bacterial clones that showed anti-B7-H3 responses in the screen were recultured and tested in triplicate. After induction of scFv-myc production, bacterial supernatants were tested in ELISAs with recombinant B7-H3 or PBS as negative controls. Commercially available anti-B7-H3 monoclonal antibodies and serum from immunized mice were used as positive controls, and secondary antibody (anti-myc) alone was used as a negative control (mean and SD, n=3). B. Structures of scFv-myc fusion proteins used in ELISAs and scFv-Fc fusion proteins used in staining experiments. C. Jurkat cells were transduced with one of three isoforms of B7-H3 shown from left to right: 1) naturally occurring human 4IgB7-H3; 2) a construct containing two membrane-proximal domains in 4Ig form, depicted as T-B7H3, which was used as an immunogen in mouse vaccination to generate a library; and 3) a naturally occurring 2xIg domain isoform for antibody production. D. Binding of five ScFv-Fc fusions identified from the library to different cell-binding isoforms of B7-H3.

[0078] Figure 3 ——Data for scFv-Fc fusions TB8, BG4, BD9, BC10, and BB5. Figure 2 D. Same method, showing the binding of scFv-Fc to different cell-binding isoforms of B7-H3. Representative 1 of 2.

[0079] Figure 4 —High level of diversity of anti-B7-H3 scFv. A. Percent similarity between 17 different anti-B7-H3 scFvs. B. By Abysis http: / / www.abysis.org / abysis / ) Analysis of the alphabetically ordered alignment of the anti-B7-H3 scFv heavy and light chains. Amino acid differences between the binding agents are shown. Dots indicate the same sequence as the binding agent above. Dashed lines indicate the absence of an amino acid.

[0080] Figure 5 Specificity of three binders, TE9, TC6, and BH6, for distinguishing mouse, nonhuman primate, and human B7H3. A. Mouse 3T3 / NA1 fibroblasts stained with TE9, TC6, and BH6 binders in whole antibody format; B. ELISA results showing binding of antibodies TE9, TC6, and BH6 to recombinant human B7 family proteins and nonhuman primate B7-H3; cynomolgus monkeys refer to macaques (Macaca fascicularis).

[0081] Figure 6——Chimeric antibodies derived from TE9 and BH6 showed specificity for human B7H3 expressed on the cell surface. TE9 and BH6 ScFv, which had been cloned into fully human IgG1 antibodies, were used in staining experiments. A. Chimeric human IgG1 whole antibodies derived from TE9 and BH6 ScFv were used to stain three neuroblastoma cell lines, namely LAN-1, Kelly, and IMR-32. The top row shows staining with a commercial directly conjugated antibody, and the bottom row represents staining with the chimeric antibody and an anti-human secondary antibody. B. Chimeric antibodies TE9 and BH6 were used to stain three B7-H3 positive Jurkat cell lines, namely 4Ig-B7-H3, 2Ig-B7-H3, and T-B7-H3 (see Figure 2 )

[0082] Figure 7 —Anti-B7-H3 CAR T cells derived from TE9 and TC6 binders exhibited similar T cell effector functions as anti-GD2 and anti-CD19 CAR T cells. A. The second-generation CAR design used in this figure combines the CD8 hinge and transmembrane (H / Tm) with the CD28-CD3z intracellular domain. B. Four-hour Cr51 cytotoxicity assay of lead CAR T cells against syngeneic B7-H3+ / - cell lines and LAN-1 cells. αGD2 CAR T cells were compared with syngeneic GD2+ / - cell lines and LAN-1. There was no significant difference in the cytotoxicity of TC6, TE9, and αGD2 CAR T cells against LAN-1 (mean and SD, n = 3). C. Cell lines used as targets have B7-H3 and GD2 expression levels. Antigen density values ​​were determined using the quantbrite antigen quantification kit (BD Bioscience). D. CAR T cells were cultured with LAN-1, Kelly, or no antigen stimulation. IL-2 and IFN-γ were measured in overnight supernatants by ELISA (mean and SD, n = 3 to 5; brackets indicate statistically significant differences using one-way ANOVA with Tukey's multiple comparison correction: ****p < 0.00001, ***p < 0.0001, **p < 0.001 and *p < 0.01). E. Antigen-specific cytokine responses at the end of the tumor re-challenge assay in experiments where irradiated target cells were added every 5 to 7 days for 4 stimulations. Data are shown on day 9 (24 hours after the second stimulation) and day 26 (24 hours after the fourth stimulation). Means and ranges are shown. Data are from two donors. Each sample was analyzed in duplicate.

[0083] Figure 8TE9-CD28z CAR T cells show superior cytokine production compared to TE9-4-1BBz CAR T cells. A. Schematic diagram of the second-generation CD28 and 4-1BB CAR constructs used in this figure. B. Transduction efficiency of TE9-CD28-CD3ζ (mean and range, n=6) or TE9-4-1BB-CD3ζ (mean and range, n=3). C. CAR T cells were cultured with cells containing antigenic targets (LAN-1 or Kelly) or without antigenic targets (unstimulated) for 18 hours. Cells were stained with CD107a, CD69, and CD25. D. 18-hour culture assay: T cells were transduced with TE9-28ζ or TE9-4-1BBζ. CAR-T cells were cultured with LAN-1 or Kelly target cells or without antigen stimulation for 18 hours. Cells were pelleted, and the supernatant was used in an ELISA to compare with standard values ​​for IL-2 or IFN-γ. (TE9-28ζ, untransduced, n=6). TE9-BBζ (mean and range, n=3). E. Repeated stimulation assay: CAR and UT T cells were cultured with LAN-1 or Kelly targets or unstimulated for 7 days, then restimulated with fresh antigen targets and incubated for another 24 hours. IFN-γ and IL-2 production were measured using ELISA (mean and range, n=3). Brackets indicate statistically significant differences using one-way ANOVA with Tukey's multiple comparison correction: ****p<0.00001, ***p<0.0001, **p<0.001 and *p<0.01.

[0084] Figure 9—Second-generation CAR T cells with CD28 H / Tm exhibit higher cytokine production and proliferation compared to CD8 H / Tm in the presence of low antigen expression. A. Schematic diagram of the second-generation TE9 CAR T cells used in this figure, which have hinge and transmembrane regions (H / Tm) for both CD8 and CD28. B. Cytokine production by T cells transduced or untransduced with second-generation CARs containing CD8 H / Tm or CD28 H / Tm. T cells were incubated overnight with varying concentrations of plate-bound B7-H3, and supernatants were analyzed for cytokine production (mean and range, n=3): St = stem and is an alternative symbol for the H / Tm used. C. Transduction efficiency of TE9-CD8 H / Tm and TE9 CD28 H / Tm (mean and range, n=6). D. TE9 CAR T cells with CD28 H / Tm or CD8 H / Tm were incubated with LAN-1, Kelly, K562, or no antigen stimulation for 18 hours. Using ELISA, the supernatant was used for quantitative cytokine production (mean and range, n=6). E. After 7 days of co-culture, T cells were restimulated with fresh target cells or antigen-free targets. Using ELISA, the supernatant from the 7-day co-culture was used for quantitative cytokine production (mean and range, n=4). F. TE9 CAR T cells with CD28 H / Tm or CD8 H / Tm were stained with CSFE and incubated with LAN-1, Kelly, K562 or no antigen stimulation for 7 days. The histogram shows the diluted CSFE (representing 1 / 3) due to proliferation for different targets. ΔMFI (change in median fluorescence intensity) was calculated as the difference between the MFI of the test condition and the unstimulated non-transduced control (mean and range, n=3). st = stem, i.e., hinge / transmembrane; brackets indicate statistically significant differences using one-way ANOVA with Tukey’s multiple comparison correction: ****p<0.00001, ***p<0.0001, **p<0.001, and *p<0.01.

[0085] Figure 10——TE9-28ζ showed superior expansion and cytokine production in long-term assays compared to GD2-28ζ. CAR T cells were transduced with TE9-28ζ (TE9), TE9-28-ILR2ζ (TE9-ILR2), or GD2-28ζ (GD2). A. Schematic diagram of the CAR T cells used in this study. B. CAR T cells or untransduced cells were cultured with LAN-1, Kelly, or no antigen stimulation. Cells were given fresh antigen stimulation every week, cultured for another 24 hours, and then analyzed. After each antigen stimulation, IFN-γ and IL-2 production were determined using ELISA (mean and range, n=4). C. Proliferation was measured by fold change in CD3+ cells measured by flow cytometry. Significance was shown between the number of cells on day 28 (mean and range, n=4). Brackets indicate statistically significant differences using one-way ANOVA with Tukey's multiple comparison correction: ****p<0.00001, ***p<0.0001, **p<0.001 and *p<0.01.

[0086] Figure 11 ——In vivo testing of TE9-28ζ, TE9-BBζ, and GD2-28ζ CAR-T constructs. Mice treated with TE9-28ζ showed increased survival and reduced tumor growth compared to other groups. A. Experimental plan. B. Survival curves. C. Main panel: Tumor diameter, where the average tumor size was evaluated on day 5 (mean, n = 6; , **p < 0.001, and *p < 0.01). D. Antigen expression after treatment, as shown by the MFI of the fluorophore used to stain the antigen in the tumor samples (mean and range, mice in the untransduced group and the αGD2-28ζ group, n = 6. Mice in the TE9-28ζ group, n = 5).

[0087] Figure 12——TE9-28ζCAR T cells show higher penetrance and survival within the tumor environment compared to GD2 CAR-T or TE9-BBζ. A. Blood, spleen, and tumor samples were collected and analyzed for the persistence of CD34+CAR T cells using flow cytometry. Although all mice were analyzed, samples with <100 cells positive for human CD45 were excluded from the analysis. Live cells were gated into 2 populations: cells positive for human CD45 and cells positive for mouse CD45. These figures show the mean and individual values ​​of the percentage of CD45-positive cells that are positive for human CD45, for all samples, n=6 unless marked with *, where n=5. B. Cells positive for human CD45 were gated based on CD3 expression. These figures show the mean and individual values ​​of the percentage of cells that are double positive for human CD45 and human CD3 (also CD34-positive), for all samples, n=6 unless marked with *, where n=5. C. Number of samples with >100 human T cells available for analysis per CAR. For all samples, n=6, unless marked with *, where n=5.

[0088] Figure 13 TE9-28ζCAR-T eradicates orthotopic medulloblastoma. Medulloblastoma MED8A cells (B7H3 positive) were injected into the hemispheres of NSG mice. 48 hours later, when small tumors were formed, 5×10 6 CAR-T cells or untransduced controls were injected into the lateral ventricle. A. Schematic diagram of the experiment. B. Bioluminescence values ​​(flux) of the mouse groups. C. Representative bioluminescence images of treated and untreated mice. At the end of follow-up, no tumors were identified in the CAR-T-treated mice.

[0089] Figure 14The reactivity of the TE9 CAR and the anti-CD33 CAR against acute myeloid leukemia cell lines was compared. The anti-CD33 CAR was constructed based on a ScFv derived from the heavy and light chain variable domains of gemtuzumab ozogamicin. The TE9 and CD33 CARs were constructed using the CD28 and CD3ζ intracellular domains, and the hinge / transmembrane domains were derived from CD8-α (denoted as CD8) or the CD28 transmembrane domain fused to the CH2 and CH3 domains of human IgG4 (denoted as CH2CH3). MV411, Nomo-1, and THP-1 are human acute myeloid leukemia cell lines. Jurkat cells and the SupT1 human leukemia cell line were engineered to express 4Ig human B7H3 or human CD33, respectively. The corresponding CAR-T cells were labeled with CellTrace Violet dye and co-cultured with the corresponding irradiated target cell line at a 1:1 ratio for 7 days. After gating on CAR-T cells, the fold proliferation of CAR-T cells was shown by dilution of CellTrace Violet dye determined by flow cytometry.

[0090] Figure 15 --TE9-28ζCAR-T cells eradicate NOMO-1 acute myeloid leukemia but do not affect normal hematopoiesis. Schematic diagram of the experiment is shown at the top. TE9-28ζCAR-T cells (CD8 to NOMO-1 acute myeloid leukemia cells or human hematopoietic stem cells in the form of umbilical cord blood) were added at an effector to target ratio of 5:1. Target and effector cells were added to methocult medium that promotes hematopoietic colony formation. After 14 days, erythroid and myeloid cell colonies were counted from umbilical cord blood progenitor cells, and leukemic cell colonies were counted from NOMO-1 progenitor cells. Three independent donors were the source of untransduced controls and TE9 CAR-T cells.

[0091] Figure 16Comparison of the TE9 second-generation CAR-T cell with equivalent CAR constructs constructed with the 376.96 and MGA271 antibodies in a restimulation assay. These three ScFv binders were cloned into the same second-generation CAR backbone, including the CD8-α hinge transmembrane and either the CD28 / CD3ζ or 41BB / CD3ζ intracellular domains. CARs and untransduced controls were incubated with irradiated B7-H3-positive neuroblastoma targets (LAN-1 or Kelly cell lines) at a 1:1 E:T ratio for 7 days, followed by restimulation with fresh antigen target and an additional 24 hours of incubation. IFN-γ and IL-2 production were measured in supernatants 24 hours after the second stimulation using ELISA. n = 3 independent donors; brackets indicate statistically significant differences using one-way ANOVA with Tukey's multiple comparison correction: ****p<0.00001, ***p<0.0001, **p<0.001, and *p<0.01.

[0092] Figure 17 and Figure 18 ——Comparison of the TE9 second-generation CAR-T and the equivalent CAR construct constructed by the 376.96-MGA271 antibody in a repeated stimulation assay involving 4 target cell re-attacks. The three ScFv binders were cloned into the same second-generation CAR backbone, including the CD8-α hinge transmembrane and CD28 / CD3ζ or 41BB / CD3ζ intracellular domains. CAR and non-transduced controls were cultured with irradiated B7-H3-positive neuroblastoma targets (LAN-1 or Kelly cell lines) at a 1:1 ratio for 7 days and then restimulated with fresh irradiated antigen targets every 7 days for a total of 4 restimulations. After each restimulation, the supernatant of the co-culture was sampled 24 hours later and cytokine secretion was evaluated (interferon gamma is shown in Figure 17 IL-2 is shown in Figure 18 (middle). Mean ± SD of 4 independent donors are shown. Brackets indicate statistically significant differences using one-way ANOVA with Tukey's multiple comparison correction: ****p < 0.00001, ***p < 0.0001, **p < 0.001, and *p < 0.01.

[0093] Figure 19——γδT cells can be transduced with TE9-28ζ and show enhanced effector function in an antigen-dependent manner. A. Schematic diagram of the experiment. After depletion of αβT cells and stimulation with anti-CD3 OKT3 monoclonal antibody, human γδT cells (mainly V-δ1 type) were expanded from peripheral blood. On day 3 after T cell stimulation, the expanded cells were transduced with the anti-B7-H3 CAR-T construct shown in the SFG skeleton; the cells were expanded in the presence of IL-15 and functionally evaluated on day 20 of expansion. B. The transduction efficiency of the expanded γδT cells was determined by flow cytometry using direct staining of CAR, and the average transduction efficiency of 9 independent donors was shown. C. The expression of human B7-H3 on target cells of the co-culture assay was evaluated by flow cytometry. D. The reactivity to the target cells was demonstrated by flow cytometry evaluation of CD107a and intracellular interferon gamma (which moved to the right after culture). After co-culture with the target overnight, the accumulation of intracellular IFN-γ and cell surface CD107a of Vδ1 cells was measured in culture medium supplemented with monensin for 4 hours. Marker accumulation was measured in CAR-transduced and non-transduced Vδ1 cells. Representative histograms of marker expression from representative donors are shown. The red line represents the median fluorescence intensity (MFI) of the individual CAR-negative effectors, while the blue line represents the MFI of the individual CAR-positive effectors.

[0094] Figure 20 ——γδT cells transduced with TE9-28ζCAR showed enhanced cytotoxicity and cytokine production in an antigen-dependent manner. γδT cells were transduced according to the figure ( Figure 19 ) were amplified and transduced and then evaluated in co-culture experiments. A. IFN-γ and CD107a expression histogram data from 3 separate donors were converted to earth mover's distance (EMD) values, which compare the expression of markers between CAR-transduced cells and non-transduced cells. A score of "0.0" indicates no difference and is represented by a dotted line (n=3; mean and distribution are shown in the figure). B. The same data were analyzed using MFI measurement to compare CAR-positive (CD34+) Vδ1 cells and non-transduced (CD34-) Vδ1 cells stratified in the same culture (n=3; mean and distribution are shown in the figure; statistical significance was determined using one-way analysis of variance).

[0095] Figure 21——γδT cells transduced with TE9-28ζCAR showed enhanced proliferation in an antigen-dependent manner after re-challenge with tumor cells. A. Schematic diagram of the experimental design: To test the persistence and proliferation of amplified CAR-Vδ1, the amplified cells were harvested and challenged twice with irradiated B7H3 antigen-positive and -negative Jurkat targets at an E:T ratio of 1:1 (B). C. After 6 days of co-culture, the dilution of CellTrace Violet dye for CAR-Vδ1 was measured. Matched data for one representative donor are shown. The right vertical line represents the edge of the undiluted dye on day 0 of the assay, the middle vertical line represents the dye MFI of CAR-Vδ1 only on day 6, indicating background proliferation, and the left vertical line represents the dye MFI of CAR-Vδ1 co-cultured with antigen-positive targets. D. To account for ongoing background proliferation, Vδ1 cells were counted before and after co-culture using flow cytometry-based counting beads, and the fold change in Vδ1 was normalized to that of the effector alone (n=3; mean ± SEM; statistical significance was determined using two-way ANOVA with Sidak's multiple comparisons).

[0096] Figure 22 ——TE9 binders show relatively high affinity in the CAR-T format. A) Schematic diagram of the construct used to determine affinity. All three binders were cloned into the same SFGγ retroviral backbone and transduced into human primary T cells. B) The relative B7H3 expression of the target cell lines used in these affinity experiments was determined using the Quantibrite Phycoerythrin Fluorescence Quantification Kit (BD Biosciences). C) Representative graphs from the Lumicks Cell Affinity Analyzer. The target cells were attached to the bottom of the microwells and CAR-T cells were added. Increasing acoustic force was applied to remove the cells. A greater force requirement indicates a greater affinity. D) Data summary of CAR-T produced by 4 independent blood donors. The percentage of cells attached at a maximum acoustic force of 1000pN is shown. Repeated measurements from 4 donors were plotted. Significant differences were depicted after passing a one-way analysis of variance.

[0097] Figure 23——TE9 anti-B7H3 binder αβ-CAR-T exhibits superior effector functionality compared to competing clinical-stage anti-B7H3 binders MGA.271 and 376.96. All data shown in this figure are from 3 independent donors, and statistical comparisons were performed using two-way analysis of variance. (a) T cells were transduced with a γ-retroviral construct encoding a second-generation 28ζCAR and RQR8 marker gene separated by a 2A cleavage sequence. (b) CAR functionality was assessed in a serial re-challenge assay (every 24 to 48 hours), comparing responses to B7H3-negative SupT1-WT and positive SupT1-B7H3. Unsorted bulk transductions of SupT1-B7H3 were used, encompassing a range of expression from zero to high. (c) Similar levels of transduction efficiency of >60% were achieved for all constructs tested (data shown as 3 independent donor transductions in series). (d) TE9-CAR-T and 376.96-CAR-T cells exhibited superior cytotoxicity against antigen-positive targets compared to MGA-CAR-T. (e, f) Tumor B7H3 expression was measured after five rounds of challenge with the SupT1-B7H3 target. TE9-CAR-T and 376.96-CAR-T cells killed all antigen-positive tumor targets, whereas MGA.271-CAR-T cells killed only tumors with medium to high B7H3 expression, enriching for antigen-intermediate tumor targets. (g) TE9-CAR-T and 376.96-CAR-T cells proliferated significantly more in response to repeated challenge with tumor targets, (h) and produced higher levels of IL-2 and IFN-γ compared to MGA.271-CAR-T cells.

[0098] Figure 24TE9-based CAR-T cells exhibit reduced basal activity in the absence of antigenic signaling. a) Three anti-B7H3 binders were evaluated in the same second-generation format and expressed from a SFGγ retroviral vector. b) Similar levels of transduction efficiency, >60%, were achieved for all constructs tested (data shown for three independent donor transductions in series). c) Three corresponding CAR-T products were prepared from three independent blood donors after stimulation with anti-CD3 and anti-CD28 antibodies in the presence of 100 units / ml IL-2. Equivalent starting numbers were plated. During preparation, 376.96-CAR-T cells proliferated significantly more than TE9-CAR-T cells, consistent with stronger autonomous signaling by 376.96-CAR-T cells. d) Following preparation, cells were seeded with target cells (see previous figure) or with control wells containing only culture medium at the indicated time points. In contrast to TE9-CAR-T, 376.96-CAR-T and MGA.271-CAR-T cells proliferated in the absence of target challenge, suggesting potential autonomous CAR-T signaling. e) Spontaneous cytokine production was measured in the supernatant of CAR-T products prepared from three identical donors in the absence of added target cells.

[0099] Figure 25—Anti-B7H3 CD28-CCR Enhances Proliferation of MART1-TCR-αβ-T Cells Against Peptide-Loaded Targets, but Does Not Enhance Cytotoxicity. (a) Schematic representation of MART1-TCR, anti-B7H3 TE9 binder CD28-CCR, and second-generation control TE9-28-ξCAR constructs in SFGγ-retroviral format. (b) Representative dot plots of transduced T cells from one donor showing RQR8 CAR / CCR marker gene and MART1-TCRvβ12 chain expression in untransduced, singly transduced, or doubly transduced αβ-T cells, gating on live, monomorphic CD3+ cells expanded on day 7. (c) Schematic representation of the combined cis signals 1+2 of the MART1 TCR-derived signal 1 and the trans-CCR-derived CD28 signal 2 with the conventional second-generation TE9-28-ξCAR. (d) Representative dot plots of the U87 glioblastoma cell line expressing HLA-A2 and B7H3, making it sensitive to targeting with both a chimeric receptor targeting MART1-TCR and a chimeric receptor targeting TE9. (E) 51Cr release over 4 hours by U87 target cells pre-pulsed with 5 μg / ml MART1 peptide or untreated U87 target cells within a specific E:T ratio range (shown are means ± SD between T cells from two independent donors; two-way ANOVA). (f) Representative histograms showing the dilution of CellTrace Violet proliferation dye in T cells after 7 days of co-culture in the presence of 100 IU / mL IL-2 following a single challenge with live U87 target cells. (g) Proliferation data were compared between four donor T cells and compared using the proliferation index, which is calculated by dividing the histogram into sixths and recording the percentage of T cells in each sixth, then multiplying this percentage by increasing increments moving to the left to give greater weight to proliferating T cells. Data were normalized to donor-matched effector-only conditions (n=4; mean ± SD; two-way ANOVA). *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001

[0100] Figure 26Coexpression of an anti-B7H3 CD28-CCR with the G115-TCR broadens cytokine responsiveness of αβ-T cells to targets that are insufficiently stimulated by the Vγ9Vδ2-TCR alone. (a) Schematic representation of the G115Vγ9Vδ2-TCR-derived signal 1 and the trans-TE9 CCR-derived CD28 signal 2 is provided. (b) Schematic representation of retroviral constructs encoding the G115-TCR alone or the G115-TCR and TE9-CCR with CD28 or 4-1BB signaling intracellular domains is shown. (c) Representative dot plots of transduced T cells from one donor showing RQR8 CCR marker gene and G115 Vδ2 chain expression in untransduced and transduced αβ-T cells, gating on viable monomorphic CD3+ cells expanded on day 7. (d) G115-TCR sensitized αβ-T cells to the target DAUDI cell line of Vγ9Vδ2-TCR with comparable cytotoxicity to unmodified naive γδ-T cells, as shown by DAUDI 51Cr release during a 4-hour co-culture (mean ± SD across T cells from four independent donors shown; two-way ANOVA). (e) PhosFlow phosphoprotein assessment of TCR-like signaling in response to stimulation with anti-CD3 mAb (clone OKT-3) or anti-Vδ2 mAb (clone B6) confirmed G115-TCR signaling in transduced αβ-T cells (mean ± SD across T cells from three independent donors shown; two-way ANOVA). (f) Schematic diagram illustrating the assay setup for factor secretion by differentially transduced T cells. (g) Target cells were either unmodified or pretreated with clone 20.1 mAb to convert cell surface CD2771 from an inactive to an active conformation, thereby enhancing signal delivery via the G115 Vγ9Vδ2-TCR. (h) The key to different target types attacked by T cells is shown, including highly sensitive DAUDI lymphoma cells, differentially sensitive AML target cells MV4-11, NOMO-1, and THP-1, and less sensitive Jurkat leukemia cells, which were either naturally B7H3-negative wild-type cells (WT cells) or transduced to express B7H3 (B7H3 cells). (ik) IL-2 production by differentially transduced T cells after overnight culture with DAUDI, AML, and Jurkat targets (E:T ratio 1:1; mean ± SD among T cells from three independent donors; two-way ANOVA). (ln) IFN-γ production by differentially transduced T cells after overnight culture with DAUDI, AML, and Jurkat targets (E:T ratio 1:1; mean ± SD among T cells from three independent donors; two-way ANOVA).*P<0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.

[0101] Figure 27 — Dependence of G115-αβ-T cell responsiveness on CD28-CCR expression increases after rechallenge with tumor targets. (a) Schematic diagram illustrating the assay setup for differentially transduced T cell functional testing. All data in this figure are from a second challenge with a tumor target, and the relevant readouts are shown. (b) The key to the different target types attacked by T cells is shown, including highly sensitive DAUDI lymphoma cells, differentially sensitive AML target cells MV4-11, NOMO-1, and THP-1, and less sensitive Jurkat leukemia cells, which are either naturally B7H3-negative wild-type cells (WT cells) or transduced to express B7H3 (B7H3 cells). (c) Unmodified, TCR-only, and CCR-TCR-modified αβ-T cells were cocultured with a range of targets, and 51Cr release was measured after 4 hours of coculture (shown are means ± SD among T cells from four independent donors; two-way ANOVA). (d) IL-2 production by differentially transduced T cells after overnight culture with DAUDI, AML, and Jurkat targets (E:T ratio of 1:1; mean ± SD between T cells from 3 independent donors; two-way ANOVA). (e) IFN-γ production by differentially transduced T cells after overnight culture with DAUDI, AML, and Jurkat targets (E:T ratio of 1:1; mean ± SD between T cells from 3 independent donors; two-way ANOVA). (f) T cells that proliferated in response to irradiated targets without exogenous IL-2 supplementation were counted over a 7-day period. This was done by inoculating 50,000 T cells with target cells at an E:T ratio of 1:1 and then calculating the number of T cells at harvest using absolute counting beads. The resulting T cell numbers are shown (mean ± SD between T cells from 3 independent donors; two-way ANOVA). *P<0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001

[0102] Figure 28 Binding specificity evaluation of TE9 and TC6 binders. The binding functionality of three lead anti-B7-H3 antibodies was evaluated using a binding ELISA with immobilized recombinant B7-H3 isoforms. (A) Structures of recombinant B7H3 proteins used to assess antibody binding. (B) Binding properties of TE9 and BH6 antibodies to the corresponding recombinant human proteins, as well as to mouse and cynomolgus monkey proteins. (C) Binding of TE9 and TC6 antibodies was compared to binding of mouse and cynomolgus monkey proteins. DETAILED DESCRIPTION

[0103] It should be understood that the different applications of the disclosed products and methods can be adjusted according to the specific needs of the art. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments of the present invention and is not intended to be limiting. All publications, patents, and patent applications cited herein, whether supra or infra, are incorporated herein by reference in their entirety.

[0104] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen-binding molecule" includes "plural antigen-binding molecules," etc.

[0105] B7H3

[0106] B7H3 can be used interchangeably with CD276. B7-H3 is a member of the immunoglobulin (Ig) superfamily. The gene is located on chromosome 15 in humans and chromosome 9 in mice and is highly conserved across species. B7H3 most commonly exists as a transmembrane protein with a residual cytoplasmic domain and no known signaling motifs. Alternative splicing results in the production of many isoforms of B7-H3. In humans and most other mammals, isoform 1 (also known as 4Ig-B7-H3) is the most common. Its extracellular domain contains four Ig subunits arranged in the pattern of Ig-V-1, Ig-C-1, Ig-V-2, and Ig-C-2. The homology between Ig-V-1, Ig-C-1 and Ig-V-2, Ig-C-2 is greater than 96%, and the duplication pattern is believed to be caused by exon duplication. The second most common 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 is composed of Ig-V-like type 2 and Ig-C-like type 2 subunits.

[0107] Antigen-binding molecules

[0108] As used herein, the term "antigen binding molecule" includes whole antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portion") or single chains. The antigen-binding molecule comprises a binding domain. The binding domain interacts with the antigen. For example, the antigen-binding molecule may comprise a binding domain that binds to B7H3.

[0109] An antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. The disclosed antibodies may be class-switched. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).

[0110] The CDRs are primarily responsible for antigen binding. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are often also identified by the chain in which a particular CDR is located. The light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3. The CDR sequences are typically arranged in an N-terminal to C-terminal direction on the light chain variable domain: LCDR1, LCDR2, and LCDR3, and in an N-terminal to C-terminal direction on the heavy chain variable domain: HCDR1, HCDR2, and HCDR3.

[0111] The framework region sequences of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework regions of the light and heavy chains, is used to position and align 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 (Clq) of the classical complement system.

[0112] Antibodies can be "monoclonal antibodies". Monoclonal antibodies are immunoglobulin molecules that are identical to each other and have a single binding specificity and affinity for a particular epitope. They are produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as those described in "Monoclonal Antibodies: A Manual of Techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982).

[0113] Antibodies can be "chimeric" antibodies, i.e., antibodies that comprise sequences from two different antibodies, typically from different species. For example, a chimeric antibody can comprise heavy and light chain variable regions derived from a first species and heavy and light chain constant regions derived from a second species. In other aspects, the variable and constant regions of the light chain can be derived from a first species, while the variable region of the heavy chain can be derived from a first species, and the constant region of the heavy chain can be derived from a second species. In other aspects, the variable and constant regions of the light chain can be derived from a first species, while the variable and constant regions of the heavy chain can be derived from a second species.

[0114] The term "fragment" of an antibody generally refers to an "antigen binding fragment" of the antibody, i.e., one or more fragments of an antibody that retains the ability to specifically bind to an antigen. The antigen binding molecules or antigen binding fragments of the present invention retain 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 antibody, trivalent antibody or tetravalent antibody, double scFv, double-chain antibody, three-chain antibody, four-chain antibody, or an epitope binding fragment of any of the above substances (see, e.g., 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 produced by modifying whole antibodies or synthesizing them de novo using recombinant DNA methods. In one embodiment, the antigen-binding molecules of the present invention are preferably scFv or scFv-Fc proteins. ScFv protein is a fusion protein in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are bound by a linker. In dsFv, these chains have been mutated to introduce disulfide bonds to stabilize the association of these chains. The term also includes genetically engineered forms such as chimeric antibodies and heterologous conjugate antibodies, such as bispecific antibodies. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Company, Rockford, IL); Kuby, Immunology, 3rd ed., WH Freeman & Company, New York, 1997.

[0115] 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 measuring the dissociation constant (Kd) of the antibody and its target. Similarly, the specificity of an antibody binding to its target can be defined by comparing the dissociation constant (Kd) of the antibody to its target with the dissociation constant of the antibody and another non-target molecule. Typically, the Kd of an antibody relative to a target is 1 / 2, preferably 1 / 5, more preferably 1 / 10 of the Kd relative to other non-target molecules. More preferably, the Kd will be 1 / 50, even more preferably 1 / 100, and even more preferably 1 / 200. The value of the dissociation constant can be directly determined by well-known methods, and even by calculating the dissociation constant of a complex mixture by methods such as, for example, those described by Caceci et al. (Byte 9:340-362, 1984). Methods for evaluating the binding affinity of the antibodies of the present invention to B7H3 preferably include ELISA or Biacore (i.e., surface plasmon resonance).

[0116] The antigen-binding molecules of the present invention bind (e.g., specifically bind) B7H3 (preferably human B7H3), i.e., preferably, they bind to B7H3 but they do not bind to other molecules or bind to other molecules with lower affinity. "Specific binding" means that the antibody binds to B7H3 with a greater affinity than it binds to another target. Specific binding can be determined by methods known in the art. The antigen-binding molecules of the present invention are preferably capable of binding to B7H3 with an affinity that is at least two times, 10 times, 50 times, 100 times greater than its affinity for binding to another non-target molecule. Preferably, the antigen-binding molecules of the present invention may have an affinity of 1×10 -9 M or lower binding affinity (i.e., K D ). In some aspects, the antigen binding molecule is at about 1×10 -9 M or lower, about 1×10 -10 M or lower, about 1×10 -11 M or lower, or about 1×10 -12 M or lower K DSpecifically bind to B7H3. The antigen binding molecules of the present invention may have a certain binding affinity to B7H3 (e.g., mouse or rat B7H3) from other mammals (e.g., primates or mice). The binding affinity of the antigen binding molecules of the present invention to B7H3 from other species gradually weakens as the binding epitope becomes less conservative as the phylogenetic distance becomes less conserved. The antigen binding molecules of the present invention may bind to (e.g., specifically bind to) any isotype of B7H3. In one embodiment, the antigen binding molecules specifically bind to human B7H3. In one embodiment, the antigen binding molecules specifically bind to human B7H3 isoform 4IgB7-H3. In one embodiment, the antigen binding molecules specifically bind to human B7H3 isoform 2IgB7-H3. In one embodiment, the antigen binding molecules have specificity for both human B7H3 isoform 4IgB7-H3 and human B7H3 isoform 2IgB7-H3. In one embodiment, the antigen binding molecules specifically bind to T-B7-H3 isoforms. In one embodiment, the antigen binding molecules are specific for T-B7-H3, human 4IgB7-H3, and human 2IgB7-H3. In one embodiment, the antigen binding molecules specifically bind to human B7H3 isoform 4IgB7-H3, and do not bind to other targets or have a lower binding affinity to other targets. In one embodiment, the antigen binding molecules specifically bind to human B7H3 isoform 2IgB7-H3, and do not bind to other targets or have a lower binding affinity to other targets. In one embodiment, the antigen binding molecules are specific for human B7H3 isoform 4IgB7-H3 and human B7H3 isoform 2IgB7-H3, and do not bind to other targets or have a lower binding affinity to other targets. In one embodiment, the antigen binding molecules specifically bind to isoform T-B7-H3, and do not bind to other targets or have a lower binding affinity to other targets. In one embodiment, the antigen binding molecule is specific for T-B7-H3, human 4IgB7-H3, and human 2IgB7-H3, and does not bind to other targets or has lower binding affinity for other targets.

[0117] The antigen-binding molecules of the present invention generally bind to the same epitope as an antigen-binding molecule having the following heavy or light chain variable region sequences: (i) SEQ ID NOs: 2 and 10, respectively, (ii) SEQ ID NOs: 18 and 26, respectively; (iii) SEQ ID NOs: 34 and 42, respectively, (iv) SEQ ID NOs: 34 and 42, respectively, (v) SEQ ID NOs: 66 and 74, respectively, (vi) SEQ ID NOs: 82 and 90, respectively, (vii) SEQ ID NOs: 98 and 106, respectively, (viii) SEQ ID NOs: 114 and 122, respectively, (ix) SEQ ID NOs: 130 and 138, respectively, (x) SEQ ID NOs: 146 and 154, respectively, (xi) SEQ ID NOs: 162 and 170, respectively, (xii) SEQ ID NOs: 178 and 186, respectively, (xiii) SEQ ID NOs: 194 and 202, respectively, and (xiv) SEQ ID NOs: 196 and 207, respectively. NO: 210 and 218, (xv) are SEQ ID NO: 226 and 234, respectively, (xvi) are SEQ ID NO: 242 and 250, respectively, or (xvii) are SEQ ID NO: 258 and 266, respectively. For example, the antigen-binding molecules of the present invention can bind to the same epitope as antigen-binding molecules having heavy chain or light chain variable region sequences of SEQ ID NO: 66 and 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 conventional methods. For example, the general position of an epitope can be determined by assessing the ability of an antibody to bind to different fragments or variant B7H3 polypeptides, for example, by measuring the binding after mutagenesis of specific residues in B7H3. In addition, the antibody and target molecule can be bound, 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, wherein the other antigen-binding molecule is preferably an antigen-binding molecule having the following heavy and light chain variable region sequences: (i) SEQ ID NOs: 2 and 10, respectively, (ii) SEQ ID NOs: 18 and 26, respectively; (iii) SEQ ID NOs: 34 and 42, respectively, (iv) SEQ ID NOs: 34 and 42, respectively, (v) SEQ ID NOs: 66 and 74, respectively, (vi) SEQ ID NOs: 82 and 90, respectively, (vii) SEQ ID NOs: 98 and 106, respectively, (viii) SEQ ID NOs: 114 and 122, respectively, (ix) SEQ ID NOs: 130 and 138, respectively, (x) SEQ ID NOs: 146 and 154, respectively, (xi) SEQ ID NOs: 162 and 170, respectively, (xii) SEQ ID NOs: 178 and 186, respectively, and (xiii) SEQ ID NOs: NO: 194 and 202, (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 following heavy and light chain variable region sequences: (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, and (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).

[0118] 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 corresponding heavy chain variable domain sequences and light chain variable domain sequences of said SEQ ID NOs) can be identified by any suitable method known in the art, for example, using any suitable antibody numbering scheme. In some aspects, CDRs are identified using any of the following schemes: the Kabat numbering scheme (Kabat et al., U.S. Department of Health and Human Services, 1991), the Chothia numbering scheme (Chothia C, Lesk A MJ 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). The skilled artisan will appreciate that these different CDR labeling systems may give slightly different results, but in each case, the skilled artisan can readily identify the CDRs. The CDR sequences set forth in SEQ ID NOs: 4, 6, and 8 and SEQ ID NOs: 12, 14, and 16 are the HCDR1-3 and LCDR1-3 sequences 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 SEQ ID NOs: 28, 30, and 32 are the HCDR1-3 and LCDR1-3 sequences 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 SEQ ID NOs: 44, 46, and 48 are the HCDR1-3 and LCDR1-3 sequences 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 SEQ ID NOs: 60, 62, and 64 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 50 and 58, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 68, 70, and 72 and SEQ ID NOs: 76, 78, and 80 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 66 and 74, respectively, as defined using the Kabat numbering scheme.The CDR sequences set forth in SEQ ID NOs: 84, 86, and 88 and SEQ ID NOs: 92, 94, and 96 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 82 and 90, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 100, 102, and 104 and SEQ ID NOs: 108, 110, and 112 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 98 and 106, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 116, 118, and 120 and SEQ ID NOs: 124, 126, and 128 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 114 and 122, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 132, 134, and 136 and SEQ ID NOs: 140, 142, and 144 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 130 and 138, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 148, 150, and 152 and SEQ ID NOs: 156, 158, and 160 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 146 and 154, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 164, 166, and 168 and SEQ ID NOs: 172, 174, and 176 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 162 and 170, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 180, 182, and 184 and SEQ ID NOs: 188, 190, and 192 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 178 and 186, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 196, 198, and 200 and SEQ ID NOs: 204, 206, and 208 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 194 and 202, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 212, 214, and 216 and SEQ ID NOs: 220, 222, and 224 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 210 and 218, respectively, as defined using the Kabat numbering scheme.The CDR sequences set forth in SEQ ID NOs: 228, 230, and 232 and SEQ ID NOs: 236, 238, and 240 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 226 and 234, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 244, 246, and 248 and SEQ ID NOs: 252, 254, and 256 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 242 and 250, respectively, as defined using the Kabat numbering scheme. The CDR sequences set forth in SEQ ID NOs: 260, 262, and 264 and SEQ ID NOs: 268, 270, and 272 are the HCDR1-3 and LCDR1-3 sequences of SEQ ID NOs: 258 and 266, respectively, as defined using the Kabat numbering scheme.

[0119] The present invention relates to antigen binding molecules comprising a binding domain that specifically binds to B7H3 (e.g., human B7H3). In some respects, the binding domain comprises a heavy chain variable domain and / or a light chain variable domain. In some respects, the binding domain comprises a heavy chain variable domain. In some respects, the binding domain comprises a light chain variable domain. In some respects, the binding domain comprises a heavy chain variable domain and a light chain variable domain. In some respects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and wherein the antigen binding molecules comprise SEQ ID NO:2 heavy chain variable domain sequence and SEQ ID NO:10 light chain variable domain sequence CDR sequence. Typically, the antigen binding molecules comprise SEQ ID NO:2 and SEQ ID NO:10 all six CDR sequences, preferably, CDR sequences will be arranged on the heavy chain and light chain of the antigen binding molecules as shown in SEQ ID NO:2 and 10, and are arranged in the same order from N-terminal to C-terminal. In some aspects, the binding domain comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and 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 aspects, 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, and 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:161.

[0120] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:18 and SEQ ID NO:26, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:18 and 26, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences may vary according to the numbering scheme used (such as Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0121] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:34 and SEQ ID NO:42, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:34 and 42, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences can be different according to the numbering scheme (such as Kabat, Chothia or IMGT) used. In some aspects, 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, and 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 aspects, 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, and 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.

[0122] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:50 and SEQ ID NO:58, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:50 and 58, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the technician, accurate CDR sequences can be different according to the numbering scheme used (such as Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0123] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:66 and SEQ ID NO:74, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:66 and 74, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences can be different according to the numbering scheme used (such as Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0124] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:82 and SEQ ID NO:90, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:82 and 90, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0125] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:98 and SEQ ID NO:106, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:98 and 106, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences can be different according to the numbering scheme used (such as Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0126] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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 molecules comprise all six CDR sequences of SEQ ID NO:114 and SEQ ID NO:122, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:114 and 122, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled artisan, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0127] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:130 and SEQ ID NO:138, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:130 and 138, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0128] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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 molecules comprise all six CDR sequences of SEQ ID NO:146 and SEQ ID NO:154, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:146 and 154, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled artisan, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0129] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:162 and SEQ ID NO:170, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:162 and 170, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0130] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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 molecules comprise all six CDR sequences of SEQ ID NO:178 and SEQ ID NO:186, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:178 and 186, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled artisan, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0131] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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 molecules comprise all six CDR sequences of SEQ ID NO:194 and SEQ ID NO:202, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:194 and 202, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled artisan, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0132] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:210 and SEQ ID NO:218, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:210 and 218, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0133] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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 molecules comprise all six CDR sequences of SEQ ID NO:226 and SEQ ID NO:234, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:226 and 234, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled artisan, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0134] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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. Generally, the antigen binding molecules comprise all six CDR sequences of SEQ ID NO:242 and SEQ ID NO:250, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:242 and 250, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled person, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0135] In some other aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and wherein the antigen binding molecules comprise 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 molecules comprise all six CDR sequences of SEQ ID NO:258 and SEQ ID NO:266, preferably, the CDR sequences will be arranged on the heavy chain and the light chain of the antigen binding molecules as shown in SEQ ID NO:258 and 266, and are arranged in the same order from N-terminal to C-terminal. These CDR sequences are defined using the Kabat numbering scheme. As understood by the skilled artisan, accurate CDR sequences may vary according to the numbering scheme used (e.g., Kabat, Chothia or IMGT). In some aspects, 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, and 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 aspects, 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, and 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.

[0136] The present invention relates to antigen binding molecules comprising a binding domain that specifically binds to B7H3 (e.g., human B7H3). In some aspects, the binding domain comprises a heavy chain variable domain and / or a light chain variable domain. In some aspects, the binding domain comprises a heavy chain variable domain. In some aspects, the binding domain comprises a light chain variable domain. In some aspects, the binding domain comprises a heavy chain variable domain and a light chain variable domain. In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and wherein HCDR1 comprises SEQ ID NO:4 sequence, HCDR2 comprises SEQ ID NO:6 sequence, HCDR3 comprises SEQ ID NO:8 sequence, LCDR1 comprises SEQ ID NO:12 sequence, LCDR2 comprises SEQ ID NO:14 sequence, and LCDR3 comprises SEQ ID NO:16 sequence. In some aspects, 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, and 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.

[0137] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0138] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0139] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0140] In some preferred aspects, the heavy chain variable domain comprises HCDR1, HCDR2 and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2 and LCDR3, and 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 preferred aspects, the binding domain comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, the light chain variable domain comprising LCDR1, LCDR2 and LCDR3, and 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.

[0141] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0142] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0143] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0144] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0145] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0146] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0147] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0148] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0149] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0150] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0151] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0152] In some aspects, the heavy chain variable domain comprises HCDR1, HCDR2, and HCDR3, and the light chain variable domain comprises LCDR1, LCDR2, and LCDR3, and 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 aspects, 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, and 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.

[0153] The present invention relates to antigen binding molecules comprising a binding domain that specifically binds to B7H3 (e.g., human B7H3). In some aspects, the binding domain comprises a heavy chain variable domain and / or a light chain variable domain. In some aspects, the binding domain comprises a heavy chain variable domain. In some aspects, the binding domain comprises a light chain variable domain. In some aspects, the binding domain comprises a heavy chain variable domain and a light chain variable domain. In some aspects, the heavy chain variable domain comprises a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.5% homogeneity to SEQ ID NO: 2, and the light chain variable domain comprises a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 99.5% homogeneity to SEQ ID NO: 10. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 2, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 10. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 2, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 10. In some aspects, 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 aspects, 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 aspects, 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: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; and wherein 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 aspects, 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: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; and wherein 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 instances, 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: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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO:2 NO:10 has 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. 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 aspects, 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 aspects, 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.

[0154] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 26. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 26. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 18, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 26. In some aspects, 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 aspects, 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 aspects, 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: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; and wherein 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 aspects, 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: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; and wherein 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 instances, 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: 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; wherein 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 wherein the light chain variable domain comprises or consists of the sequence of SEQ ID NO: 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 aspects, 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 aspects, 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.

[0155] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 42. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 42. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 34, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 42. In some aspects, 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 aspects, 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 aspects, 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: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; and wherein 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 aspects, 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: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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:42 has 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. 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 aspects, 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 aspects, 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.

[0156] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 58. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 58. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 50, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 58. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:58 has 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. 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 aspects, 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 aspects, 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.

[0157] In some preferred aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 74. In some preferred aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 74. In some preferred aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 66, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 74. In some preferred aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 cases, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:74 has 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. 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 aspects, 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 aspects, 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.

[0158] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 90. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 90. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 82, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 90. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: 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 aspects, 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 aspects, 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.

[0159] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 106. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 106. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 98, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 106. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:106 has 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. 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 aspects, 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 aspects, 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.

[0160] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 122. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 122. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 114, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 122. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO: 122 has 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. 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 aspects, 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 aspects, 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.

[0161] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 138. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 138. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 130, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 138. In some aspects, 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 aspects, 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 aspects, 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, and LCDR3 comprises the sequence of SEQ ID NO: 144; and wherein 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 aspects, 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, and LCDR3 comprises the sequence of SEQ ID NO: 144; and wherein 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 instances, 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, and LCDR3 comprises the sequence of SEQ ID NO: 144; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: In some aspects, the heavy chain variable domain comprises or consists of the sequence of SEQ ID NO: 130, and the light chain variable domain comprises or consists of the sequence of SEQ ID NO: 138. In some aspects, 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 aspects, 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.

[0162] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 154. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 154. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 146, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 154. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO: 154 has 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. 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 aspects, 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 aspects, 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.

[0163] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 170. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 170. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 162, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 170. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: In some aspects, the heavy chain variable domain comprises or consists of the sequence of SEQ ID NO: 162, and the light chain variable domain comprises or consists of the sequence of SEQ ID NO: 170. In some aspects, 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 aspects, 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.

[0164] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 186. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 186. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 178, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 186. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: 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 aspects, 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 aspects, 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.

[0165] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 202. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 202. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 194, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 202. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:202 has 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. 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 aspects, 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 aspects, 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.

[0166] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 218. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 218. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 210, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 218. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:218 has 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. 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 aspects, 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 aspects, 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.

[0167] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 234. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 234. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 226, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 234. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: NO:234 ​​has 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. 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 aspects, 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 aspects, 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.

[0168] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 250. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 250. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 242, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 250. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein 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 99.5% sequence identity to the sequence of SEQ ID NO: 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 aspects, 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 aspects, 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.

[0169] In some aspects, the heavy chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the sequence of SEQ ID NO: 266. In some aspects, the heavy chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 266. In some aspects, the heavy chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 258, and the light chain variable domain comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO: 266. In some aspects, 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 aspects, 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 aspects, 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: 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; and wherein 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 aspects, 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: 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; and wherein 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 instances, 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: 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; wherein 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 wherein the light chain variable domain comprises or consists of the sequence of SEQ ID NO: NO:266 has 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. 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 aspects, 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 aspects, 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.

[0170] 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. In order to determine the percent identity of two nucleic acid sequences (or polynucleotide sequences), the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in the first sequence or the second sequence for optimal comparison). The nucleotides at corresponding nucleotide positions are then compared. When the 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 percent identity between the 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 a penalty is optionally imposed on the score of the number of spaces introduced and / or the length of the spaces introduced.

[0171] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment is performed on a specific portion of the aligned sequences that has sufficient identity, but not on portions with a lower degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm 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. This algorithm is incorporated into the BLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0172] In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity is determined based on the length of the aligned sequences (i.e., gapped alignment). To obtain gapped alignments for comparison purposes, the gapped BLAST described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402 can be used. In another embodiment, the alignment is optimized by introducing appropriate gaps, and the percent identity is determined based on the full length of the aligned sequences (i.e., overall alignment). A preferred, non-limiting example of a mathematical algorithm for overall comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.

[0173] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids (including glycine, both the D optical isomer and the L optical isomer), amino acid analogs, and peptidomimetics.

[0174] Alternatively, the antigen binding molecules of the present invention can comprise one or more variants in the specified sequence." variant " can be any one replacement, deletion or addition variant in the above-mentioned amino acid sequence. Variant can comprise 1, 2, 3, 4, 5, at most 10, at most 20, at most 30 or more amino acid replacements and / or deletions from the above-mentioned specific sequence and fragment, while maintaining the activity of antigen binding molecules as described herein. " deletion " variant can comprise the deletion of, for example, 1, 2, 3, 4 or 5 independent amino acids. " substitution " variant preferably relates to one or more amino acids replaced with the same number of amino acids, and carries out conservative amino acid substitution. For example, amino acid can be replaced by an alternative amino acid with similar properties, for example, 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 characteristics of the 20 main amino acids that can be used to select suitable substituents are as follows:

[0175]

[0176] The substituents may also be selected from the amino acids selenocysteine ​​and pyrrolysine.

[0177] Preferred "derivatives" or "variants" include those in which the amino acids appearing in the sequence are not naturally occurring amino acids but structural analogs thereof. The amino acids used in the sequence may also be derivatized or modified, for example, labeled, provided that the function of the antigen binding molecule is not significantly adversely affected. Derivatives and variants as described above may be prepared during the synthesis of the antigen binding molecule or by post-production modification, or when the antigen binding molecule is in recombinant form, may be prepared using known site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or nucleic acid ligation techniques. For example, the antigen binding molecule may be labeled with radiolabeled amino acids. Examples of radiolabels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125I. 131 I. Radiolabeling can be used for diagnostic and therapeutic purposes.

[0178] Antigen binding molecules as described herein can be derivatized or connected to another molecule (such as another peptide or protein). Generally, antigen binding molecules are derivatized so that derivatization or labeling does not adversely affect the combination with B7H3. For example, antigen binding molecules can be functionally connected with one or more other molecular entities, such as another antibody (such as bispecific antibody or diabody), detection agent, pharmaceutical agent and / or can mediate the protein or peptide that antibody or antibody portion associates with another molecule (such as streptavidin core region or polyhistidine tag), for example by chemical coupling, gene fusion, non-covalent association or other means. Antigen binding domains that specifically bind to B7H3 can be labeled with a detectable portion or a marker as described herein.

[0179] Methods for detecting labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect the emitted illumination. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0180] 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 used to improve the biological characteristics of the antigen binding domain, such as increasing serum half-life or increasing tissue binding.

[0181] Other Features of the Antigen-Binding Molecules of the Present Invention

[0182] Antigen binding molecules can be human or humanized. "Humanized" antigen binding molecules include human framework regions and one or more CDRs from non-human antigen binding molecules such as antibodies (e.g., monkeys, mice, rats, or synthetic antibodies). The non-human antigen binding molecules providing CDR are "donors," and the human antigen binding molecules providing the framework are "acceptors." Preferably, all six CDR sequences in the humanized antigen binding molecules are from antigen binding molecules. Humanized antigen binding molecules may not include constant regions. If constant regions are present in humanized antigen binding molecules, they are generally substantially identical to human antigen binding molecule constant regions, such as having at least 85%, at least 90%, at least 95%, at least 98% or about 100% sequence identity with human constant regions; preferably having at least 90% sequence identity with human constant regions, or most preferably having at least 95% sequence identity. Therefore, in preferred aspects, all parts of the humanized antigen binding molecules except CDR are substantially identical to the corresponding parts of the natural human antigen binding molecule sequences (i.e., having at least 90% and preferably at least 95% sequence identity). "Humanized antigen binding molecules " can include humanized light chain and humanized heavy chain.Humanized antigen binding molecules are combined with identical antigens to the donor antigen binding molecules providing CDR. The acceptor framework of humanized antigen binding molecules can have a limited number of amino acid replacements (generally about 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 replacements, preferably 1 to 20 and most preferably 1 to 10 replacements) taken from the donor framework. Humanized antigen binding molecules or other monoclonal antibodies can have additional conservative amino acid replacements that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulin can be constructed (for example, referring to U.S. Patent number 5,585,089) by genetic engineering. In some respects, the binding domains of the antigen binding molecules of the present invention can be people's or humanized. In some respects, heavy chain variable domains and / or light chain variable domains can be people's or humanized. In some respects, antigen binding domains are humanized antigen binding domains and include one or more human framework regions.

[0183] In some aspects, the antigen binding molecule can be an antibody fragment or a single-chain antibody, optionally wherein the fragment is a Fab fragment, a Fab' fragment, a 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), a scFv-Fc protein, a bivalent antibody, a trivalent antibody or a tetravalent antibody, a double scFv, a double-chain antibody, a three-chain antibody, a four-chain antibody, or an epitope-binding fragment of any of the above substances (see, 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 generating and manufacturing 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 aspects, antigen binding molecules are scFv or scFv-Fc. ScFv protein is the fusion protein that wherein the light chain variable region (LCVR) of immunoglobulin and the heavy chain variable region (HCVR) of immunoglobulin are combined by joint. In some aspects, scFv has the joint sequence (SEQ ID NO:273) of GGGGSGGGGSGGGGS. In some aspects, scFv includes SEQ ID NO:273 joint, and heavy chain variable domain is connected with light chain variable domain by joint. In some aspects, the components of scFv are arranged in the order of 5'-HCVR-joint-LCVR-3'. In some aspects, scFv has the heavy chain variable domain comprising SEQ ID NO:2 sequence, and this heavy chain variable domain is connected with the light chain variable domain comprising SEQ ID NO:10 sequence by the joint with SEQ ID NO:273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 18, which is connected to a light chain variable domain comprising the sequence of SEQ ID NO: 26 via a linker having the sequence of SEQ ID NO: 273. In some aspects, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 34, which is connected to a light chain variable domain comprising the sequence of SEQ ID NO: 42 via a linker having the sequence of SEQ ID NO: 273. In some aspects, the scFv has a heavy chain variable domain comprising the sequence of SEQ ID NO: 50, which is 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 some preferred aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 66 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 74 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 82 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 90 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 98 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 106 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 114 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 122 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 130 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 138 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 146 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 154 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 162 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 170 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO: 178 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO: 186 sequence via a joint having a SEQ ID NO: 273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising SEQ ID NO: 194, connected to a light chain variable domain comprising SEQ ID NO: 202 via a linker comprising SEQ ID NO: 273. In some aspects, the scFv has a heavy chain variable domain comprising SEQ ID NO: 210, connected to a light chain variable domain comprising SEQ ID NO: 218 via a linker comprising SEQ ID NO: 273.In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO:226 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO:234 ​​sequence via a joint having a SEQ ID NO:273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO:242 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO:250 sequence via a joint having a SEQ ID NO:273 sequence. In some aspects, the scFv has a heavy chain variable domain comprising a SEQ ID NO:258 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO:266 sequence via a joint having a SEQ ID NO:273 sequence. In a preferred aspect, the scFv has a heavy chain variable domain comprising a SEQ ID NO:50 sequence, which is connected to a light chain variable domain comprising a SEQ ID NO:58 sequence via a joint having a SEQ ID NO:273 sequence. In another preferred aspect, the scFv comprises a heavy chain variable domain comprising SEQ ID NO: 258, connected to a light chain variable domain comprising SEQ ID NO: 266 via a linker comprising SEQ ID NO: 273. In a more preferred aspect, the scFv comprises a heavy chain variable domain comprising SEQ ID NO: 66, connected to a light chain variable domain comprising SEQ ID NO: 74 via a linker comprising SEQ ID NO: 273. Optionally, the components of the scFv can be arranged in the order 5'-LCVR-linker-HCVR-3'.

[0184] In some respects, antigen binding molecules are multispecific molecules.In some respects, antigen binding molecules are bispecific molecules, such as comprising the first antigen binding domain and the second antigen binding domain, wherein the first antigen binding domain corresponds to the antigen binding molecules of the present invention and specifically binds B7H3, and wherein the second antigen binding domain specifically binds different target antigens.In some respects, antigen binding molecules can be biparatope molecules, such as comprising the first antigen binding domain and the second antigen binding domain, wherein the first antigen binding domain corresponds to the antigen binding molecules of the present invention, and wherein the second antigen binding domain is from the second different antigen binding molecules of the present invention, wherein the first antigen binding domain and the second antigen binding domain specifically bind B7H3 and identify different, non-overlapping epitopes.In some respects, antigen binding molecules are trispecific molecules, such as comprising the first antigen binding domain, the second antigen binding domain and the third antigen binding domain, wherein the first antigen binding domain corresponds to the antigen binding molecules of the present invention and specifically binds B7H3, and wherein the second antigen binding domain (and the third antigen binding domain) specifically binds different target antigens.

[0185] In some aspects, the antigen binding molecules are bispecific T cell adaptors (BiTEs). In some aspects, BiTEs include a first binding domain and a second binding domain, wherein the first antigen binding domain corresponds to the antigen binding molecules of the present invention and specifically binds B7H3, and wherein the second antigen binding domain specifically binds the CD3 on the T cell surface. For example, the second antigen binding domain specifically binds the CD3 subunit of the T cell receptor (TCR). Antigen binding molecules comprising such binding domains are well known to those skilled in the art. For example, anti-CD3 monoclonal antibodies contain this binding domain and are easily obtained. In some aspects, the second antigen binding domain specifically binds other T cell specific surface molecules.

[0186] In one aspect, the antigen binding molecule of the present invention is a bispecific 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, and wherein the first antigen binding domain comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains a HCDR1 comprising the sequence of SEQ ID NO: 68, a HCDR2 comprising the sequence of SEQ ID NO: 70, and a HCDR3 comprising the sequence of SEQ ID NO: 72, and wherein the light chain variable domain contains a LCDR1 comprising the sequence of SEQ ID NO: 76, a LCDR2 comprising the sequence of SEQ ID NO: 78, and a LCDR3 comprising the sequence of SEQ ID NO: 80; preferably, wherein 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.

[0187] Bispecific molecules, trispecific molecules or multispecific molecules can be produced by cross-linking two or more antigen binding domains. Suitable cross-linking agents include those cross-linking agents with two distinct reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those cross-linking agents with the same bifunctionality (such as disuccinimidyl suberate). In some aspects, the bispecific molecules (e.g., BiTE) or biparatope molecules described herein comprise two scFvs. For example, for a BiTE molecule, the first antigen binding domain can be an scFv of the present invention that specifically binds to B7H3, and the second CD3 antigen binding domain can also be an scFv. For example, two scFv antibodies can be covalently linked using a short peptide linker with 5 to 20 amino acids.

[0188] In some aspects, antigen binding molecules can be connected to effector molecules; for example, antigen binding molecules that specifically bind to B7H3 can be covalently linked to effector molecules or toxins. Connection can be carried out by chemical or recombinant means (e.g., peptide linkers). When the connection is chemical, a reaction may occur to produce a covalent bond connecting an antibody or its fragment to an effector molecule. The connection may include a peptide linker comprising, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20 or preferably 1 to 10 amino acids. Antigen binding molecules, optionally linked to effector molecules, may also be linked to lipids, proteins, polypeptides or carbohydrates to increase or preferably reduce their half-life in vivo. Effector molecules may be selected from the group consisting of anticancer agents, cytotoxic agents, cytostatic agents, drugs, radioisotopes, detectable labels, enzymes, fluorophores, fluorescent proteins, chemiluminescent agents, radiolabels, heavy metals, tracer molecules or any other detectable compounds known to technicians. In preferred aspects, antibodies may be coupled to anticancer agents, cytotoxic agents or cytostatic agents. In some aspects, the antigen binding molecule can be conjugated to pyrrolobenzodiazepine (PBD) or monomethyl auristatin E (MMAE). MMAE is a synthetic anti-tumor agent with the following structure:

[0189]

[0190] PBD has the following general structure:

[0191]

[0192] PBDs may differ in the number, type, and position of substituents in the aromatic A ring and the pyrrolo C ring, as well as the degree of saturation of the C ring. In the B ring, an imine (N=C), methanolamine (NH—CH(OH)), or methanolamine methyl ether (NH—CH(OMe)) is present at the N10-C11 position. PBDs typically have an (S)-configuration at the chiral C11a position, which provides a right-handed twist when viewed from the C ring toward the A ring. Many naturally occurring PBDs have been identified, and more than 10 synthetic pathways have been developed to synthesize a variety of analogs (see, for example, Thurston et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. and Thurston, DE, Chem. Rev. 2011 111 (4), 2815-2864). The term "PBD" should be understood to include PBD dimers.

[0193] Chimeric antigen receptors (CARs), chimeric costimulatory receptors (CCRs), and cells containing them

[0194] The present invention provides CAR, which includes an antigen binding molecule as described herein that specifically binds to B7H3. CAR is an engineered receptor that can transplant any specificity onto immune effector cells. In classical CAR, the specificity of the antibody is transplanted onto T cells. For example, a retroviral vector can be used to transfer the nucleic acid encoding CAR to T cells. In this way, a large number of cancer-specific T cells can be produced for adoptive cell transfer.

[0195] CAR is modular and generally 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 intracellular domains). The antigen binding ability of CAR is limited by the extracellular target antigen binding domain. Typically, the extracellular target antigen binding domain is a scFv. The transmembrane domain anchors CAR to the cell membrane, and the intracellular signaling domain transmits activation signals. The intracellular signaling domain may include one or more costimulatory domains. In some aspects, CAR also includes a hinge region, a transmembrane domain and an intracellular signaling domain.

[0196] The extracellular target antigen binding domain of CAR is usually fused with an intracellular signaling domain (or intracellular domain) through a spacer (or hinge) and a transmembrane domain, and the intracellular signaling domain comprises or associates with an intracellular T cell signaling domain. When CAR binds to a target antigen, this results in an activation signal being transmitted to, for example, a T cell expressing the activation signal. The hinge provides flexibility close to the target antigen for CAR (eg, scFv). Longer hinges provide additional flexibility and allow better access to membrane proximal epitopes, while short hinges allow more effective binding of membrane distal epitopes. It will be understood by the skilled artisan that any suitable hinge or spacer sequence can be used. The hinge can be an IgG-based hinge derived from IgG1, IgG2, or IgG4. The hinge can be derived from natural CD28 or CD8. The hinge or spacer sequence can, for example, comprise a short flexible linker, IgG1 Fc region, IgG1 hinge, or CD8 stem, or a combination thereof. Alternatively, the linker may comprise an alternative linker sequence having similar length and / or domain spacing characteristics to an IgG1 Fc region, an IgG1 hinge, or a CD8 stem. In some aspects, the hinge region of the CAR of the present invention is derived from CD8.

[0197] CAR may also include a membrane-spanning transmembrane domain. It may include a hydrophobic alpha 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 an ICOS transmembrane domain. The transmembrane domain closest to the membrane-end component from the intracellular signaling domain is often used, but different transmembrane domains may be used. In some aspects, the transmembrane domain of the CAR of the present invention is derived from CD8 (sometimes referred to herein as CD8α) or CD28.

[0198] CAR may include an intracellular signaling domain (or intracellular domain), which is a part of the CAR involved in signal transmission. The intracellular signaling domain includes or associates with an intracellular T cell signaling domain. After antigen recognition, the receptors aggregate and transmit the activation signal to the cell. The most commonly used T cell signaling component is the signaling component of CD3-ζ (or CD3ζ), which contains 3 ITAMs (immunoreceptor tyrosine activation motifs). After antigen binding, this activates the signal to the T cell. In some aspects, the intracellular signaling domain includes CD3-ζ. CD3-ζ alone may not provide a fully effective activation signal and may require additional costimulatory signaling. One or more costimulatory molecules can be used. Costimulatory molecules can be from the CD28 family (including CD28 and ICOS) or the tumor necrosis factor receptor family (including 4-1BB, OX40 or CD27). It will be understood by those skilled in the art that any suitable costimulatory domain can be used. According to the number of costimulatory domains, CAR can be divided into first generation CAR (only CD3ζ), second generation CAR (one costimulatory domain + CD3ζ) or third generation CAR (more than one costimulatory domain + CD3ζ). In some aspects, the intracellular signaling domain includes a costimulatory domain. In some aspects, the intracellular signaling domain includes a costimulatory domain derived from CD28 or 4-1BB.

[0199] The present invention also provides a chimeric costimulatory receptor (CCR), which includes an antigen binding molecule as described herein that specifically binds B7H3. CCR is similar to CAR, and generally includes an extracellular target antigen binding domain, a hinge region (or spacer), a membrane-spanning domain, and one or more intracellular signaling domains (or intracellular domains, or intracellular domains). The antigen binding ability of CCR is limited by the extracellular target antigen binding domain. Typically, the extracellular target antigen binding domain is scFv. The membrane-spanning domain anchors CCR to the cell membrane, and the intracellular signaling domain transmits activation signals. However, in contrast to CAR, the intracellular signaling domain of CCR cannot carry out T cell signaling. That is, the intracellular signaling domain of CCR does not give signal 1 (the signal generated after endogenous TCR interacts with its ligand, i.e., T cell activation signal). On the contrary, the intracellular signaling domain of CCR gives signal 2 (the signal generated by the interaction between the cognate receptor on the co-stimulatory molecule on the antigen presenting cell and the T cell). Therefore, CCR provides signal 2 but does not provide signal 1. CCR provides costimulation but does not provide TCR signaling. Therefore, the intracellular signaling domain of CCR lacks the intracellular T cell signaling domain and does not associate with the intracellular T cell signaling domain. For example, the intracellular signaling domain of CCR may lack CD3-ζ and may not associate with CD3-ζ. The intracellular signaling domain of CCR may include or consist of one or more costimulatory domains. One or more costimulatory domains may be any costimulatory domains known in the art. For example, the intracellular signaling domain of CCR may include or consist of (i) a costimulatory domain derived from CD28 and / or (ii) a costimulatory domain derived from 4-1BB. That is, the intracellular signaling domain of CCR may include or consist of a CD28 costimulatory domain and / or a 4-1BB costimulatory domain.

[0200] The extracellular target antigen binding domain of CCR is usually fused with the intracellular signaling domain (or intracellular domain) through a spacer (or hinge) and a transmembrane domain. When CCR binds to the target antigen, this causes the costimulatory signal to be transmitted to, for example, the T cell expressing the costimulatory signal. Hinge provides flexibility close to the target antigen for CCR (e.g., scFv). Longer hinges provide additional flexibility and allow better access to membrane proximal epitopes, while short hinges allow more effective binding of membrane distal epitopes. It will be appreciated by those skilled in the art that any suitable hinge or spacer sequence can be used. Hinge can be an IgG-based hinge derived from IgG1, IgG2 or IgG4. Hinge can be derived from natural CD28 or CD8. Hinge or spacer sequence can, for example, include short flexible joints, IgG1 Fc regions, IgG1 hinges or CD8 stems, or combinations thereof. Alternatively, the linker can include an alternative linker sequence having a length and / or domain spacing characteristic similar to IgG1 Fc regions, IgG1 hinges or CD8 stems. In some aspects, the hinge region of the CAR of the invention is derived from CD8.

[0201] CCR may also include a membrane-spanning transmembrane domain. It may include a hydrophobic alpha 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 an ICOS transmembrane domain. The transmembrane domain closest to the membrane-end component from the intracellular signaling domain is often used, but different transmembrane domains may be used. In some aspects, the transmembrane domain of the CCR of the present invention is derived from CD8 (sometimes referred to herein as CD8α) or CD28.

[0202] The CAR or CCR of the present invention as described herein may include antigen binding molecules as described herein that specifically bind B7H3. In some aspects, the CAR or CCR of the present invention comprising an extracellular target antigen binding domain is an antigen binding molecule as described herein. All descriptions of the antigen binding molecules of the present invention can be directly applied to CAR and CCR as described herein. Any features of the antigen binding molecules of the present invention as described herein are applicable to CAR and CCR as described herein. Specifically, any antigen binding molecules as described herein can be incorporated into CAR or CCR as described herein. CAR or CCR comprising an extracellular target antigen binding domain is an antigen binding molecule of the present invention.

[0203] The CAR or CCR of the invention described herein may comprise a signal peptide such that when the CAR or CCR is expressed in a cell (such as a T cell), the nascent protein is directed to the endoplasmic reticulum and then to the cell surface where it is expressed.

[0204] For example, the CAR or CCR of the present invention may preferably comprise an antigen binding molecule of the present invention, which antigen binding molecule comprises a binding domain that specifically binds to B7H3, and wherein the binding domain comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains a HCDR1 comprising a sequence of SEQ ID NO: 68, a HCDR2 comprising a sequence of SEQ ID NO: 70, and a HCDR3 comprising a sequence of SEQ ID NO: 72, and wherein the light chain variable domain contains a LCDR1 comprising a sequence of SEQ ID NO: 76, a LCDR2 comprising a sequence of SEQ ID NO: 78, and a LCDR3 comprising a sequence of SEQ ID NO: 80; more preferably, wherein 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 as described herein, the scFv having a heavy chain variable domain comprising a sequence of SEQ ID NO: 66, the heavy chain variable domain being connected to a light chain variable domain comprising a sequence of SEQ ID NO: 74 via a linker having a 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-ζ, and a costimulatory domain derived from CD28.

[0205] The present invention also provides cells comprising CAR described herein, preferably T cells (i.e., CAR-T cells). In some aspects, the T cells are α-β T cells. In some aspects, the T cells are γ-δ T cells. In some aspects, the cells may be NK cells or iPS cells. The present invention also provides cells comprising CCR described herein, preferably T cells. In some aspects, the T cells are α-β T cells. In some aspects, the T cells are γ-δ T cells. In some aspects, the cells may be NK cells or iPS cells.

[0206] The T cell can be a T cell or T lymphocyte of the lymphocyte type that plays a major role in cell-mediated immunity.They can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of T cell receptors (TCR) on the cell surface.There are various types of T cells, such as helper T cells, cytolytic T cells, memory T cells and regulatory T cells.Any type of T cell can be used to produce CAR-T cells or the T cells expressing CCR.

[0207] T helper T cells (TH cells) assist other white blood cells in the immune process, including maturing B cells into plasma cells and memory B cells, and activating cytotoxic T cells and macrophages. TH cells express CD4 on their surface. When TH cells are presented together with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs), TH cells are activated. 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.

[0208] Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens that associate with the MHC class I present on the surface of all nucleated cells. IL-10, adenosine, and other molecules secreted by regulatory T cells can inactivate CD8+ cells to an anergic state, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0209] Memory T cells are a subpopulation of antigen-specific T cells that persist for a long time after the infection subsides. Memory T cells rapidly expand into a large number of effector T cells after being re-exposed to their cognate antigens, thereby providing the immune system with "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 CD4+ or CD8+. Memory T cells usually express the cell surface protein CD45RO.

[0210] Regulatory T cells (Treg cells) (formerly known as suppressor T cells) are crucial for maintaining immune tolerance. The primary role of Tregs is to shut down T cell-mediated immunity at the end of an immune response and to suppress autoreactive T cells that evade the negative selection process in the thymus. Two main types of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are involved in interactions between developing T cells and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with 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 can prevent the development of Tregs, leading to the fatal autoimmune disease IPEX. Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can be generated during normal immune responses.

[0211] Cell can be natural killer cell (or NK cell).NK cell forms a part of innate immune system.NK cell provides rapid response to the innate signal of the cell from virus infection in MHC independent mode.NK cell (belonging to innate lymphocyte group) is defined as large granular lymphocyte (LGL), and NK cell constitutes the third kind of cell differentiated by common lymphocyte progenitor cell that produces B lymphocyte and T lymphocyte. Known NK cell is differentiated and matured in bone marrow, lymph node, spleen, tonsil and thymus, and then NK cell enters circulation therein.NK cell can use the CAR construct designed for CAR-T cell, and forms CAR-NK cell.NK cell can use the CCR construct designed for T cell, and forms the NK cell expressing CCR.

[0212] T cells (or NK cells) comprising the CAR or CCR of the present invention can be prepared by the following steps:

[0213] (i) isolating a sample containing T cells (or NK cells) from a subject or from other sources listed below; and

[0214] (ii) transducing or transfecting T cells (or NK cells) with a nucleic acid sequence encoding the CAR or CCR of the present invention.

[0215] The T cells (or NK cells) comprising or expressing the CAR or CCR of the present invention can be produced in vitro, and these cells can be derived from the patient's own peripheral blood (first party, autologous treatment), or produced in the case of hematopoietic stem cell transplantation from donor peripheral blood (second party) or from unrelated donor peripheral blood (third party, allogeneic treatment).Expression of the T cells (or NK cells) according to the CAR or CCR of the present invention can also be derived from inducible progenitor cells or embryonic progenitor cells and differentiate into T cells (or NK cells) in vitro.It is possible to use an immortalized T cell line that maintains its lysis function and can be used as a therapeutic agent.

[0216] The leukocytes in the blood sample can be separated using, for example, a blood cell separator (leukocyte separation). Peripheral blood mononuclear cells (PMBC) can be separated and collected from the sample. Before transduction or transfection with nucleic acid encoding the CAR or CCR according to the present invention, the T cells (or NK cells) in PMBC can be activated and / or amplified, for example, by being treated with anti-CD3 monoclonal antibodies or IL-2.

[0217] Cells comprising CAR or CCR of the present invention can be produced by introducing DNA or RNA encoding CAR or CCR by one of a variety of methods, including transduction with a viral vector or transfection with DNA or RNA. For example, the amplified T cells are purified and then transduced or transfected with nucleic acid sequences encoding CAR or CCR of the present invention, such as by retroviral vectors (such as integrative gamma retrovirus (RV) or slow virus (LV) vectors) or by using CRISPR / Cas9 systems.

[0218] T cells (or NK cells) can then be purified, for example, by selection based on expression of CAR or CCR.

[0219] Nucleic acid molecules, vectors and host cells

[0220] A nucleic acid molecule is provided, comprising a nucleotide sequence encoding an antigen binding molecule or CAR according to the present invention. A nucleic acid molecule is also provided, comprising a nucleotide sequence encoding a heavy chain variable domain or a light chain variable domain as described herein.

[0221] Nucleic acid molecules (or polynucleotides) can encode all or part of the antigen binding molecules of the present invention. Therefore, the nucleic acid molecules of the present invention can encode "all", i.e., full length, of any antigen binding molecules, variants or fragments as described herein. "Part" of an antigen binding molecule generally refers to a heavy chain or a light chain, or any region thereof, such as a variable region. For example, "part" of an antigen binding molecule can refer to a heavy chain variable domain or a light chain variable domain. The terms "nucleic acid molecules" and "polynucleotides" are used interchangeably herein and refer to a polymeric form of any length of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs). 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. Nucleic acid molecules of the present invention can be provided in the form of separation or purification.

[0222] A polynucleotide sequence that "encodes" a selected polypeptide 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 a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. For purposes of the present invention, such polynucleotide sequences may include, but are not limited to, cDNAs from viral, prokaryotic, or eukaryotic mRNAs, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0223] In one aspect, the nucleic acid molecules of the present invention comprise a nucleotide sequence encoding a heavy chain variable domain or a light chain variable domain sequence as described herein. Such nucleic acid molecules may comprise or consist of the following nucleotide sequences: SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241 or 257, or SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249 or 265, corresponding to VH and VL, respectively. The nucleic acid molecules of the present invention may comprise or consist of the nucleotide sequences of both SEQ ID NO: 1 and 9. The nucleic acid molecules of the present invention may comprise or consist of the nucleotide sequences of both SEQ ID NO: 17 and 25. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:33 and 41. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:49 and 57. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:65 and 73. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:81 and 89. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:97 and 105. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:113 and 121. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:129 and 137. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:145 and 153. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:161 and 169. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs:177 and 185. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs: 193 and 201. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs: 209 and 217. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs: 225 and 233. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs: 241 and 249. The nucleic acid molecule of the present invention may comprise or consist of both the nucleotide sequences of SEQ ID NOs: 257 and 265.The nucleic acid molecule of the present invention may preferably encode an antigen binding molecule comprising, for example, a heavy chain variable domain amino acid sequence of SEQ ID NO: 66 and a light chain variable domain amino acid sequence of SEQ ID NO: 74 as described above, or a variant or fragment thereof. The nucleic acid molecule of the present invention may comprise a nucleotide sequence encoding a CAR according to the present invention.

[0224] In order to produce scFv, the DNA fragment encoding heavy chain variable domain and light chain variable domain can be operably connected with another fragment encoding flexible joint (for example, encoding amino acid sequence (Gly4-Ser) 3) so that heavy chain variable domain and light chain variable domain sequence can be expressed as continuous single chain protein, wherein the heavy chain variable domain and the light chain variable domain are connected by a flexible joint. Optionally, a cleavage site can be included in a joint, such as a furin cleavage site. The nucleic acid encoding VH and / or VL optionally encodes an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fc domain. The Fc domain can be an optimized Fc domain. In one example, the immunoadhesin is IgG1 Fc.

[0225] Alternatively, a suitable polynucleotide sequence can be a variant of one of these specific polynucleotide sequences. For example, a variant can be a substitution, deletion or addition variant of any of the above-mentioned nucleic acid sequences. A variant polynucleotide can comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90 or up to 100 or more nucleic acid substitutions and / or deletions from a sequence given in the sequence listing.

[0226] Suitable variants can be at least 70% homologous to the polynucleotides of any one of the nucleotide sequences disclosed herein, preferably at least 80% or 90% homologous thereto, and more preferably at least 95%, 97% or 99% homologous thereto. Preferably, at least for the coding region of the polynucleotides, there are homology and identity at these levels. The method for measuring homology is well known in the art, and it will be understood by those skilled in the art that in this article, homology is calculated based on nucleic acid identity. The calculation of homology is also described in the previous chapters.

[0227] Due to the redundancy of the genetic code, variant sequences may be different from the specific sequences given in the sequence table. The DNA code has four main nucleic acid residues (A, T, C and G) and uses these residues to "spell" three-letter codons that represent the amino acids of the proteins encoded in the genes of the organism. The linear sequence of codons along the DNA molecule is translated into a linear sequence of amino acids in the proteins encoded by these genes. The code is highly degenerate, with 61 codons encoding 20 natural amino acids and 3 codons representing "stop" signals. Therefore, most amino acids are encoded by more than one codon - in fact, several amino acids are encoded by four or more different codons. Therefore, the variant polynucleotide of the present invention can encode the same polypeptide sequence as another polynucleotide of the present invention, but due to the use of different codons to encode the same amino acid, it is possible to have different nucleic acid sequences.

[0228] Polynucleotide " fragment " according to the present invention can be by truncation, for example, by removing one or more nucleotides from one end or both ends of polynucleotide and prepare.In this way, can remove at most 10, at most 20, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90 or at most 100 or more amino acids from 3 ' and / or 5 ' end of polynucleotide.Fragment can also be produced by one or more internal deletions.Such fragment can be derived from sequence as described herein or can be derived from variant polynucleotide as described herein.Preferably, the length of such fragment is between 90 to 1000 residues, for example, between 90 to 300, 90 to 500, 100 to 800, 200 to 900 or 300 to 100 residues.Alternatively, fragment of the present invention can be a longer sequence, for example, comprising at least 50%, at least 60%, at least 70%, at least 80% or at least 90% full-length polynucleotide of the present invention.

[0229] Therefore, the antigen binding molecules of the present invention can be produced by polynucleotide or delivered in the form of polynucleotide, and these polynucleotide encodings and can express the antigen binding molecules of the present invention.When antibody comprises two or more chains, the polynucleotide of the present invention can encode one or more antibody chains.For example, the polynucleotide of the present invention can encode antibody light chain variable domain, antibody heavy chain variable domain or both.Two kinds of polynucleotides can be provided, wherein a kind of polynucleotide encoding antibody light chain variable domain, and another kind of polynucleotide encoding corresponding antibody heavy chain variable domain.This polynucleotide or polynucleotide pair can be expressed together, thereby produce antigen binding molecules of the present invention.

[0230] The polynucleotides of the present invention can be synthesized according to methods well known in the art, such as those described in the examples of Sambrook et al. (1989, "Molecular Cloning - a laboratory manual"; Cold Spring Harbor Press).

[0231] The nucleic acid molecules of the present invention can be provided in the form of an expression cassette, which comprises a control sequence operatively connected to an insertion sequence, thereby allowing the antigen binding molecules of the present invention to be expressed in vivo. These expression cassettes are typically provided in a carrier (e.g., a plasmid or a recombinant viral vector) in sequence. This expression cassette can be directly applied to a host subject. Alternatively, a carrier comprising a polynucleotide of the present invention can be applied to a host subject. Preferably, gene vectors are used to prepare and / or administer polynucleotides. Suitable carriers can be any carriers that can carry sufficient amounts of genetic information and allow expression of polypeptides of the present invention.

[0232] Therefore, the present invention also provides expression vectors comprising the nucleic acid molecules described herein. Such expression vectors are conventional construction methods in the field of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as polyadenylation signals, which may be necessary and positioned in the correct orientation to allow expression of the polypeptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, we can refer to the research of Sambrook et al.

[0233] The present invention also provides host cells comprising nucleic acid molecules or expression vectors described herein. These cells are modified to express the antigen binding molecules 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 inserting vectors or expression cassettes encoding the antigen binding molecules of the present invention include mammalian HEK293T, CHO, HeLa, NS0 and COS cells. Host cell lines that can be used to express antigen binding molecules are well known in the art. Preferably, the selected cell line will be a cell line that is not only stable but also allows mature glycosylation. Such cell lines of the present invention can be cultivated using conventional methods to produce antigen binding molecules of the present invention.

[0234] Antibody-drug conjugates (ADCs)

[0235] The present invention provides an antibody-drug conjugate (ADC), the ADC comprising an antigen binding molecule as described herein connected to a drug. In some aspects, the drug is an anticancer agent, a cytotoxic agent, or a cytostatic agent. In some aspects, the drug is selected from pyrrolobenzodiazepine (PBD) and monomethyl auristatin E (MMAE). In some aspects, the drug is a fluorophore or a tracer molecule.

[0236] Also described herein are antibody-drug conjugates of formula (I):

[0237] Ab–(L–D)p(I)

[0238] wherein Ab is an antigen-binding molecule of the present invention as described herein;

[0239] Wherein L is a linker connecting Ab to D;

[0240] wherein D is an anticancer agent, a cytotoxic agent, or a cytostatic agent; and

[0241] Here, p is preferably 1 to 8.

[0242] In some aspects, L may be absent or may simply be a covalent bond between the antibody (Ab) and the drug (D).

[0243] Antibody-drug conjugates (i.e., immunoconjugates) allow targeted delivery of cytotoxic agents or cytostatic agents (i.e., drugs that kill cells or inhibit cell growth and division, such as drugs that can be used to treat cancer) to cells, such as cancer cells. The antigen binding molecule portion allows selective binding to target tumors. Then, ADC can be internalized by target cells (typically cancer cells), thereby causing drug accumulation in the cell. ADC can include a joint connecting the antibody and drug payload. Once ADC is internalized by the target cell, the joint can be cut, thereby releasing the payload into the cytoplasm. Systemic administration of uncoupled drugs typically causes unacceptable toxicity levels to normal cells or non-target cells. Therefore, the present invention provides antibody-drug conjugates that deliver targeted drugs to B7H3 positive cells. B7H3 positive cells can be cancer cells. Therefore, the present invention provides antibody-drug conjugates comprising antigen binding molecules of the present invention that specifically bind to B7H3 as defined above, and drugs, wherein the drugs are anticancer agents, cytotoxic agents, or cytostatic agents.

[0244] The antibody-drug conjugates described herein can have a variety of advantageous features. For example, after administration to a subject in need, the antibody-drug conjugate can be rapidly cleared from the subject's body to minimize residual toxicity. The Ab can be selected, modified, or engineered in other ways to have a shorter half-life in the target subject. The antibody-drug conjugates described herein are highly specific, targeted, and effective agents.

[0245] Antibodies (Ab)

[0246] In the antibody-drug conjugates described herein, Ab can be any antigen-binding molecule of the present invention as described herein. All descriptions of the antigen-binding molecules of the present invention can be directly applied to the Ab in the antibody-drug conjugates described herein. Any features of the antigen-binding molecules of the present invention described herein are applicable 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 conjugate of the present invention is an antigen-binding molecule of the present invention.

[0247] For example, the Ab can be an antigen-binding molecule of the present invention, which comprises a binding domain that specifically binds to B7H3, and wherein the binding domain comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises a HCDR1 comprising the sequence of SEQ ID NO: 68, a HCDR2 comprising the sequence of SEQ ID NO: 70, and a HCDR3 comprising the sequence of SEQ ID NO: 72, and wherein the light chain variable domain comprises a LCDR1 comprising the sequence of SEQ ID NO: 76, a LCDR2 comprising the sequence of SEQ ID NO: 78, and a LCDR3 comprising the sequence of SEQ ID NO: 80; preferably, wherein 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.

[0248] Drug Unit

[0249] Antibody-drug conjugates can comprise any antibody (Ab) described herein coupled (i.e., connected or fused) to an anticancer agent, cytotoxic agent, or cytostatic agent (D). Thus, D is an anticancer agent, cytotoxic agent, or cytostatic agent. An anticancer agent (also referred to as an antitumor agent) is any agent, small molecule, or biological agent that is effective in treating cancer. A cytotoxic agent is any agent that causes cell killing (preferably cancer cell killing) and tumor reduction. A cytostatic agent is any agent that inhibits, reduces, or prevents cell (preferably cancer cell) growth or division and inhibits tumor growth. D can be a known anticancer therapeutic agent that has proven anticancer, cytotoxic, or cytostatic properties.

[0250] Drug loading is the average drug unit (D) quantity of every antibody (Ab), and is represented by p.The average drug quantity of every antibody in the antibody drug conjugate prepared by coupled reaction can be characterized by conventional methods (such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA determination and electrophoresis).In some cases, separation, purification and the characterization of homogeneous antibody-drug conjugate can be realized by method such as reversed-phase HPLC or electrophoresis, wherein p is the specific value from the antibody-drug conjugate with other drug loadings.Drug loading (p) is usually subject to the restriction of the drug and joint attachment site quantity on the antibody.Attachment site can be understood as meaning that the drug unit on the antibody is usually attached by a joint.For example, the antibody can have 1,2,3,4,5,6,7 or 8 attachment sites that can be attached to a drug linker.In some aspects, antibody has 1 to 8,1 to 6,1 to 4 or 1 to 2 such attachment sites, preferably 1 to 8, and most preferably 1 to 4 such attachment sites. Typically, during the coupling reaction, the drug moiety less than the theoretical maximum is coupled to the antibody. The loading capacity (drug / antibody ratio) of ADC can be controlled in several different ways, including: (i) limiting the molar excess of drug-linker intermediate (DL) or linker reagent relative to the antibody, and (ii) limiting the coupling reaction time or temperature. Antibody-drug conjugate compositions are described herein, which include a mixture of antibody-drug conjugates, wherein the antibody has one or more attached drug units, and wherein these drug units can be attached to the antibody at various sites (such as at different amino acid residues). The drug unit is typically attached to the antibody via a joint. Suitable joints are further described herein. Suitable methods for attaching or coupling antibodies to joints are also further described herein.

[0251] In some aspects, the average number of drug units (D) per antibody (Ab) in the antibody drug conjugates of the present invention is in the range of 1 to 8. In some aspects, 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 aspects, in the antibody drug conjugates of the present invention, there are one or two drug units (D) per antibody (Ab). In some aspects, p is 1 to 8, preferably 1 to 4. In some aspects, p is about 2.

[0252] In some aspects, each D is independently selected from: an anticancer agent, a cytotoxic agent, a cytostatic agent, a drug, a radioisotope, a detectable label, an enzyme, a fluorophore, a fluorescent protein, a chemiluminescent agent, a radiolabel, a heavy metal, or any other detectable compound known to a skilled artisan. In preferred aspects, D is an anticancer agent, a cytotoxic agent, or a cytostatic agent. In further preferred aspects, D is a pyrrolobenzodiazepine (PBD) or monomethyl auristatin E (MMAE).

[0253] connector

[0254] L is a linker connecting the antibody Ab and the drug D. L can be any linker suitable for connecting, covalently linking, or coupling the antibody Ab to the drug D. The linker L can be cleavable or non-cleavable. The linker L is preferably stable outside the cell. Therefore, before transport or delivery into the cell, the antibody-drug conjugate of the present invention is preferably stable and remains intact, i.e., the antibody Ab remains connected to the drug D. In some aspects, the linker L is stable outside the target cell (i.e., in the extracellular environment), but is cut inside the cell (i.e., in the intracellular environment) to release the drug D from the antibody Ab. Therefore, the antibody Ab targets the drug D with anticancer, cytotoxic and / or cell growth inhibitory effects to target cells expressing B7H3. Generally, the cutting of the linker occurs at a sufficiently fast rate, thereby allowing the drug to have an anticancer, cytotoxic or cytostatic effect on the target cell. After the target cell internalizes the antibody-drug conjugate, the linker can be cut at any point. In some aspects, the linker can be preferentially cut in a specific intracellular compartment within the target cell. For example, the linker L can be preferentially cut in lysozyme. An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate and / or drug; (iii) remain stable and intact (i.e., not cleaved) until the conjugate and / or drug has been delivered or transported to its target site; and (iv) maintain the cytotoxic, anticancer, cell-killing and / or cytostatic effects of drug D. The stability of the antibody-drug conjugate can be measured by standard analytical techniques such as mass spectrometry, HPLC, and separation / analysis techniques LC / MS.

[0255] The linker can be a non-cleavable linker, i.e., a linker that is not easily cleaved, for example, by enzymatic activity (such as protease activity) or under specific conditions (such as acidic conditions). In some preferred aspects, L is a cleavable linker, i.e., a linker that is easily cleaved when there is a suitable cleavage portion or under specific conditions. L can be selected from an acid-cleavable linker, a protease-cleavable linker, a disulfide bond 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 allows the drug unit to be targeted for delivery to B7H3-positive cells. Suitable linkers are described in, for example, Yang et al., Med Res Rev. 2020; 1–32, and the skilled person will be able to select a suitable linker.

[0256] The conjugate of the antibody and cytotoxic agent can be prepared using any suitable method as disclosed in the art, for example, "Bioconjugate Techniques", GT Hermanson, 3rd edition Elsevier, 2013. The linker can be coupled to the antibody (Ab) using, for example, a cleavable disulfide bond or a non-cleavable thioether linker chemical method. The linker can be attached to a lysine residue in the antibody, which can be natural or engineered. The linker can be attached to a cysteine ​​residue in the antibody. The cysteine ​​can be natural, such as the cysteine ​​of one of the interchain disulfide bridges in the antibody, or the cysteine ​​can be engineered, i.e., site-specifically inserted into the desired coupling site in the antibody sequence. The linker can be attached to the non-natural amino acid (such as acetyl-phenylalanine, p-acetyl-L-phenylalanine (pAcF), selenocysteine ​​or p-azidomethyl-L-phenylalanine) in the antibody, for example, via an oxime bond. The antibody can be engineered to include a non-natural amino acid at the desired coupling site. Chemical enzyme sites can also be used for direct coupling.For example, an azido group can be formed at the asparagine residues in, for example, the constant region of an antibody, and connected to the drug unit using, for example, a copper-mediated click reaction. An azido group can be formed in a selective hydrolysis reaction mediated by endo-β-N-acetylglucosaminidase (EndoS) chemical enzyme. Other strategies for site-specific coupling of a joint and its attached drug unit are known in the art and are widely covered in GT Hermanson, " Bioconjugation Technology ", 2013, Elsevier.

[0257] Pharmaceutical compositions, methods and uses

[0258] The present invention also provides pharmaceutical compositions comprising antigen binding molecules, CARs, cells (e.g., T cells) or ADCs as described herein. Preferably, these pharmaceutical compositions further comprise a pharmaceutically acceptable carrier.

[0259] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for parenteral (e.g., intravenous, intramuscular or subcutaneous) administration (e.g., by injection or infusion). " Remington's Pharmaceutical Sciences " compiled by EWMartin (Mack Publishing Co., Easton, PA 19th edition, 1995) describes compositions and preparations suitable for drug delivery of antigen binding molecules as described herein, CAR, cells comprising CAR or ADC.

[0260] Preferably, pharmaceutically acceptable carrier comprises aqueous carrier or diluent.The example of suitable aqueous carrier that can be used for pharmaceutical composition of the present invention comprises water, buffered water and saline.The example of other carriers comprises glucose aqueous solution, glycerol, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol etc.) and their suitable mixture, vegetable oil (such as olive oil) and injectable organic ester such as ethyl oleate.For example, by using coating material such as lecithin, by keeping required particle diameter in the case of dispersion, and by using surfactant, suitable mobility can be kept.In many cases, preferably in composition, comprise isotonic agent, for example sugar, polyol (such as mannitol, sorbitol) or sodium chloride.

[0261] The therapeutic composition must generally be sterile and stable under manufacturing and storage conditions. The composition can be formulated into a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. Sterile injectable solution can be prepared by the following method: the required amount of active agent (such as antibody) is mixed with a combination of one of the above-mentioned ingredients or a plurality of the above-mentioned ingredients (as needed) in an appropriate solvent, followed by sterilization microfiltration. The pharmaceutical composition of the present invention may include additional active ingredients and the antigen binding molecules of the present invention, CAR, cells comprising CAR or ADC. For example, the pharmaceutical composition may also include additional therapeutic agents or preventive agents.

[0262] The present invention also provides a method for treating cancer, wherein the method includes administering an antigen binding molecule as described herein, CAR, a cell comprising CAR, ADC or a pharmaceutical composition to a subject in need. An effective amount of an antigen binding molecule as described herein, CAR, a cell comprising CAR, ADC or a pharmaceutical composition can be administered. An "effective amount" refers to an amount that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. The present invention also provides an antigen binding molecule as described herein, CAR, a cell comprising CAR, ADC or a pharmaceutical composition for use in a method for treating cancer.

[0263] In some instances, the cancer is selected from solid tumors, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma,

[0264] Rhabdomyosarcoma, haematological malignancies, acute myeloid leukaemia, desmoplastic small round cell tumour (DSRCT), melanoma, breast, prostate, colon, lung, kidney or pancreatic cancer, or oral squamous cell carcinoma (SCC).

[0265] When a disease or condition (e.g., cancer, such as a solid tumor) is "treated" as described herein (e.g., in the methods or uses of the present invention), this means that one or more symptoms of the disease or condition (e.g., cancer, such as a solid tumor) are improved. This does not mean that the symptoms of the disease or condition (e.g., cancer, such as a solid tumor) are completely cured so that they are no longer present in the patient's body, although in some methods, this may be the case. Therefore, in all cases, the term "treatment" or "treating" may be replaced by the term "amelioration" or "ameliorating," respectively. The methods or uses of the present invention (such as methods of treatment or therapy) can reduce one or more symptoms of a disease or condition (e.g., cancer, such as a solid tumor) compared to before treatment.

[0266] One or more methods in a variety of methods known in the art can be used to administer the antigen binding molecules of the present invention, CAR, cells or ADC comprising CAR, or pharmaceutical compositions comprising antigen binding molecules of the present invention, CAR, cells or ADC comprising CAR by one or more routes of administration. As will be appreciated by those skilled in the art, route of administration and / or mode will vary according to the desired result. Preferably, the antigen binding molecules of the present invention, CAR, cells or ADC comprising CAR or pharmaceutical compositions can be administered by parenteral administration. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, typically by injection. The preferred route of administration of the antigen binding molecules of the present invention, CAR, cells, ADC comprising CAR or compositions includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, for example, by injection or infusion. Alternatively, the antigen binding molecules of the present invention, CAR, cells, ADC comprising CAR or pharmaceutical compositions can be administered by non-parenteral routes (such as topical, epidermal or mucosal administration routes). Local administration is also possible, including peritumoral, paratumoral, intratumoral, intralesional, perilesional, intracavitary infusion, intravesical administration, and inhalation.

[0267] The suitable dosage of the antigen binding molecules of the present invention, CAR, the cell comprising CAR or ADC can be determined by skilled medical practitioners.The actual dosage level of active ingredient in the pharmaceutical composition of the present invention can be varied, to obtain the specific amount of active ingredient effective for specific patients, compositions and modes of administration, so as to achieve the desired therapeutic response without causing toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific antibody used, route of administration, administration time, excretion rate of the antibody, treatment duration, other drugs used in combination with the specific composition used, compounds and / or materials, age, sex, body weight, illness, general health and previous medical history of the treated patient and similar factors known in the medical field.

[0268] The suitable dosage range of the antigen binding molecules, CARs, cells comprising CARs or ADCs of the present invention can be, for example, in the range of about 100 ng / kg of the patient's body weight / day to be treated to about 25 mg / kg of the patient's body weight / day. For example, a suitable dosage can be about 1 μg / kg body weight / week to about 10 mg / kg body weight / week, about 100 μg / kg body weight / week to about 10 mg / kg body weight / week, or about 10 μg / kg body weight / week to about 5 mg / kg body weight / week. A suitable dosage can be about 1 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, or about 10 μg / kg body weight / day to about 5 mg / kg body weight / day. In some aspects, 1×10 6 CAR T cells / kg or 5×10 6 CAR T cells / kg.

[0269] The dosage regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the needs of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate the composition for parenteral administration in dosage unit form. As used herein, dosage unit form refers to a physically discrete unit that is suitable as a unit dose for a subject to be treated; each unit contains a calculated predetermined amount of active compound that, in combination with a desired pharmaceutical carrier, will produce the desired therapeutic or conditioning effect.

[0270] Antigen binding molecules as described herein, CAR, cells comprising CAR or ADC can be administered in single dose or multiple doses.These multiple doses can be administered by the same or different approaches and applied to the same or different positions.Alternatively, antigen binding molecules can be used as sustained release formulations, in which case less frequent administration is required.Dosage and frequency can vary according to the half-life of antigen binding molecules in the patient and the desired treatment duration.

[0271] The pharmaceutical composition may include any antigen binding molecules of the present invention as described herein, CAR, cells or ADC comprising CAR. In some aspects, the pharmaceutical composition may include a single antigen binding molecule of the present invention, CAR, cells or ADC comprising CAR. In some aspects, the pharmaceutical composition may include two or more different types of antigen binding molecules of the present invention, CAR, cells or ADC comprising CAR within the same composition. The present invention also provides for the simultaneous administration of two different pharmaceutical compositions, each of which comprises a single but different type of antigen binding molecules of the present invention, CAR, cells or ADC comprising CAR.

[0272] Methods for detecting cancer

[0273] The present invention also provides a method for detecting cancer in a subject, the method comprising:

[0274] A biological sample from the subject is contacted with an antigen binding molecule described herein, and the antigen binding molecule bound to the sample is detected, wherein binding of the antigen binding molecule to the sample indicates that the subject has cancer. In some cases, the cancer is selected from solid tumors, neuroblastoma, medulloblastoma, glioblastoma, DIPG, osteosarcoma,

[0275] 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). In some cases, the antigen binding molecule specifically binds to human B7H3, and wherein binding of the antigen binding molecule indicates that the subject has cancer.

[0276] The present invention is illustrated by the following examples:

[0277] Example

[0278] Example 1 - Materials and Methods

[0279] Cells and culture conditions

[0280] The following cell lines were used in this study: Jurkat, 293T, CHO, MEXi 293E, 293F, LAN-1, Kelly, SupT1, and K562. CHO cells were cultured in CHO medium (Gibco) supplemented with 8 mM GlutaMAX (Gibco) supplemented with 0.4 mM hypoxanthine and 0.32 mM thymidine (Gibco). MEXi 293E cells were cultured in MEXi medium (IBA) supplemented with 50 mg / l geneticin and 8 mM GlutaMAX. 293F cells were cultured in Freestyle 293 Expression Medium (Thermofisher). All three cell lines were cultured in an orbital shaker at 37°C with 5% CO2. The temperature used for protein production was 32°C. 293T cells were grown in IMDM (Sigma) supplemented with 10% FCS (Gibco) and 100 U penicillin / 0.1 mg streptomycin / l. These 293T cells were cultured at 37° C. and 5% CO 2 . The remaining cells were grown in RPMI (Sigma) + 10% FCS 100 U penicillin / 0.1 mg streptomycin / l at 37° C. and 5% CO 2 .

[0281] γ-retroviral transduction

[0282] 24 hours before transfection, 293T cells were plated at 1.5 × 10 6 Cells were transduced using GeneJuice (Merck) with a GOI (gene of interest) expression cassette and the helper plasmids env (RD114) and gagpol (PegPam-env). Retroviral supernatant was harvested at 48 and 72 hours. To ensure stable transduction, target cells were plated on 24-well plates coated with Retronectin (Takara) and incubated with retroviral supernatant for 72 hours.

[0283] Preparation of B7-H3 phage display library

[0284] 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 on a protein A column. Three BALB / cJ mice were injected with the recombinant protein ( Figure 1A). Serum was extracted continuously and seroconversion was confirmed by flow cytometry. Spleen mRNA was extracted using the RNeasy Mini kit (QIAGEN). The mRNA was reverse transcribed (Superscript III reverse transcriptase, Invitrogen) and then amplified by PCR (Amplitaq polymerase, Applied Biosystems). A further PCR reaction was used to connect the heavy and light chain DNA with a serine glycine linker. The amplified DNA was first cloned into the intermediate pSP73 vector before being cloned into the pHEN vector. E. coli ( Figure 1 ).

[0285] Panning of B7-H3 library

[0286] 4Ig-B7-H3 cDNA was purchased from Sinobiological and cloned into two vectors to produce B7-H3-Histag and B7-H3-Streptag. CHO cells and MEXi293E cells were transiently transduced with these constructs, respectively. Cells were cultured until viability decreased, and proteins were filtered from the cell supernatant using a HiTrap MabSelect Protein-A column (Cytiva) or a Strep-Tactin XT:Twin Strep-tag purification column (IBA).

[0287] 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 incubated overnight. The bacteria were removed by centrifugation, and the phage particles in the supernatant were precipitated with PEG 600 / 2.5M NaCl. After washing, the phage were resuspended in sterile water.

[0288] The immunotubes were incubated with the B7-H3-Histag and MagStrep "Type 3" XT beads (IBA) were incubated with the B7-H3-Streptag overnight at 4°C to coat the tubes / beads. The tubes / beads were washed 3 times in PBS and blocked by incubation with Marvel Milk for 2 hours at room temperature. The precipitated phages were blocked separately with Marvel Milk. The tubes / beads were washed 3 times with PBS. The blocked phages were applied to the coated and blocked tubes and incubated for 2 hours at room temperature. The tubes / beads were washed and the bound phages were eluted with 1 ml of 100 μM triethylamine. The eluted phages were incubated with TG1 E. coli at an OD value of 0.5 for 40 minutes. The bacteria were pelleted and plated on agar plates ( Figure 1 B).

[0289] Selection of anti-B7-H3 scFv

[0290] Selected colonies from the panning library were grown in 96-well plates. Positive binders were identified using an 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 pcDNA3.1 expression vector in scFv-Fc format. 293F cells were transiently transfected with PEI (Sigma). Cells were cultured until their viability decreased and the supernatant was harvested. Protein was purified on a HiTrap MabSelect Protein-A column (Cytiva). The purified and diluted protein was used to stain cell-bound B7-H3 on Jurkat cells and binding was analyzed by flow cytometry.

[0291] Generation of B7-H3-positive Jurkat cells

[0292] Truncated B7-H3 (T-B7-H3) in an SFG gamma-retroviral expression cassette was used. The 4Ig-B7-H3 isoforms of B7-H3 were purchased (Sinobiological) and cloned into a gamma-retroviral expression cassette. 4Ig-B7-H3 was digested to produce 2Ig-B7-H3. Jurkat cells were stably transduced with each of the three isoforms of B7-H3 using retroviral transduction technology.

[0293] Isolation of PBMCs and T cells

[0294] Leukapheresis cones were obtained from NHS blood and transplants. PBMCs were separated by Ficoll centrifugation using Lymphoprep (Stemcell Technologies). PBMCs 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).

[0295] Generation of CAR-T constructs

[0296] The gene module for each cell in the anti-B7-H3 CAR T cells was designed and cloned into the previously described expression vector scFv-CH2-CH3-CD28-CD3zγ-retroviral CAR expression cassette with the RQR8 marker gene for selection / elimination using restriction sites at the 3' and 5' primer ends of CAR (Thermofisher) (Philip et al., 2014). These gene modules include 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, and TE9-CD8H / Tm-CD28-ILR2-CD3z.

[0297] CAR T cell transduction

[0298] PBMCs were cultured at 1×10 6 PBMCs were suspended in RPMI containing FCS and L-glutamine at a concentration of 10 cells / ml. PBMCs were activated with 0.5 μg / ml anti-CD3 (Miltenyi Biotec) and anti-CD28 antibodies (Miltenyi Biotec). 100 IU / ml recombinant human IL-2 (Proleukin, Novartis) was added 48 hours before and on the day of transduction. T cells were transduced using γ-retroviral transduction.

[0299] T cell functional assay

[0300] In co-culture assays with CAR-T cells, targets were LAN-1, Kelly, K562, or AML target cells (MV411, NOMO1, THP1) or no antigen stimulation. Assays were performed in 48-well plates at an effector-to-target ratio of 2:1. In most experiments, control target cells were Jurkat or SupT1 cells stably transduced to express 4xIg of the target antigen, human B7H3. In co-culture assays for 18 hours, CAR T cells were co-cultured with the target. After 18 hours, the supernatant was removed for ELISA, and the cells were incubated with monensin (BioLegend). Activation markers CD69 and CD25, as well as the degranulation marker CD107a, were measured by flow cytometry. During the 7-day co-culture, 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 antigen-free target cells in 24-well plates for 6 days at an effector-to-target ratio of 2:1. On day 6, the plates were centrifuged to pellet the cells, 1 ml of medium was removed, and 1 ml of medium containing fresh target cells was added. After another 24 hours, the supernatant was removed for ELISA, the cells were pelleted, and the levels of depletion markers Tim3, Lag-3, and PD-1 were measured by flow cytometry, as well as proliferation determined by CSFE dilution. To evaluate the proliferation capacity of IL-2Rβ-modified CAR constructs, CAR T cells were labeled with Cell Trace Violet (ThermoFisher) and co-cultured with wild-type Jurkat, B7-H3-expressing Jurkat, or antigen-free target cells in 48-well plates for 6 days at an effector-to-target ratio of 1:1. The cells were seeded in the absence of cytokines, 70 ng / mL IL-15 (PeproTech) or 100 IU / mL IL-2 (Proleukin, Novartis), and fresh target cells were supplemented on the 2nd and 4th days of co-culture. On the 6th day, cell proliferation and multiplication were evaluated by flow cytometry using absolute counting beads (BioLegend). In the 28-day co-culture assay, CAR T cells were co-cultured with irradiated LAN-1, Kelly or antigen-free target cells in 24-well plates with an effector-target ratio of 2: 1. Cell culture medium was replenished every 2 to 3 days. CAR T cells were attacked with irradiated target cells every 6 days, cultured for another 24 hours and analyzed. Cells were precipitated weekly, and the supernatant was removed for ELISA. CAR-T cell proliferation was measured by flow cytometry using absolute counting beads (BioLegend) every week.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). Cr was used. 51 Release cytotoxicity assay was used to test cytotoxicity. Target cells were incubated with Cr 51 Incubate for 1 hour, then wash and seed in 96-well plates. CAR T cells or untransduced cells were seeded 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, then the supernatant was removed and incubated overnight with scintillation fluid (Perkin Elmer) at room temperature. Cr released into the supernatant was measured using a 1450 MicroBeta TriLux (Perkin Elmer). 51 A plate-based assay was used to measure the activity of CAR T cells in response to decreasing concentrations of B7-H3 protein. 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 untransduced cells were added. The plates were incubated overnight at 37°C, the cells were pelleted, and the supernatant was removed for ELISA.

[0301] Hematopoietic colony assay

[0302] 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 to study hematopoietic stem cells. The assay is based on the ability of single hematopoietic progenitor cells, called colony-forming units (CFU), to proliferate and differentiate into colonies in response to cytokine stimulation in semisolid culture medium. The colonies formed can be counted and characterized based on their unique morphology. The assay was used to study colony formation of cord blood (CB) and NOMO-1 leukemia cells after treatment with TE9-CD8-28ζCAR T cells. Untransduced T cells were used as a control.

[0303] H4434 Classical Methocult medium (STEMCELL Technologies) was used as a semisolid matrix in this assay: this medium contains rh SCF (stem cell factor), rh GM-CSF (granulocyte macrophage colony-stimulating factor), rh IL-3, rh EPO, and allows the growth of CFU-E (erythroid progenitors), BFU-E (erythroid burst-forming units), CFU-GM (granulocyte and / or macrophage progenitors) and CFU-GEMM (multipotent progenitors) in CB as well as the growth of leukemic colonies.

[0304] Before the experiment, the Methocult medium was aliquoted into the Sterilin TM 7 ml Bijou (Thermo Scientific) and stored at -20 ° C. Briefly, effector cells (untransduced T cells and TE9-CD8-28ζCAR T) and target cells (CB and NOMO-1) were co-cultured in tissue culture-treated 48-well plates at 37 ° C for 18 hours at an effector:target ratio of 5:1.

[0305] After incubation, cells from each co-culture condition were collected separately and washed using Iscove's MDM medium (STEMCELL Technologies) containing 2% FBS, which is the recommended medium for preparing and washing samples for CFU determination. The cells were then resuspended in Iscove's medium at the calculated desired concentration, which was estimated to be 20,000 CB cells in 40 μl of cell suspension and 2,000 NOMO-1 cells in 40 μl of cell suspension without any lysis.

[0306] 40 μl of the cell suspension was then transferred to a 2 ml aliquot of Methocult and shaken vigorously to evenly distribute the cells in the Methocult. 1 ml of Methocult containing the desired cell number (10,000 CB and 1,000 NOMO-1) was then inoculated into a well of a 6-well tissue culture treated plate using a 16-gauge blunt-end needle (STEMCELL Technologies).

[0307] These seeding densities were chosen to make the NOMO-1 and CB conditions as comparable as possible, approximating that only 1% of the cells in the CB were hematopoietic progenitor colony-forming units. Other densities were also tested, but these either resulted in overcrowding of cells in the wells or did not generate enough colonies for accurate quantification (data not shown).

[0308] PBS was added around the plate to maintain a high humidity level and prevent the Methocult from drying out, and the plates were incubated for 14 days at 37° C. The number and morphology of the colonies were assessed microscopically on day 14, and then, to visualize the colonies on photographs, the colonies were stained dark purple with p-iodonitrotetrazolium violet (Sigma).

[0309] Antibody and flow cytometric analysis

[0310] The following antibodies were used in this study: 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), and anti-PD-1. Biosciences), anti-mouse CD45 (30-F11, BioLegend), anti-human CD45 (HI30, BioLegend), GhostRed TM 780 (Tonbo Biosciences), Zombie Yellow Viability Dye (BioLegend), Propidium Iodide (Gibco), Cell Trace Violet (ThermoFisher), Absolute Counting Beads (BioLegend).

[0311] Cross-reactivity of TC6, TE9, and BH6 intact antibodies

[0312] TC6, TF9, and BH6 were prepared as chimeric antibodies with a human IgG1 Fc domain. Antibodies were purified on a protein A column (Cytiva) and tested against plate-bound antigen by ELISA using goat anti-human IgG (H+L) (SeraCare) for detection. Cross-reactivity with mouse B7-H3 was tested by flow cytometry using the mouse cell line 3T3 / NA1.

[0313] In vivo LAN-1 neuroblastoma and Med8A medulloblastoma models

[0314] Animal protocols were approved by the local institutional research committee and conformed to UK Home Office guidelines. Male NSG mice aged 6 to 8 weeks were provided by UCL. All experiments were performed under UK Home Office license, project license number 15981 / 01 and personal license number 12972. For neuroblastoma LAN-1 experiments, Geltrex was used. TM1×10 in (Thermofisher) 6 LAN-1-BFP / Luc were injected subcutaneously into the flank of NSG mice. 6 CAR T cells were injected intravenously into the tail vein. Tumor size was monitored twice a week with a digital caliper. 200 μl of luciferin was administered to the back of the neck of the mice weekly and imaged using a PhotonIMAGERTM optical imaging system (BiospaceLab). When the tumors reached a threshold size, the mice were sacrificed and blood, spleen, and tumor samples were collected. Cells were dissociated using a cell strainer and residual red blood cells were removed using ACK lysis buffer (ThermoFisher). The cells were stained and markers were analyzed using flow cytometry. In the Med8A medulloblastoma experiment, 1 × 10 cells stably transduced with luciferase were injected into the mouse body. 6 med8A medulloblastoma cells were stereotaxically implanted into the hemisphere in a volume of 3 to 5 μl. 48 hours later, 5 × 10 6 CAR-T cells or non-transduced controls were injected into the lateral ventricle. Tumor growth was assessed by bioluminescence imaging.

[0315] γδT cell expansion

[0316] PBMCs were isolated from purchased whole blood leukocyte cones by density gradient centrifugation using Lymphoprep (Stemcell) according to the manufacturer's instructions. PBMCs were either cryopreserved in 90% FBS, 10% DMSO or resuspended in complete T cell culture medium for further processing. Complete T cell culture medium consisted of xeno-free and serum-free CTS-OpTmizer (ThermoFisher) containing 10% synthetic serum replacement (Thermo Fisher) and GlutaMAX (Thermo Fisher), all of which were provided in research and GMP grades. The Thermo Fisher catalog numbers for these products are as follows: research grade CTS-OpTmizer (A1048501) and GMP-compliant alternative GMP grade OpTmizer-CTS (A3705003), synthetic immune cell serum replacement (A2596101) that meets both production standards, and GlutaMAX (35050061) that also meets both standards. If starting with cryopreserved material, thaw the PBMCs and culture at 10 × 10 6 The cells were then allowed to rest overnight in pre-warmed complete medium to avoid excessive stress on the lymphocytes and improve the quality of depletion. They were then immediately stimulated at a density of 2 × 10 cells / mL in standard cell culture plates. 6 cells / mL to 4×106 PBMCs were either cultured at a concentration of 10 cells / mL or first depleted of αβ T cells using the TCRα / β product line (Miltenyi Biotec) according to the manufacturer's instructions, and CD56-positive cells were simultaneously depleted 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 MACS cell separation LD columns (Miltenyi Biotec). If cultured in G-Rex tubes (Wilson Wolf), cells were cultured at a concentration of 2×10 6 cells / cm 2 to 4×10 6 cells / cm 2 Depleted PBMCs were seeded. Thus, prepared PBMCs 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 and a subsequent second culture in 70 ng / mL were stimulated with 1 μg / mL OKT-3 (Miltenyi Biotec, catalog number 130-093-387, RRID: AB_1036144) or with 1 μg / mL PHA (Merck) and various cytokine combinations, including: (i) 100 IU / mL IL-2 aldesleukin (Proleukin; Novartis); (ii) 70 ng / mL IL-15 (Peprotech); (iii) 20 ng / mL rhIL-7 (Peprotech); or (iv) a “DOT protocol” cytokine cocktail. rIL-15 and 30ng / mL IFN-γ (all cytokines were from Peprotech). When comparing the complete "DOT protocol" and the test expansion protocol, the method described by Almeida et al. (2016) was used, but the step of positive selection with OKT-3 after αβTCR depletion was omitted. Briefly, the first cytokine culture of depleted PBMCs was stimulated with 70ng / mL OKT-3, and the second cytokine culture was stimulated with 1μg / mL OKT-3. Viable cells were counted before and during expansion using trypan blue exclusion, an automated cell counter (Invitrogen), and flow cytometry-based absolute counting beads (Biolegend).

[0317] PBMCs were depleted of Vδ2γδT cells at one of three expansion stages (pre-initiation, mid-cycle split, or harvest). Anti-TCR / Vδ2 mAb clone B6 (BioLegend, catalog number 331404, RRID: AB_1089228) was used at 0.5 μg / 10 6 All depletions were performed at a concentration of 100 PBMC. When depletion was performed at the start, the Vδ2 cell start was incorporated into the αβTCR / CD56 depletion process. This was done as follows: PBMC were incubated with αβTCR-biotin mAb and Vδ2 (clone: ​​B6)-biotin mAb, washed, and then incubated with anti-biotin and anti-CD56 microbeads according to the manufacturer's protocol. After washing again, depletion was performed using Miltenyi LD magnetic columns as described above and according to the manufacturer's protocol. If depletion was performed at mid-term split or final harvest, the expanded cells were harvested, washed, and treated with 0.5 μg of clone B6 / 10 6 PBMCs were labeled and incubated for 20 minutes, washed, and then incubated and depleted using Miltenyi anti-biotin microbeads and LD columns.

[0318] Viral transduction of γδ T cells using CAR-T

[0319] 293T cells (ATCC catalog number CRL-3216, RRID: CVCL_0063) were plated at 10 cm 2 Plate (Corning) 1.5×10 6 Cells were seeded in 10 mL of Gibco IMDM (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS). At 70% confluency, 293T cells were transfected using GeneJuice (Merck) according to the manufacturer's protocol. Triple plasmid transient transfection was performed using the SFG-γ retroviral vector (RRID: Addgene_22493), including equimolar ratios of B7H3-CAR, gag+pol (RRID: Addgene_8449) and RD114 envelope (RRID: Addgene_17576) plasmids. Retroviral supernatants were harvested 48 hours and 72 hours after transfection and immediately used for T cell transduction. Briefly, non-tissue culture treated 24-well plates (Costar) were coated with RetroNectin (Takara) dissolved in PBS (final concentration of 1 mg / mL) and incubated at 4°C for 24 hours. Remove the Retronectin and add 1.5 mL of retroviral supernatant to each Retronectin-coated well. Then, add 3 × 10 5The plate was centrifuged at 1000 x g for 40 minutes at room temperature and then incubated at 37°C in complete T cell culture medium supplemented with IL-15 to a final concentration of 70 ng / mL (approximately 140 IU / mL). Three days later, the transduced T cells were harvested, washed, and resuspended in complete T cell culture medium supplemented with specific cytokines for expansion. Transduction efficiency was assessed by flow cytometry detection of the CD34 marker gene.

[0320] Statistical analysis

[0321] All statistical analyses were performed in GraphPad Prism v8. Unless otherwise stated, data are presented as mean ± range. 51 Cytotoxicity assays were analyzed using two-way ANOVA. Statistical analysis for in vitro assays was performed using one-way ANOVA with Tukey's multiple comparisons. 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.

[0322] Example 2 - Development of Novel Anti-B7-H3 Antibodies in Single-Chain Format

[0323] Targeting of the B7-H3 cancer antigen with T cells engineered to express CARs has shown great promise in preclinical models and is being translated into clinical studies. To date, most studies have used repurposed antibodies, where the scFv was derived from an existing monoclonal antibody. To generate novel B7-H3 binders that could potentially be more finely tuned for CAR-T applications, mice were immunized with recombinant B7H3-Fc fusion protein, and spleen RNA from the immunized mice was used as a substrate for the generation of ScFv libraries in phage ( Figure 1 A). Single scFvs were isolated from a phage library by panning with human B7-H3 and screened as CAR-T binding elements by direct cloning into a CAR-T format for empirical comparison of CAR-T effector function ( Figure 1 B). 17 binding agents were identified by ELISA screening ( Figure 2 Based on ELISA and genetic heterogeneity of the clones, ten scFvs were selected for production in the scFv-Fc format. Based on the strength and specificity of binding to B7-H3 isoforms (isoform 1, isoform 2, or an artificially truncated isoform T-B7-H3 that has been used as an immunogen), five of the ten scFv-Fc fusion proteins (TE9, TC6, BH6, TF9, and BF9) were selected for further evaluation in the CAR-T format by flow cytometry ( Figure 2 Four of the anti-B7-H3 binders (TE9, TC6, TF9, and BF9) bound to both human isoforms of B7-H3, but BH6 showed specificity for 4Ig-B7-H3 ( Figure 2 D). Five other binders (TB8, BG4, BD9, BC10, BB5) showed weaker overall binding and lacked reactivity against the physiological target isoforms 4Ig B7H3 or 2Ig B7H3 ( Figure 3 All binders from the library selected by the original ELISA were sequenced and evaluated for their degree of sequence similarity, revealing a high degree of diversity among the selected binders ( Figure 4 ). The binders TE9, TC6, and BH6 were generated in whole antibody format. In ELISA assays, these antibodies showed specific binding to B7-H3 but not to other members of the human B7 family. BH6 bound to both human B7-H3 and mouse B7-H3, but TE9 and TC6 were specific for humans and cynomolgus monkeys ( Figure 5 ) and showed similar antigenic specificity to neuroblastoma and synthetic cell lines as a commercially available anti-B7-H3 monoclonal antibody ( Figure 6 ).

[0324] Example 3 - B7-H3 binders in a CAR-T format exhibit a range of antigen-specific effector functions The ability of five candidate scFv sequences to confer antigen-specific T cell functions in a second-generation CD8H / Tm-CD28-CD3ζ (28ζ) CAR format, comprising the CD8-α hinge and transmembrane (H / Tm) sequences, with CD28 and CD3ζ signaling domains ( Figure 7 The effector function of CAR was evaluated by culturing with neuroblastoma cells that naturally express B7-H3 ( Figure 7 C). All five CAR-T constructs showed similar transduction efficiency in human T cells (data not shown). In a four-hour killing assay, two binders (TE9, TC6) showed significant cytotoxicity specific for target cells expressing B7-H3 ( Figure 7 B), and both binders also showed the greatest cytokine response to the neuroblastoma target. However, in the 24-hour assay, binder BH6 showed lower production of B7-H3-specific cytokines ( Figure 7 D).

[0325] To determine the performance of the CAR during long-term co-culture, the CAR in CAR-T format was evaluated in a repeated antigen challenge assay during which the CAR T cells were stimulated with irradiated tumor cells four times over a four-week period. Three of the anti-B7-H3 binders were compared with the FMC63 anti-CD19 CAR-T construct. These experiments demonstrated that TE9-28ζ and CD19-28ζ CAR-T cells maintained a sustained IL-2 production capacity in response to a fourth re-challenge with B7-H3-positive leukemia cells ( Figure 7 E). Therefore, TE9 binders were selected for further optimization of CAR-T function.

[0326] Example 4 - CD28 co-stimulation and CD8 hinge / transmembrane provide optimal long-term persistence for TE9 CAR-T cells

[0327] We next compared the CD28 and 4-1BB intracellular domains with the CD8 hinge and transmembrane (H / Tm) domains by assaying their effector functions against B7-H3-expressing neuroblastoma cells. Figure 8 A). Similar levels of transduction efficiency were observed with both constructs ( Figure 8 B) Cytotoxic degranulation as measured by CD107a and upregulation of activation markers CD25 and CD69 after target addition were slightly higher in the CD28ζ construct than in the 4-1BBζ construct, but the differences were not significant ( Figure 8 C). In short-term co-culture, TE9-28ζ produced more interferon-γ (IFN-γ) and significa...

Claims

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, wherein the heavy chain variable domain comprises a heavy chain complementary determining region (HCDR) 1, HCDR2 and HCDR3, and the light chain variable domain comprises a light chain complementary determining region (LCDR) 1, LCDR2 and LCDR3, wherein the antigen binding molecule comprises the following complementary determining region (CDR) sequences: (a) the heavy chain variable domain sequence of SEQ ID NO: 66 and the light chain variable domain sequence of SEQ ID NO: 74; or (b) the heavy chain variable domain sequence of SEQ ID NO: 2 and the light chain variable domain sequence of SEQ ID NO: 10; or (c) the heavy chain variable domain sequence of SEQ ID NO: 18 and the light chain variable domain sequence of SEQ ID NO: 26; or (d) the heavy chain variable domain sequence of SEQ ID NO: 34 and the 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) 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) 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) 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) 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) 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) the heavy chain variable domain sequence of SEQ ID NO:258 and the light chain variable domain sequence of SEQ ID NO:

266.

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, wherein the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, and the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3, and wherein: (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) 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; or (h) 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; or (i) 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; or (j) 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; or (k) 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; or (l) 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; or (m) 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; or (n) 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; or (o) 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; or (p) 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; or (q) 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.

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) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 66, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 74, or a sequence with at least 90% identity thereof; or (b) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 2, or a sequence that is at least 90% identical thereto, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 10, or a sequence that is at least 90% identical thereto; or (c) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 18, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 26, or a sequence with at least 90% identity thereof; or (d) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:34, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO:42, or a sequence with at least 90% identity thereof; or (e) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 50, or a sequence that is at least 90% identical thereto, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 58, or a sequence that is at least 90% identical thereto; or (f) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 82, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 90, or a sequence with at least 90% identity thereof; or (g) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 98, or a sequence with at least 90% identity thereto, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 106, or a sequence with at least 90% identity thereto; or (h) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 114, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 122, or a sequence with at least 90% identity thereof; or (i) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 130, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 138, or a sequence with at least 90% identity thereof; or (j) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 146, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 154, or a sequence with at least 90% identity thereof; or (k) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 162, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 170, or a sequence with at least 90% identity thereof; or (l) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 178, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 186, or a sequence with at least 90% identity thereof; or (m) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 194, or a sequence with at least 90% identity thereto, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 202, or a sequence with at least 90% identity thereto; or (n) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 210, or a sequence with at least 90% identity thereto, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 218, or a sequence with at least 90% identity thereto; or (o) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 226, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 234, or a sequence with at least 90% identity thereof; or (p) wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 242, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises a sequence of SEQ ID NO: 250, or a sequence with at least 90% identity thereof; or (q) wherein the heavy chain variable domain comprises the sequence of SEQ ID NO: 258, or a sequence with at least 90% identity thereof, and wherein the light chain variable domain comprises the sequence of SEQ ID NO: 266, or a sequence with at least 90% identity thereof.

4. The antigen binding molecule according to claim 1 or 3, wherein the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, and the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3, and wherein: (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) 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; or (h) 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; or (i) 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; or (j) 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; or (k) 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; or (l) 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; or (m) 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; or (n) 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; or (o) 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; or (p) 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; or (q) 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.

5. The antigen binding molecule according to any one of the preceding claims, wherein the antigen binding molecule specifically binds to human B7H3. The antigen binding molecule according to claim 5 , wherein the antigen binding molecule specifically binds to the human B7H3 isoform 4IgB7-H3 or 2IgB7-H3.

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

8. An antigen binding molecule according to any one of the preceding claims, wherein: (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, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain Fab (scFab) fragment, a single-chain Fv protein (scFv), a tandem scFv protein, a disulfide-stabilized Fv protein (dsFv) or a scFv-Fc protein.

9. The antigen binding molecule of claim 8(iii), wherein the antigen binding molecule is a scFv, optionally wherein the scFv further comprises a linker having a sequence of SEQ ID NO: 273, wherein the heavy chain variable domain is connected to the light chain variable domain via the linker, and wherein: (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 comprises the sequence of SEQ ID NO: 18, and the light chain variable domain 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 comprises the sequence of SEQ ID NO: 82, and the light chain variable domain 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 comprises the sequence of SEQ ID NO: 130, and the light chain variable domain 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 comprises the sequence of SEQ ID NO: 178, and the light chain variable domain 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 comprises the sequence of SEQ ID NO: 226, and the light chain variable domain 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.

10. The antigen binding molecule according to any one of the preceding claims, wherein the antigen binding molecule is a multispecific molecule, optionally wherein the antigen binding molecule is a bispecific or trispecific molecule.

11. An antigen binding molecule according to claim 10, wherein the antigen binding molecule comprises a first binding domain that specifically binds to B7H3 and an additional binding domain that specifically binds to a second antigen, optionally wherein the additional binding domain specifically binds to CD3 on the surface of a T cell. The antigen binding molecule according to claim 11 , wherein the antigen binding molecule comprises two scFvs.

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

14. A chimeric antigen receptor (CAR) or a chimeric co-stimulatory receptor (CCR), said CAR or CCR comprising an antigen binding molecule according to any one of the preceding claims that specifically binds to B7H3.

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

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

17. A cell comprising a CAR or CCR according to claim 15 or 16, wherein the cell is a T cell, optionally wherein the T cell is an α-β T cell or a γ-δ T cell.

18. A nucleic acid molecule comprising a nucleotide sequence encoding: (i) an antigen binding molecule according to any one of claims 1 to 13 or a CAR or CCR according to any one of claims 14 to 16, or (ii) a heavy chain variable domain or a 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), the 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 anticancer agent, a cytotoxic agent, a cytostatic agent, 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. The antigen binding molecule according to any one of claims 1 to 13, the CAR or CCR according to any one of claims 14 to 16, the cell according to claim 17, or the ADC according to claim 20 or 21, or the pharmaceutical composition according to claim 22 for use in a method for treating cancer.

24. An antigen binding molecule for use according to claim 23, wherein the cancer is selected from a solid tumor, 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, renal cancer or pancreatic cancer or oral squamous cell carcinoma (SCC).

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

Images