Cross-species b7-h3-binding molecules and uses thereof

Abstract

The present disclosure relates generally to the development of immuno-reagents, including antigen-binding molecules and chimeric antigen receptors (CARs) that have cross-species binding affinity for use in diagnosing and treating cancers. Some embodiments of the disclosure provide recombinant nucleic acids and recombinant cells, e.g., T cells that have been engineered to express an antigen-binding molecule and / or a CAR as disclosed herein. Further provided are methods for generating a population of recombinant cells, e.g., engineered T cells with improved therapeutic properties, pharmaceutical compositions containing such a population of recombinant cells, and methods and kits for the diagnosis, prevention, and / or treatment of a health condition in subjects in need thereof.

Description

Attorney Docket No.: 078430-546001 WOCROSS-SPECIES B7-H3-BINDING MOLECULES AND USES THEREOFSTATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with Government support under contract R35 CA263500-04 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 761,773; filed on February 21, 2025; and U.S. Provisional Patent Application Serial No. 63 / 792,092; filed on April 21, 2025. The disclosures of the above-referenced applications are herein expressly incorporated by reference it their entireties, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing XML file, named “078430-54600 IWO Sequence Listing_ST26.xml,” was created on February 18, 2026, and is 44,741 bytes in size.SUMMARY

[0004] The present disclosure relates generally to, inter alia, novel compositions and methods for the prevention and / or treatment of various health conditions. In particular, some embodiments of the disclosure relate to the development of immuno-reagents, including antigenbinding molecules and chimeric antigen receptors (CARs) that have a binding affinity for both human B7-H3 (hB7-H3) and a murine B7-H3 (mB7-H3) (e.g, cross-species binding affinity) for use in diagnosing and treating cancers that express B7-H3. Some embodiments of the disclosure provide recombinant nucleic acids encoding an antigen-binding molecule and / or a chimeric antigen receptor (CAR) as disclosed herein. Also provided, in some embodiments, are recombinant cells, e.g., T cells that have been engineered to express an antigen-binding molecule and / or a CAR as disclosed herein. Further provided are methods for generating a population of recombinant cells, e.g., engineered T cells with improved therapeutic properties for adoptive T-cell therapy, and pharmaceutical compositions containing such a population of engineered TAttorney Docket No.: 078430-546001 WOcells with enhanced therapeutic properties, as well as methods and kits for the diagnosis, prevention, and / or treatment of a health condition in subjects in need thereof.

[0005] In one aspect of the disclosure, provided herein are antigen-binding molecules having a binding affinity to a human B7-H3 (hB7-H3) and a murine B7-H3 (mB7-H3), the antigenbinding molecule including: (a) a variable light chain (VL) domain including complementarity determining regions LCDR1, LCDR2, and LCDR3 including the amino acid sequences of SEQ ID NO: 21, 22, and 23, respectively; and (b) a variable heavy chain (VH) domain including complementarity determining regions HCDR1, HCDR2, and HCDR3 that include the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively; and further including one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. As described in greater detail below, for the purpose of this application, all amino acid numbering is based on the full-length sequence of the 376.96 scFv (SEQ ID NO: 11).Accordingly, residues A173, Y180, and T181 in the full-length sequence of SEQ ID NO: 11 correspond to residues A50, Y57, and T58 of SEQ ID NO: 2.

[0006] Non-limiting exemplary embodiments of the antigen-binding molecules of the disclosure can include one or more of the following features. In some embodiments, the VL domain includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VH domain includes the amino acid sequence of SEQ ID NO: 2, and further include one or more amino acid substitutions at a position selected from the group consisting of Al 73, Y180, and T 181. In some embodiments, the B7-H3 binding molecules of the disclosure include (a) a VL domain including the amino acid sequence of SEQ ID NO: 1; and (b) a VH domain including the amino acid sequence of SEQ ID NO: 2, and further include one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181. In some embodiments, the antigen-binding molecule is or includes a full-length antibody. In some embodiments, the antigen-binding molecule is or includes an antigen-binding fragment. In some embodiments, the antigen-binding fragment is a single-chain antibody fragment (scFv), a F(ab), a F(ab'), a Fab'-SH, a F(ab')2, or a Fv fragment. In some embodiments, the antigen-binding fragment is or includes a scFv. In some embodiments, the scFv is or includes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-16. In some embodiments, the VL and VH domains of theAttorney Docket No.: 078430-546001 WOscFv are operably linked to one another via a polypeptide linker. In some embodiments, the polypeptide linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0007] In some embodiments of the disclosure, the substitution at position Al 73 of the VH domain is an Alanine-to-Threonine (A173T) substitution. In some embodiments, the substitution at position Y50 is a Tyrosine -to-Serine (Y180S) substitution. In some embodiments, the substitution at position T181 is a Threonine-to-Isoleucine (T181I) substitution. In some embodiments, the antigen-binding molecule includes the following substitutions: A173T and Y180S. In some embodiments, the antigen-binding molecule includes the following substitutions: A173T and T181I. In some embodiments, the antigen-binding molecule includes the following substitutions: A173T, Y180S, and T181I.

[0008] In some embodiments, the antigen-binding molecule binds or is capable of binding mB7-H3 with an equilibrium dissociation constant (KD) value lower than the KD of the monoclonal antibody 376.96. In some embodiments, the antigen-binding molecule binds or is capable of binding mB7-H3 with a KD value of less than 2.34 pM. In some embodiments, the antigen-binding molecule binds or is capable of binding mB7-H3 with a KD value of less than 8.2 nM, less than 1.3 nM, less than 840 pM, less than 810 pM, or less than 190 pM. In some embodiments, the antigen-binding molecule binds or is capable of binding hB7-H3 with a KD value of less than 180 pM, less than 150 pM, or less than 110 pM.

[0009] In one aspect of the disclosure, provided herein are chimeric antigen receptors (CARs) including an antigen-binding moiety derived from an antigen-binding molecule as disclosed herein, or an antigen-binding fragment thereof.

[0010] Non-limiting exemplary embodiments of the CARs of the disclosure can include one or more of the following features. In some embodiments, the antigen-binding moiety includes a scFv including: (a) a variable light chain (VL) domain including the amino acid sequence of SEQ ID NO: 1; and (b) a variable heavy chain (VH) domain including the amino acid sequence of SEQ ID NO: 2, and further including one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CARs disclosed herein further include one or more of the following domains: (a) a signal peptide; (b) polypeptide linker inserted between the VH and VL domains; (a) aAttorney Docket No.: 078430-546001 WOtransmembrane domain (TMD); (d) a hinge domain; (e) a costimulatory domain; and (e) a CD32 chain. In some embodiments, the signal peptide is from a human GM-CSF receptor (GMCSR), CD45, CD8ct, Ig-Kappa, or IL-2. In some embodiments, the signal peptide is from GM-CSF receptor ct (GMCSR2).

[0011] In some embodiments, the TMD of the CARs disclosed herein is derived from a protein selected from a T-cell receptor (TCR) alpha chain, a TCR beta chain, a TCR zeta chain, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD27 (TNFRSF19), CD28, CD33, CD45, CD80, CD83, CD86, CD134, CD137, CD152 (CTLA4), CD154, CD279, and PD-1. In some embodiments, the TMD is a CD8 TMD or a CD28 TMD. In some embodiments, the TMD is a CD8 TMD. In some embodiments, the TMD is a CD28 TMD.

[0012] In some embodiments, the hinge domain of the CARs disclosed herein is derived from a protein selected from the group consisting of LFA-1 (CD1 la / CD18), LFA-2 (CD2), CD4, CD5, CD8, CD27 (TNFRSF7), CD28, CD70, 4-1BB, 0X40 (CD134), CD152 (CTLA4), ICOS (CD278), IgGl Fc region, and IgG4 Fc region. In some embodiments, the hinge domain is derived from CD8 or CD28. In some embodiments, the hinge domain is derived from CD8. In some embodiments, the hinge domain is derived from CD28.

[0013] In some embodiments, the costimulatory domain of the CARs disclosed herein is derived from a protein selected from the group consisting of 4- IBB (CD 137), CD27 (TNFRSF7), CD28, 0X40 (CD134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CD1 la / CD18), DAP10, DAP12, and a co-stimulatory inducible T-cell costimulatory (ICOS) protein. In some embodiments, the costimulatory domain is a CD28 costimulatory domain or a 4-1BB costimulatory domain. In some embodiments, the costimulatory domain is a CD28 costimulatory domain. In some embodiments, the costimulatory domain is a 4- IBB costimulatory domain.

[0014] In some embodiments of the disclosure, the VL and VH domains of the scFv are operably linked to one another via a polypeptide linker. In some embodiments, the polypeptide linker includes or consists of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the scFv is operably linked to the hinge domain via a polypeptide connector. In some embodiments, the polypeptide connector includes or consists of the amino acid sequence Ala-Ala- Ala (AAA).Attorney Docket No.: 078430-546001 WO

[0015] In some embodiments, the substitution at position A 173 is an Alanine -to-Threonine (A173T) substitution. In some embodiments, the substitution at position Y50 is a Tyrosine-to-Serine (Y180S) substitution. In some embodiments, the substitution at position T181 is an Threonine -to-Isoleucine (T181I) substitution. In some embodiments, the antigen-binding molecule includes the A173T / Y180S substitutions. In some embodiments, the antigen-binding molecule includes the A173T / T181I substitutions. In some embodiments, the antigen-binding molecule includes the A173T / Y180S / T181I substitutions.

[0016] In some embodiments, the CARs of the disclosure include, in N-terminus to C-terminus direction: (a) GMCSFR signal peptide; (b) an antigen-binding moiety derived from an antigenbinding molecule as disclosed herein, or an antigen-binding fragment thereof; (c) a CD8 hinge domain; (d) a CD8 TMD; (e) a 4-1BB costimulatory domain; and (I) a CD3^ chain.

[0017] In some embodiments, the CARs of the disclosure include, in N-terminus to C-terminus direction: (a) GMCSFR signal peptide; (b) an antigen-binding moiety derived from an antigenbinding molecule, as disclosed herein, or an antigen-binding fragment thereof; (c) a CD28 hinge domain; (d) a CD28 TMD; (e) a CD28 costimulatory domain; and (I) a CD3^ chain.

[0018] In some embodiments, the CAR disclosed herein binds or is capable of binding mB7-H3 with a KD value lower than the KD of the monoclonal antibody 376.96, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9. In some embodiments, the CAR disclosed herein binds or is capable of binding mB7-H3 with a KD value lower than the KD of the monoclonal antibody 376.96, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CAR disclosed herein includes an antigen-binding molecule capable of binding mB7-H3 with a KD value lower than the KD of the monoclonal antibody 376.96, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0019] In some embodiments, the CAR disclosed herein includes an antigen-binding molecule capable of binding mB7-H3 with a KD value of less than 2.34 pM. In some embodiments, the CAR disclosed herein includes an antigen-binding molecule capable of binding mB7-H3 with aAttorney Docket No.: 078430-546001 WOKD value of less than 8.2 nM, less than 1.3 nM, less than 840 pM, less than 810 pM, or less than 190 pM. In some embodiments, the CAR disclosed herein includes an antigen-binding molecule capable of binding hB7-H3 with a KD value of less than 180 pM, less than 150 pM, or less than HO pM.

[0020] In another aspect, provided herein are recombinant nucleic acid molecules including a nucleic acid sequence encoding: (a) an antigen-binding molecule as disclosed herein; and / or (b) a chimeric antigen receptor (CAR) as disclosed herein. Non-limiting exemplary embodiments of the recombinant nucleic acid molecules of the disclosure can include one or more of the following features. In some embodiments, the recombinant nucleic acid molecule is operably linked to one or more heterologous nucleic acid sequences. In some embodiments, the one or more heterologous nucleic acid sequences includes a promoter sequence. In some embodiments, the recombinant nucleic acid molecule is incorporated into an expression cassette or a vector.

[0021] In yet another aspect, provided herein are recombinant cells including: (a) an antigenbinding molecule as disclosed herein; (b) a CAR as disclosed herein; and / or (c) a recombinant nucleic acid as disclosed herein. Non-limiting exemplary embodiments of the recombinant cell of the disclosure can include one or more of the following features. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a non-human primate cell. In some embodiments, the mammalian cell is a T lymphocyte. In some embodiments, the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell. In some embodiments, the T cell was obtained leukapheresis of a sample obtained from a subject.

[0022] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.Attorney Docket No.: 078430-546001 WO

[0023] In yet another aspect, provided herein are pharmaceutical compositions including a pharmaceutically acceptable excipient and one or more of the following: (a) an antigen-binding molecule as disclosed herein; (b) a CAR as disclosed herein; (c) a recombinant nucleic acid as disclosed herein; and / or (d) a recombinant T cell as disclosed herein. In some embodiments, the composition includes a recombinant T cell as disclosed herein, and a pharmaceutically acceptable carrier.

[0024] In another aspect, provided herein are methods for diagnosing, preventing, and / or treating a health condition in a subject in need thereof, including administering to the subject a composition including one or more of the following: (a) an antigen-binding molecule as disclosed herein; (b) a CAR as disclosed herein; (c) a recombinant nucleic acid as disclosed herein; (d) a recombinant T cell as disclosed herein; and / or (e) a pharmaceutical composition as disclosed herein.

[0025] Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the recombinant cells are allogeneic relative to the subject. In some embodiments, the recombinant cells are autologous relative to the subject. In some embodiments, the health condition is a proliferative disorder. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer is positive for B7-H3 expression. In some embodiments, the B7-H3 -positive cancer is selected from the group consisting of nervous system cancer, cervical cancer, sarcoma, neuroblastoma, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, and hepatocellular carcinoma. In some embodiments, the B7-H3-positive cancer is an adult malignancy or a pediatric cancer. In some embodiments, the pediatric cancer is osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, atypical teratoid rhabdoid tumor, medulloblastoma, or neuroblastoma. In some embodiments, the T cells are obtained from tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs).

[0026] In some embodiments, the administered composition confers an enhanced effector function of the recombinant T cells. In some embodiments, the enhanced effector function of the recombinant T cells is selected from the group consisting of growth rate (proliferation), cytokine production, target cell inhibition (e.g., anti-cancer cytotoxicity), macrophage activation, maintenance of enhanced effector cell function, NK cell activation, exhaustion resistance, and inAttorney Docket No.: 078430-546001 WOvivo persistence (e.g., survival). In some embodiments, the enhanced effector function includes increased production of one or more cytokines. In some embodiments, the one or more cytokines includes interferon gamma (IFNy), tumor-necrosis factor a (TNFa), and / or interleukin-2 (IL-2). In some embodiments, the composition is administered to the subject individually (monotherapy) or in combination with a second therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, or surgery.

[0027] In yet another aspect, provided herein are kits for the diagnosis, prevention, and / or treatment of a health condition in a subject in need thereof, the kit including one or more of the following: (a) an antigen-binding molecule as disclosed herein; (b) a CAR as disclosed herein; (c) a recombinant nucleic acid as disclosed herein; (d) a recombinant T cell as disclosed herein; and / or (e) a pharmaceutical composition as disclosed herein.

[0028] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGS. 1A-1B schematically summarize the results of experiments performed to express 376.96 scFv using yeast surface display. In these experiments, yeast display was carried out with standard pCTCON2 vector. An FXa site was incorporated between Aga2p and HA. The light chain variable (VL) domain, heavy chain variable (VH) domain, and (G4S linker are shown. VL domain was in N-terminal orientation. Gly-Ser linkers were incorporated between VH and VL domains. A C-terminal c-myc tag was used for protein expression detection.

[0030] FIGS.2A-2C schematically summarize the results of experiments performed to illustrate that yeast surface displayed 376.96 scFv binding to human and mouse B7-H3. In these experiments, Macrogenics was a different B7-H3 targeting scFv that is known to only target human B7-H3.

[0031] FIG. 3 schematically summarizes the sorting strategy for 376.96 scFv library. In these experiments, the library was generated using Agilent GeneMorphll Kit. Each 50pl reaction contained 5 pl of Mutazyme II Buffer, 1 pl of 40mM dNTP mix, 125ng each ofAttorney Docket No.: 078430-546001 WOoJLS244 / oJLS245, 50ng / 100ng / 250ng 376.96 pCT, and 5pl of Mutazyme II Buffer. In average, 3.2 nucleotide changes / kb and 2.3 amino acid changes / kb were observed.

[0032] FIGS.4A-4B schematically summarize the sorting of library of 376.96 scFv variants for higher affinity to mouse B7-H3.

[0033] FIGS. 5A-5B depict the consensus mutations identified in complementary determining regions (CDRs) after Round 3 of sorting. The sequences of the CDRs of heavy chain variable region HCDR1, HCDR2, and HCDR3 are provided in the Sequence Listing.

[0034] FIGS. 6A-6B depict the consensus mutations identified in CDR regions after Round 4 of sorting.

[0035] FIGS. 7A-7C summarize the results of experiments performed to evaluate the binding affinity of yeast displayed scFv variants to murine B7-H3.

[0036] FIGS. 8A-8C summarize the results of experiments performed to evaluate the binding affinity of yeast displayed scFv variants to human B7-H3.

[0037] FIGS.9A-9B summarize the results of experiments performed to evaluate the binding affinity of yeast displayed scFv variants to a non-target, isotype control.

[0038] FIGS. 10A-10C summarize the results of experiments illustrating the expression of engineered scFv variants as full length 4-lBBz CARs on primary human T cells.

[0039] FIGS. 11A-11B summarize the results of experiments performed to evaluate the binding affinity for murine B7-H3 of engineered CAR variants expressed on human T cells .

[0040] FIGS. 12A-12B summarize the results of experiments to investigate the expression of activation / exhaustion markers on CAR T variants with or without mB7-H3+tumor stimulation. In these experiments, B7-H3.BBz CAR T cell variants were incubated with or without mB7-H3-high mouse group 3 medulloblastoma tumorspheres (mouse G3MB) at a 1:1 ratio overnight. T cell expression of activation / exhaustion markers (e.g., CD25, CD69, CD39, LAG3, and / or PD1) was determined at baseline and after exposure to tumor cells.

[0041] FIG. 13 depicts Incucyte images of killing of GFP+ / mB7-H3hlmouse group 3 medulloblastoma tumorspheres (mouse G3MB) by CAR T variants at various effector : target (E : T) ratios.

[0042] FIGS. 14A-14C depict Incucyte killing of GFP+ / mB7-H3111mouse group 3 medulloblastoma tumorspheres (mouse G3MB) by CAR T variants at E:T ratios of 2:1, 1:4, andAttorney Docket No.: 078430-546001 WO

[0043] FIG. 15 summarizes the results of experiments illustrating the expression of scFv variants as full length 4-lBBz (BB) or CD28z (28) CARs on primary human T cells.

[0044] FIGS. 16A-16B summarize the results of experiments to investigate the expression of activation / exhaustion markers on CAR T variants with or without hB7-H3+Nalm6 tumor stimulation.

[0045] FIGS. 17A-17C depict Incucyte killing of GFP+ / hB7-H3+Nalm6 tumor cells by CAR T variants at E:T ratios of 1:2, 1:4, and 1:8.

[0046] FIG. 18 summarizes the results of experiments illustrating the expression ofY180S variant (mut) as full length mCD28z CARs on murine T cells.

[0047] FIGS. 19A-19B summarize the results of experiments to investigate the expression of activation / exhaustion markers on CAR T variants expressed on mouse T cells at rest.

[0048] FIGS.20A-20B depict fL-2 cytokine production after coculture of CAR variants with murine osteosarcoma cells with varying expression of mB7-H3 (F331 : mB7-H3hl; F420: mB7-H3mid; K7M2: mB7-H310). In these experiments, it was observed that engineered B7-H3 murine CAR-T cells (MUT) produced IL-2 in response to mouse osteosarcoma tumor cells while parent mouse B7-H3 CAR-T did not produce IL-2 after co-culture with osteosarcoma cells lines.

[0049] FIGS.21A-21B depict IFN-gamma (IFNy) cytokine production after coculture of CAR variants with murine osteosarcoma cells with varying expression of mB7-H3 (F331 : mB7-H3hl; F420: mB7-H3mid; K7M2: mB7-H310). In these experiments, it was observed that engineered B7-H3 murine CAR-T (MUT) produced IFNy in response to mouse osteosarcoma cells and correlated with antigen density. Parental B7-H3 CAR-T produced significative lower or no IFNy in response to same cell lines.

[0050] FIGS.22A-22C depict Incucyte killing of murine GFP+ osteosarcoma tumor cells (F331, F420 and K7M2) by mB7-H3 CAR variants. In these experiments, it was observed that engineered B7-H3 murine CAR-T (MUT) showed improved killing compared with original CAR versus mouse osteosarcoma cell lines.

[0051] FIGS.23A-23C summarize the results of experiments to evaluate CAR T cell persistence in vivo in F331 osteosarcoma tumor bearing mice. In these experiments, it was observed that infused mouse T cells (mCD45.1+) transduced with engineered B7-H3 CAR-T (MUT) persist better than wildtype.Attorney Docket No.: 078430-546001 WO

[0052] FIGS.24A-24B summarize the results of experiments to evaluate CAR T cell antitumor efficacy in F331 osteosarcoma tumor bearing mice. In these experiments, it was observed that engineered B7-H3 mouse CAR-T cell (mut) delayed tumor growth in comparison with wild-type mouse CAR-T cells (wt).

[0053] FIGS.25A-25B summarize the results of experiments illustrating that no toxicity was evident after a high dose of engineered B7-H3 CAR T (MUT) in the F331 osteosarcoma model. In these experiments, it was observed that engineered B7-H3 CAR-T cells showed no toxicity after administered 10 million CAR T cells intravenously in the F331 osteosarcoma mouse model.

[0054] FIG. 26 summarizes the results of experiments to evaluate CAR T cell antitumor efficacy in medulloblastoma (mouse G3MB overexpressing mB7-H3 and sorted for high expression of mB7-H3) tumor bearing mice. In these experiments, it was observed that engineered B7-H3 CAR-T cells (MUT) led to improved survival compared to treatment with wild-type CAR T cells (WT).DETAILED DESCRIPTION OF THE DISCLOSURE

[0055] Provided herein are, inter alia, novel compositions and methods for the diagnosis, prevention and / or treatment of various health conditions. In particular, some embodiments of the disclosure relates to the development of immuno-reagents, including antigen-binding molecules and chimeric antigen receptors (CARs) that have a binding affinity for both human B7-H3 (hB7-H3) and a murine B7-H3 (mB7-H3) (e.g., cross-species binding affinity) for use in detecting and treating cancers that express B7-H3. In particular, some embodiments of the disclosure relate to novel anti-B7-H3 scFv molecules with robustly enhanced affinity for mB7-H3, while maintaining affinity for hB7-H3 and specificity for B7-H3 binding. Experimental data presented herein demonstrated that CAR constructs comprising these anti-B7-H3 scFv molecules possess the same ability to inhibit (e.g., kill) human tumors but also the functionality to also inhibit (e.g., kill) murine tumors through the expression of these binders.

[0056] As described in greater detail below, the present disclosure generally relates to a novel strategy aimed to overcome a critical problem in the development of cellular cancer immunotherapies: current models of preclinical efficacy are poor predictors of clinical efficacy. This is in part due to a reliance on exclusive testing of human T cells engineered with human protein targeting CARs in immunocompromised mouse models. Thus, while these data canAttomey Docket No.: 078430-546001 WOindicate basal antitumor efficacy, they fail to account for other factors that hinder CAR T efficacy in the clinic; notably a hostile tumor immune microenvironment and off-target toxicities. The development of CARs that allow for detailed pre-clinical testing and mitigation of the challenges prior to clinical administration is an unmet need in the field, and part of the reason why many cellular therapies fail in the clinic, especially when targeting solid tumors. This is especially important for pan-cancer antigens, such as B7-H3, which are expressed on most solid tumors, with limited expression on healthy tissues. In particular, B7-H3 is a pan-cancer antigen with limited expression in healthy tissue, making it an ideal CAR target. Thus, as B7-H3 CAR T cells may serve as pan-cancer therapies, it is particularly important to develop B7-H3 CAR T cells in the most representative pre-clinical models possible, to best position these therapies for clinical success.

[0057] Anti-B7-H3 monoclonal antibody “376.96” has been used as a monoclonal antibody and incorporated into CARs previously to target human B7-H3+tumor cells. However, 376.96 was previously reported to have weak affinity for mB7-H3, and has some activity when incorporated into CARs. However, as described in greater detail below, 376.96 has limited efficacy as a CAR (mB7-H3.28z) in mediating mB7-H3+tumor killing, especially against tumors expressing low levels of mB7-H3. Therefore, there remains a need for the development of additional anti-B7-H3 binders with improved properties for immunotherapies.

[0058] To address this need, the inventors have employed directed evolution to engineer scFv molecules that are species cross-reactive and able to target both mouse and human B7-H3 with similar potency. A single binder that can target both species streamlines therapeutic development by allowing for the use of a single sequence for pre-clinical and clinical testing. For example, species cross-reactivity will allow for interrogation of efficacy, toxicity, and effect on the tumor microenvironment in immunocompetent contexts that better replicate tumors found in the clinic. Using a cross-reactive binder (as opposed to two distinct CARs) will allow for development and validation of a single CAR construct that can be used in pre-clinical models and clinical trials. In addition, barriers to efficacy can be better determined and overcome, as opposed to relying on less realistic pre-clinical models. Thus, improvements in efficacy derived from pre-clinical testing in the immunocompetent murine setting would be expected to translate to the clinic. Furthermore, without being bound to any particular theory, a de-risked and validated cross-reactive CAR could be a valuable commercial asset, because a pre-clinically de-risked pan-Attorney Docket No.: 078430-546001 WOcancer CAR could be of great interest to researchers and medical practitioners seeking to expand their cellular therapy platforms, which would benefit cancer patients, as they provide improved therapeutic options for several tumor types.

[0059] As described in greater detail the Examples below, an existing scFv, derived from the B7-H3 -targeting monoclonal antibody 376.96, has been engineered for species cross-reactivity using yeast surface display based directed evolution. 376.96 had previously been developed as a CAR that effectively targeted human B7-H3 (hB7-H3) and had been shown to have very weak affinity for mouse B7-H3 (mB7-H3) (Du et al. Cancer Cell 35, 221-237, 2019; which is incorporated herein by reference). As demonstrated in experimental data described below, despite its weak affinity for mB7-H3, when converted into a fully murine CAR, the 376.96-based CAR was not able to mediate an antitumor response and thus was not suitable for modeling in the murine immunocompetent setting. Given that this scFv had a weak, but existing affinity for mB7-H3, the inventors designed an affinity maturation process to improve the affinity to mB7-H3, while maintaining the affinity to hB7-H3, thus developing a species cross-reactive binder. In particular, some experiments described herein demonstrated the identification of numerous anti-B7-H3 scFv variants with robustly enhanced affinity for mB7-H3, while maintaining affinity for hB7-H3 and specificity for B7-H3 binding. Some other experiments described herein demonstrated that CAR constructs engineered to include these anti-B7-H3 scFv molecules possess the same ability to inhibit (e.g., kill) human tumors but also the functionality to also inhibit (e.g., kill) murine tumors through the expression of these binders. In particular, experimental studies described herein demonstrated that the newly engineered CARs are efficacious in controlling both murine and human B7-H3+ tumor growth in vitro and in vivo. This efficacy was also further demonstrated with limited toxicity in vivo against multiple mB7-H3+ tumors.

[0060] The compositions and methods disclose herein have several advantages and improvements over existing methods. In particular, most existing B7-H3 -targeting CAR T cells are not species cross-reactive. These CAR T cells have followed the development pipeline used for other types of CAR T cells, with pre-clinical testing solely conducted against human tumors in immunocompromised mice, with an inability to account for the impact of other immune cells or off-target toxicities. While clinical testing of these CARs is in its early stages, early results have been mixed, with limited efficacy and some toxicities. These results reinforce the need forAttorney Docket No.: 078430-546001 WOrelevant pre-clinical development in immunocompetent models, as can only be done with a species-cross reactive CAR. Without being bound to any particular theory, it is believed the cross-species B7-H3-binding molecules disclosed herein have significant advantages compared to other B7-H3 targeting CARs in existence which position these new B7-H3-binding molecules for clinical success. In particular, the Y180S-based CAR disclosed herein possesses near identical affinity for mB7-H3 and hB7-H3, an ideal property that allows for equitable comparisons in both human and murine tumor models. Furthermore, the engineered CARs disclosed herein display very favorable signaling properties, with minimal tonic signal and potent induction of T cell activation upon antigen stimulation. This is in contrast to other B7-H3-targeting CARs, and notably the MGA B7-H3.BBz CAR previously developed. This B7-H3 CAR produces a high level of tonic signal and is difficult to pair with other CARs in a single cell, limiting its potential utility and further underscoring the importance of the newly engineered B7-H3 -targeting CARs disclosed herein.DEFINITIONS

[0061] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0062] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”

[0063] The terms “cell,” “cell culture,” “cell line,” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers, or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate orAttorney Docket No.: 078430-546001 WOinadvertent mutations) or environmental influences (e.g. , methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell, cell culture, or cell line.

[0064] The term “effective amount”, “therapeutically effective amount”, or “pharmaceutically effective amount” of a subject recombinant polypeptide of the disclosure generally refers to an amount sufficient for a composition to accomplish a stated purpose relative to the absence of the composition (e.g. , achieve the effect for which it is administered, treat a disease, reduce a signaling pathway, or reduce one or more symptoms of a disease or health condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0065] The term “operably linked”, as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. For example, an operably linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is functional link that allows for expression of the polynucleotide of interest. In this sense, the term “operably linked” refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. Thus, a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence, ft should be understood that, operably linked elements may be contiguous or non-contiguous. In the context of a polypeptide, “operably linked” refers to a physical linkage (e.g. , directly or indirectly linked) between amino acid sequences (e.g. , different segments, modules, or domains) to provide for a described activity ofAttomey Docket No.: 078430-546001 WOthe polypeptide. In the present disclosure, various segments, region, or domains of the recombinant polypeptides of the disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the recombinant polypeptides in the cell. Unless stated otherwise, various modules, domains, and segments of the recombinant polypeptides of the disclosure are operably linked to each other. Operably linked modules, domains, and segments of the recombinant polypeptides of the disclosure may be contiguous or non-contiguous (e.g., linked to one another through a linker).

[0066] The term “percent identity,” as used herein in the context of two or more proteins (or nucleic acids), refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acids that are the same, e.g., about 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9%9, 99.5%, 99.9%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. In some embodiments, this definition also includes sequences that have modifications such as deletions and / or additions (e.g., insertions), as well as those that have substitutions. Such modifications can occur naturally or synthetically. In some embodiments, sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol (1990) 215:403). In some embodiments, sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine structural similarity or identity amino acid sequences include those relying on position-specific structure-scoring matrix (P3SM) that incorporates structure -prediction scores from Rosetta, as well as those based on a length-Attorney Docket No.: 078430-546001 WOnormalized edit distance as described previously in, e.g., Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0067] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions.

[0068] The term “recombinant” when used with reference to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been altered or produced through human intervention such as, for example, has been modified by or is the result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of the protein or can be include amino acid residues that have been modified, e.g., labeled. The term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more of amino acids in the peptide or protein; creation of a fusion protein, e.g., a fusion protein comprising an antibody fragment; and addition, deletion, and / or substitution of one or more of nucleic acids in the nucleic acid sequence. The term “recombinant” when used in reference to a cell is not intended to include naturally-occurring cells but encompass cells that have been engineered / modified to include or express a polypeptide or nucleic acid that would not be present in the cell if it was not engineered / modified.

[0069] A “variant” of a polypeptide, such as an immunoglobulin chain or an antigen-binding fragment thereof, e.g., a scFv, refers to a polypeptide comprising an amino acid sequence that has at least about 70-99.9% (e.g, 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%,Attorney Docket No.: 078430-546001 WO85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity or similarity to a referenced amino acid sequence that is set forth herein. Similarly, a “variant” of a nucleic acid molecule refers to a nucleic acid molecule comprising a nucleic acid sequence that has at least about 70-99.9% (e.g., 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9%9, 99.5%, 99.9%) sequence identity or similarity to a referenced nucleic acid sequence that is set forth herein.

[0070] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0071] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0072] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.Attorney Docket No.: 078430-546001 WO

[0073] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0074] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by.” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0075] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0076] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the presentAttorney Docket No.: 078430-546001 WOdisclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.B7 HOMOLOGY 3 PROTEIN (B7-H3)

[0077] The B7 homology 3 protein (B7-H3) (also known as CD276 and B7RP-2) is a type I transmembrane glycoprotein of the immunoglobulin superfamily with its sequence similar to the extracellular domain of PD-L1 (also known as B7-H1) and belongs to the B7 superfamily, a group of molecules that costimulate or downmodulate T-cell responses.

[0078] In mice, the extracellular domain of B7-H3 consists of a single pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like domains, whereas in humans it consists of one pair (2Ig-B7-H3) or two highly similar pairs (4Ig-B7-H3) due to exon duplication.

[0079] B7-H3 mRNA is widely expressed in most normal tissues, e.g., in lymphoid tissues and non-lymphoid organs at the transcription level (mRNA). B7-H3 mRNA is expressed in most normal tissues. In contrast, B7-H3 protein expression is very limited on normal tissues because of its post-transcriptional regulation by microRNAs. B7-H3 protein is mainly expressed in activated dendritic cells, monocytes, T lymphocytes, B lymphocytes, and NK lymphocytes, while the expression yield is very low in other normal tissues. However, B7-H3 is highly expressed in a variety of solid tumors, such as lung cancer, gastric cancer, pancreatic cancer, prostate cancer, kidney cancer, ovarian cancer, endometrial cancer, colorectal cancer, liver cancer, and breast cancer, and its overexpression is closely associated with survival, prognosis, or tumor grade. In addition to being highly expressed in tumors, B7-H3 may have a function similar to PD-L1 -mediated T cell inhibitory signals. It has been proposed that B7-H3 has costimulatory and co-inhibitory functions, depending on tumor specificity, microenvironment factors, and signal intensity. In addition to its role as an immunomodulatory agent, B7-H3 has been implicated in enhancing cancer metastasis and angiogenesis.

[0080] Since B7-H3 expression is mainly restricted to tumors, B7-H3 is a very important tumor-associated antigen that can be used as a target for potential broad-spectrum immunotherapy.COMPOSITIONS OF THE DISCLOSUREAttorney Docket No.: 078430-546001 WO

[0081] As described in greater detail below, some aspects of the present disclosure relate to novel antigen-binding molecules and CARs capable of binding human B7-H3 (hB7-H3) and a murine B7-H3 (mB7-H3). Also provided, in some embodiments, are (i) recombinant nucleic acids encoding such antigen-binding molecules and / or CARs, (ii) recombinant cells (e.g., T cells) that have been engineered to express an antigen-binding molecule and / or CAR as disclosed herein, and are directed against a cell of interest, e.g. , a cancer cell. Further provided are pharmaceutical compositions containing one or more of the antigen-binding molecules, CARs, nucleic acids, and / or recombinant cells as disclosed herein.Antigen-binding molecules

[0082] As outlined above, some embodiments of the disclosure relate to new series of B7-H3 binding molecules that are species cross-reactive and able to target both mouse and human B7-H3 (e.g., having a binding affinity for both human and a mouse B7-H3) with similar potency.

[0083] In one aspect of the disclosure, provided herein are antigen-binding molecules having a binding affinity to a human B7-H3 (hB7-H3) and a murine B7-H3 (mB7-H3). In some embodiment, the B7-H3 binding molecules of the disclosure include all six CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) of the 376.96 scFv (SEQ ID NO: 11) and further include one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181. In some embodiments, the B7-H3 binding molecules include (a) a VL domain including LCDR1 , LCDR2, and LCDR3 that include the amino acid sequences of SEQ ID NO: 21, 22, and 23, respectively; and (b) a VH domain including HCDR1, HCDR2, and HCDR3 that include the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively; and further include one or more amino acid substitutions at a position selected from the group consisting of Al 73, Y 180, and T181, wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antigenbinding molecule including: (a) a VL domain including the amino acid sequence of SEQ ID NO: 1; and (b) a VH domain including the amino acid sequence of SEQ ID NO: 2, and further including one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. The amino acid sequences of exemplary scFv variants and engineered CAR variants described herein can be found in the Sequence Listing, including the sequences for the signal peptide, variable light chain (VL) domains, variable heavy chain (VH)Attorney Docket No.: 078430-546001 WOdomains, linkers, amino acid substitutions, peptide connectors, CD8 hinge, CD8 transmembrane domain (TMD), CD28 hinge, CD28 TMD, CD28 costimulatory domain, 4- IBB costimulatory domain, and CD32 domain. As discussed above, for the purpose of this application, all amino acid numbering is based on the full-length sequence of the 376.96 scFv of SEQ ID NO: 11. Accordingly, residues A173, Y180, and T181 in the full-length sequence of the 376.96 scFv (SEQ ID NO: 11) correspond to residues A50, Y57, and T58 of SEQ ID NO: 2.

[0084] Non-limiting exemplary embodiments of the antigen-binding molecules of the disclosure can include one or more of the following features. In some embodiments, the antigenbinding molecule is a full-length antibody or an antigen-binding fragment. Non-limiting examples of antigen-binding fragments suitable for the compositions and methods of the disclosure include single-chain antibody fragment (scFv), F(ab), F(ab'), Fab'-SH, F(ab')2, and Fv fragment. In some embodiments, the antigen-binding fragment is a scFv. In some embodiments, the scFv is or includes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-16. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments of the disclosure, the VL and VH domains of the scFv are operably linked to one another via a polypeptide linker. In some embodiments, the polypeptide linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0085] In some embodiments, the B7-H3 binding molecules disclosed herein can include conservative modifications and substitutions at one or more of positions A173, Y180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. Such conservative substitutions include those described by Dayhoff in The Atlas ofAttorney Docket No.: 078430-546001 WOProtein Sequence and Structure 5 (1978), and by Argos in EMBO J, 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Vai, He, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0086] In some embodiments, the amino acid substitution(s) at one or more of positions Al 73, Y180, and T181 of the antigen-binding molecules disclosed herein is independently selected from the group consisting of an alanine (A) substitution, an arginine (R) substitution, an asparagine (N) substitution, an aspartic acid (D) substitution, a leucine (L) substitution, an isoleucine (I), a lysine (K) substitution, a phenylalanine (F) substitution, a tyrosine (Y) substitution, a glutamine (Q) substitution, a glutamic acid (E) substitution, a serine (S) substitution, a threonine (T) substitution, a cysteine (C) substitution, and combinations of any thereof. Non-limiting examples of the amino acid substitutions in the antigen-binding molecules disclosed herein are provided in Tables 1 below. In some embodiments, the amino acid substitution at position 173 is A173T, A173P, A173S, A173D, A173G, or A173V. In some embodiments, the amino acid substitution at position 173 is A173T, A173S, A173Y, or A173C. In some embodiments, the amino acid substitution at position 180 is Y180S, Y180C, Y180F, Y180D, Y180N, or Y180H. In some embodiments, the amino acid substitution at position 180 is Y180S, Y180T, Y180C, Y180A, Y180L, Y180G, Y180I, or Y180H. In some embodiments, the amino acid substitution at position 181 is T 18 II, T181N, T181S, T181P, or T181A. In some embodiments, the amino acid substitution at position 181 is T18 II, T181L, T181A, T181G, or T181V.TABLE 1 : Exemplary amino acid substitutions in the antigen-binding molecules of the disclosure.

[0087] In some embodiments of the disclosure, the substitution at position Al 73 of the VH domain is an Alanine-to-Threonine (A173T) substitution. In some embodiments, the substitution at position Y50 is a Tyrosine -to-Serine (Y180S) substitution. In some embodiments, theAttorney Docket No.: 078430-546001 WOsubstitution at position T181 is a Threonine-to-Isoleucine (T181I) substitution. In some embodiments, the antigen-binding molecule includes the following substitutions: A173T and Y180S. In some embodiments, the antigen-binding molecule includes the following substitutions: A173T and T181I. In some embodiments, the antigen-binding molecule includes the following substitutions: A173T, Y180S, and T181I.

[0088] In some embodiments, the B7-H3 binding molecules of the disclosure include all six CDRs (LCDR1, LCDR2, LCDR3, HCDR1, and HCDR3) of the 376.96 scFv (SEQ ID NO: 11) and further include a HCDR2 including the following sequence:A(T / S / Y / C)ISGGGRY(S / T / C / A / L / G / I / V)T(I / L / A / G / V)YYPDSMKG (SEQ ID NO: 18), wherein one, two, or three of the variable positions are not the wild-type amino acid that occurs in the corresponding position in SEQ ID NO: 18 and wherein alternatives at positions are shown in parentheses.

[0089] In some embodiments, the antigen-binding molecule binds or is capable of binding mB7-H3 with a KD value lower (i.e., binds more tightly) than the KD of the monoclonal antibody 376.96.

[0090] Generally, binding affinity can be used as a measure of the strength of a non-covalent interaction between two molecules, e.g., an antigen-binding molecule disclosed herein and an antigen (e.g., B7-H3). In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules can be quantified by determination of the equilibrium dissociation constant (KD). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation using, e.g. , the surface plasmon resonance (SPR) method (Biacore, Carterra, ForteBio). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or kon) and dissociation rate constant ka (or koff), respectively. KD is related to ka and ka through the equation KD = ka / ka. The value of the dissociation constant can be determined directly by various methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the KD can be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). As shown in Example 2 and FIGS. 7A-7C through 9A-9B below, binding affinity of the antigenbinding molecules described herein can also be assayed using yeast-surface display flowAttorney Docket No.: 078430-546001 WOcytometry based binding assays as described by Hunter & Cochran (2016, Meth. In Enzym. 580: 21-44).

[0091] Other assays to evaluate the binding ability (e.g., binding affinity and / or specificity) of the antigen-binding molecules of the present disclosure towards target antigens (e.g., hB7-H3 and mB7-H3) include, for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics and binding affinity of the antigen-binding molecules also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of antigen-binding molecule for a target antigen (e.g., hB7-H3 or mB7-H3) can be calculated by the Scatchard method described by Frankel et al., Mol. Immunol, 16: 101-106, 1979. It will be understood that the binding affinity of antigen-binding molecule for a target antigen is the strength of interaction between the antigen-binding molecule with the target antigen, whereas the binding specificity of an antigen-binding molecule for a target antigen relates to the affinity to the target antigen relative to other antigens. It will also be understood that an antigen-binding molecule that “specifically binds” a target antigen (such as hB7-H3 or mB7-H3) is an antigen-binding molecule that binds the target antigen but does not significantly bind non-target antigens. In some embodiments, the antigen-binding molecule “specifically binds” a target antigen if it does not significantly bind other antigens (e.g. , non-target antigens) but binds the target antigen with high affinity, e.g., with a KD value of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less.

[0092] In some embodiments, the antigen-binding molecule of the disclosure has a higher affinity for mB7-H3 as compared to the wild-type (WT) 376.96 parent scFv sequence, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% higher affinity for mB7-H3 as compared to the wild-type (WT; parent) 376.96 scFv sequence, while maintaining high affinity for hB7-H3. In some embodiments, the antigen-binding molecule disclosed herein binds or is capable of binding mB7-H3 with a KD value lower than the KD of the monoclonal antibody 376.96. In some embodiments, the antigen-binding molecule of the disclosure binds or is capable of binding mB7-H3 with a KD value of less than 2.34 pM. In some embodiments, the antigen-binding molecule binds or is capable of binding mB7-H3 with a KD value of less than 8.2 nM, less thanAttorney Docket No.: 078430-546001 WO1.3 nM, less than 840 pM, less than 810 pM, or less than 190 pM. In some embodiments, the antigen-binding molecule binds or is capable of binding hB7-H3 with a KD value of less than 180 pM, less than 150 pM, or less than 110 pM.Chimeric antisen receptors (CARs) tarsetins B7-H3

[0093] In one aspect of the disclosure, provided herein are chimeric antigen receptors (CARs) including an antigen-binding moiety derived from an antigen-binding molecule as disclosed herein, or an antigen-binding fragment thereof.

[0094] Non-limiting exemplary embodiments of the CARs of the disclosure can include one or more of the following features. In some embodiments, the antigen-binding moiety of the CARs includes all six CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) of the 376.96 scFv (SEQ ID NO: 11) and further include one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181. In some embodiments, the antigenbinding moiety of the CARs includes (a) a VL domain including LCDR1, LCDR2, and LCDR3 that include the amino acid sequences of SEQ ID NO: 21, 22, and 23, respectively; and (b) a VH domain including HCDR1, HCDR2, and HCDR3 that include the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively; and further include one or more amino acid substitutions at a position selected from the group consisting of Al 73, Y 180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antigen-binding moiety of the CARs includes a scFv including a VL domain that includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, In some embodiments, the antigen-binding moiety of the CARs includes a scFv including a VH domain that includes the amino acid sequence of SEQ ID NO: 2, and further including one or more amino acid substitutions at a position selected from the group consisting of Al 73, Y180, and T 181. In some embodiments, the antigen-binding moiety of the CARs includes a scFv including: (a) a VL domain including the amino acid sequence of SEQ ID NO: 1; and (b) a VH domain including the amino acid sequence of SEQ ID NO: 2, and further including one or more amino acid substitutions at a position selected from the group consisting of A173, Y180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the scFv is or includes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-16. In some embodiments, the scFv is or includes an amino acid sequence having atAttorney Docket No.: 078430-546001 WOleast 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the scFv is or includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the CARs disclosed herein further include one or more of the following domains: (a) a signal peptide; (b) polypeptide linker inserted between the VH and VL domains; (a) a transmembrane domain (TMD); (d) a hinge domain; (e) a costimulatory domain; and (e) a CD3^ chain.

[0095] Suitable signal peptides include, but are not limited to, those from human GM-CSF receptor (GMCSR), CD45, CD8ct, Ig-Kappa, and IL-2. In some embodiments, the signal peptide is from GM-CSF receptor ct (GMCSR2).

[0096] Non-limiting examples of TMDs suitable for the CARs disclosed herein include those derived from a T-cell receptor (TCR) alpha chain, a TCR beta chain, a TCR zeta chain, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, and CD27 (TNFRSF19). Additional examples of TMDs suitable for the CARs disclosed herein include, but are not limiting to, those from CD28, CD33, CD45, CD80, CD83, CD86, CD134, CD137, CD152 (CTLA4), CD154, CD279, and PD-1. In some embodiments, the TMD is a CD8 TMD. In some embodiments, the TMD is a CD28 TMD. In some embodiments, the TMD is a CD8 TMD.

[0097] Suitable hinge domains for the CARs disclosed herein include, but are not limited to, those derived from LFA-1 (CD1 la / CD18), LFA-2 (CD2), CD4, CD5, CD8, CD27 (TNFRSF7), CD28, CD70, 4-1BB, 0X40 (CD134), CD152 (CTLA4), ICOS (CD278), IgGl Fc region, and IgG4 Fc region. In some embodiments, the hinge domain is derived from CD8. In some embodiments, the hinge domain is derived from CD28.

[0098] Non-limiting examples of costimulatory domains of the CARs disclosed herein include those derived from 4-1BB (CD137), CD27 (TNFRSF7), CD28, 0X40 (CD134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54). Also suitable are costimulatory domains derived from LFA-1 (CD1 la / CD18), DAP10, DAP12, and a co-stimulatory inducible T-cell costimulatory (ICOS)Attorney Docket No.: 078430-546001 WOprotein. In some embodiments, the costimulatory domain is a CD28 costimulatory domain. In some embodiments, the costimulatory domain is a 4- IBB costimulatory domain.

[0099] In some embodiments of the disclosure, the VL and VH domains of the scFv are operably linked to one another via a linker polypeptide sequence (e.g., peptidal linkage). In principle, there are no particular limitations to the length and / or amino acid composition of the linker polypeptide sequence. In some embodiments, any single-chain peptide comprising about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a polypeptide linker. In some embodiments, the linker polypeptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 12 to 18 amino acid residues.

[0100] In certain embodiments, the polypeptide linker contains only glycine and / or serine residues (e.g., glycine- serine linker). Examples of such polypeptide linkers include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser; Ser Gly Gly Gly; Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly; Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly; Gly Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly Gly; (Gly Gly Gly Gly Ser)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of one or more. In some embodiments, the polypeptide linkers are modified such that the amino acid sequence GSG (that occurs at the junction of traditional Gly / Ser linker peptide repeats) is not present. For example, in some embodiments, the polypeptide linker includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, where X is any amino acid that can be inserted into the sequence and not result in a polypeptide comprising the sequence GSG, and n is 0 to 4. In some embodiments, the sequence of a polypeptide linker is (GGGXlX2)nGGGGS and XI is P and X2 is S and n is 0 to 4. In some other embodiments, the sequence of a polypeptide linker is (GGGXlX2)nGGGGS and Xi is G and X2 is Q and n is 0 toAttorney Docket No.: 078430-546001 WO4. In some other embodiments, the sequence of a polypeptide linker is (GGGXlX2)nGGGGS and XI is G and X2 is A and n is 0 to 4. In yet some other embodiments, the sequence of a polypeptide linker is GGGGS(XGGGS)n, and X is P and n is 0 to 4. In some embodiments, a polypeptide linker of the disclosure comprises or consists of the amino acid sequence (GGGGA)2GGGGS. In some embodiments, the polypeptide linker comprises or consists of the amino acid sequence (GGGGQ)2GGGGS. In another embodiment, a polypeptide linker comprises or consists of the amino acid sequence (GGGPS)2GGGGS. In another embodiment, the polypeptide linker comprises or consists of the amino acid sequence GGGGS(PGGGS)2. In some embodiments, the polypeptide linker is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In some embodiments, the polypeptide linker is (G4S)2. In some embodiments, the polypeptide linker includes the sequence GGGSGGGSGGGSG-. In some embodiments, the polypeptide linker includes or consists of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

[0101] In some embodiments, the scFv is operably linked to the hinge domain via a polypeptide connector. In principle, there are no particular limitations to the length and / or amino acid composition of the polypeptide connector. In some embodiments, any single-chain peptide comprising about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a polypeptide connector. In some embodiments, the polypeptide connector includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the polypeptide connector includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the polypeptide connector includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the polypeptide connector includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the polypeptide connector includes about 12 to 18 amino acid residues. In some embodiments, the polypeptide connector includes or consists of the amino acid sequence Ala-Ala-Ala (AAA).

[0102] In some embodiments, the substitution at position A 173 is an Alanine -to-ThreonineAttorney Docket No.: 078430-546001 WO(A173T) substitution. In some embodiments, the substitution at position Y50 is a Tyrosine-to-Serine (Y180S) substitution. In some embodiments, the substitution at position T181 is an Threonine -to-Isoleucine (T181I) substitution. In some embodiments, the antigen-binding molecule includes the A173T / Y180S substitutions. In some embodiments, the antigen-binding molecule includes the A173T / T181I substitutions. In some embodiments, the antigen-binding molecule includes the A173T / Y180S / T181I substitutions.

[0103] In some embodiments, the CARs of the disclosure include, in N-terminus to C-terminus direction: (a) GMCSFR signal peptide; (b) an antigen-binding moiety derived from an antigenbinding molecule as disclosed herein, or an antigen-binding fragment thereof; (c) a CD8 hinge domain; (d) a CD8 TMD; (e) a 4-1BB costimulatory domain; and (I) a CD3^ chain.

[0104] In some embodiments, the CARs of the disclosure include, in N-terminus to C-terminus direction: (a) GMCSFR signal peptide; (b) an antigen-binding moiety derived from an antigenbinding molecule, as disclosed herein, or an antigen-binding fragment thereof; (c) a CD28 hinge domain; (d) a CD28 TMD; (e) a CD28 costimulatory domain; and (I) a CD3^ chain.

[0105] In some embodiments, the CAR disclosed herein binds or is capable of binding mB7-H3 with an a KD value lower than the KD of the monoclonal antibody 376.96, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9. In some embodiments, the CAR disclosed herein binds or is capable of binding mB7-H3 with a KD value lower than the KD of the monoclonal antibody 376.96, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CAR disclosed herein includes an antigen-binding molecule binds or capable of binding mB7-H3 with a KD value lower than the KD of the monoclonal antibody 376.96, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0106] In some embodiments, the CAR disclosed herein includes an antigen-binding molecule that binds or is capable of binding mB7-H3 with a KD value of less than 2.34 pM. In some embodiments, the CAR disclosed herein includes an antigen-binding molecule that binds or is capable of binding mB7-H3 with a KD value of less than 8.2 nM, less than 1.3 nM, less than 840Attorney Docket No.: 078430-546001 WOpM, less than 810 pM, or less than 190 pM. In some embodiments, the CAR disclosed herein includes an antigen-binding molecule that binds or is capable of binding hB7-H3 with a KD value of less than 180 pM, less than 150 pM, or less than 110 pM.Nucleic acids

[0107] In another aspect, provided herein are (i) recombinant nucleic acid molecules encoding the antigen-binding molecules and / or CARs disclosed herein, (ii) expression cassettes encoding the antigen-binding molecules and / or CARs disclosed herein, and (iii) expression vectors containing nucleic acids encoding the antigen-binding molecules and / or CARs disclosed herein. In some embodiments, recombinant nucleic acid molecules can be operably linked to regulator sequences which facilitate expression of the antigen-binding molecules and / or CARs in a host cell.

[0108] Accordingly, some embodiments of the disclosure provide recombinant nucleic acid molecules including a nucleic acid sequence encoding: (a) an antigen-binding molecule as disclosed herein; and / or (b) a chimeric antigen receptor (CAR) as disclosed herein.

[0109] The terms “nucleic acid” and “polynucleotide” can be used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acids comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid can contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.

[0110] The nucleic acids of the present disclosure can be nucleic acids of any length, including nucleic acids that are generally between about generally between about 0.5 Kb and about 20 Kb, for example between about 0.5 Kb and about 20 Kb, between about 1 Kb and about 15 Kb, between about 2 Kb and about 10 Kb, or between about 5 Kb and about 25 Kb, for example between about 10 Kb to 15 Kb, between about 15 Kb and about 20 Kb, between about 5 Kb and about 20 Kb, about 5 Kb and about 10 Kb, or about 10 Kb and about 25 Kb.[OHl] In some embodiments, the recombinant nucleic acid molecules of the disclosure include a nucleic acid sequence encoding an antigen-binding molecule that includes an amino acidAttorney Docket No.: 078430-546001 WOsequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of an antigenbinding molecule or CAR as disclosed herein.

[0112] In some embodiments, the recombinant nucleic acids as disclosed herein can be incorporated into an expression cassette or an expression vector. Accordingly, some embodiments disclosed herein relate to vectors or expression cassettes including the recombinant nucleic acids as disclosed herein. It will be understood that an expression cassette generally includes a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and / or into a subject. As such, in some embodiments, an expression cassette of the disclosure include a coding sequence for the an antigen-binding molecule or CAR as disclosed herein, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0113] In some embodiments, the recombinant nucleic acids of the disclosure can be incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that can be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector. Accordingly, also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acids encoding any of the chimeric polypeptides disclosed herein. The recombinant nucleic acids described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al. , "Molecular Cloning: A Laboratory Manual'' 2nd ED. (1989).Attorney Docket No.: 078430-546001 WO

[0114] It should be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art can make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector’s copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the multivalent polypeptides and multivalent antibodies of the present disclosure to be amplified in copy number. Such amplifiable vectors are known in the art.

[0115] Accordingly, in some embodiments, the antigen-binding molecules and CARs of the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g. , non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included.

[0116] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0117] Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0118] The nucleic acid sequences encoding the chimeric polypeptides of the present disclosure can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. Codon usages within the coding sequence of the chimeric polypeptides disclosed herein can be optimized to enhance expression in the host cell, such that about 1%,Attorney Docket No.: 078430-546001 WOabout 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a host cell.

[0119] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject chimeric polypeptides, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this disclosure, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.

[0120] Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, lentiviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0121] The recombinant nucleic acids are not limited to sequences that encode the antigenbinding molecules and / or CARs; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecules are ribonucleic acids (RNAs), molecules can be produced, for example, by in vitro transcription.Recombinant cells and cell culture

[0122] The nucleic acid molecules encoding the antigen-binding molecules and / or CARs present disclosure can be introduced into a host cell, such as, for example, a human T lymphocyte, to produce a recombinant cell (e.g., engineered T cell) containing the nucleic acid molecules. Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calciumAttorney Docket No.: 078430-546001 WOphosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like. Accordingly, in another aspect of the present disclosure, provided herein are recombinant cells including one or more of the following: (a) an antigen-binding molecule as disclosed herein; (b) a CAR as disclosed herein; and (c) a recombinant nucleic acid as disclosed herein.

[0123] In some embodiments, host cells (e.g., T cells) can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells (e.g., T cells) can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0124] Non-limiting exemplary embodiments of the recombinant cell of the disclosure can include one or more of the following features. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a non-human primate cell. In some embodiments, the mammalian cell is a T lymphocyte. In some embodiments, the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell. In some embodiments, the T cell was obtained leukapheresis of a sample obtained from a subject.

[0125] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.Pharmaceutical compositions

[0126] The antigen-binding molecules, CARs, recombinant nucleic acids, recombinant cells (e.g., engineered T cells), and / or cell cultures of the disclosure can be incorporated intoAttorney Docket No.: 078430-546001 WOcompositions, including pharmaceutical compositions. Such compositions generally include antigen-binding molecules, CARs, recombinant nucleic acids, recombinant cells, and / or cell cultures as described herein and a pharmaceutically acceptable carrier. Accordingly, in one aspect, some embodiments of the disclosure relate to pharmaceutical compositions for treating, preventing, ameliorating, reducing or delaying the onset of health condition, for example a proliferative disease (e.g., cancer). In some embodiments, the pharmaceutical composition includes at least one antigen-binding molecule, CAR, recombinant nucleic acid, recombinant cell, and / or cell culture as disclosed herein, in an admixture with a pharmaceutically acceptable carrier.

[0127] In certain embodiments, the pharmaceutical compositions in accordance with some embodiments disclosed herein include cell cultures that can be washed, treated, combined, supplemented, or otherwise altered prior to administration to an individual in need thereof. Furthermore, administration can be at varied doses, time intervals or in multiple administrations.

[0128] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual. In some embodiments, the individual is a human. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0129] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to an individual. For example,Attorney Docket No.: 078430-546001 WOthe pharmaceutical composition may be formulated to be suitable for parenteral, intrapleural, inhalation, intraperitoneal, oral, intradermal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, pulmonary, or intratumoral administration.METHODS OF THE DISCLOSURE

[0130] Administration of any one of the therapeutic compositions described herein, e.g., antigen-binding molecules, CARs, recombinant cells (e.g., engineered T cells), recombinant nucleic acids, and / or pharmaceutical compositions, can be used in the treatment of relevant conditions, such as health disorders and proliferative diseases (e.g, cancer). In some embodiments, the antigen-binding molecules, CARs, recombinant cells, recombinant nucleic acids, and / or pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more relevant health conditions associated with B7-H3, such as health disorders and proliferative diseases. In some embodiments, the health disorder or proliferative disease is a cancer. In some embodiments, the subject is a mammalian subject. In some embodiments, the individual is a patient under the care of a physician.

[0131] Accordingly, in another aspect of the disclosure, provided herein are methods for diagnosing, preventing, and / or treating a health condition in a subject in need thereof, including administering to the subject a composition including one or more of the following: (a) an antigen-binding molecule as disclosed herein; (b) a CAR as disclosed herein; (c) a recombinant nucleic acid as disclosed herein; (d) a recombinant T cell as disclosed herein; and / or (e) a pharmaceutical composition as disclosed herein.

[0132] Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the recombinant cells are allogeneic relative to the subject. In some embodiments, the recombinant cells are autologous relative to the subject. In some embodiments, the health condition is a proliferative disorder. In some embodiments, the proliferative disorder is a cancer. In some embodiments, the cancer is positive for B7-H3 expression, e.g, a B7-H3 -positive cancer. In some embodiments, the cancer overexpresses the B7-H3 antigen. In principle, any B7-H3 -positive cancer can be suitablyAttorney Docket No.: 078430-546001 WOdiagnosed, prevented, and / or treated by the compositions and methods disclosed herein.Examples of suitable cancers include, but are not limited to, nervous system cancer, cervical cancer, sarcoma, neuroblastoma, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, and hepatocellular carcinoma. In some embodiments, the B7-H3 -positive cancer is an adult malignancy. In some embodiments, the B7-H3 -positive cancer is a pediatric cancer. In some embodiments, the pediatric cancer can be a cancer that develops in children ages 0 to 14. In some embodiments, the pediatric cancer is osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, atypical teratoid rhabdoid tumor, medulloblastoma, or neuroblastoma. In some embodiments, the T cells are obtained from tumor infiltrating lymphocytes (TILs). In some embodiments, the T cells are obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells are obtained leukapheresis of a sample obtained from a subject.

[0133] In some embodiments, the administered composition inhibits proliferation of a target cancer cell, and / or inhibits tumor growth of the cancer in the subject. For example, the target cell can be inhibited if its proliferation is reduced, if its pathologic or pathogenic behavior is reduced, if it is destroyed or killed, etc. Inhibition includes a reduction of the measured pathologic or pathogenic behavior of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the methods include administering to the individual an effective number of the engineered T cells disclosed herein, wherein the engineered T cells inhibit the proliferation of the target cell and / or inhibit tumor growth of a target cancer in the subject compared to the proliferation of the target cell and / or tumor growth of the target cancer in subjects who have not been administered with the engineered T cells.

[0134] Administration of the compositions described herein, e.g., engineered T cells, nucleic acids, and pharmaceutical compositions, can be used in the stimulation of an immune response. In some embodiments, one or more of engineered immune cells, nucleic acids, and / or pharmaceutical compositions as described herein are administered to an individual after induction of remission of cancer with chemotherapy, or after autologous or allogeneic hematopoietic stem cell transplantation. In some embodiments, compositions described hereinAttorney Docket No.: 078430-546001 WOare administered to a subject in need of increasing the production of interferon gamma (IFNy), tumor-necrosis factor alpha (TNFa), and / or interleukin-2 (IL-2) in the treated subject relative to the production of these molecules in subjects who have not been administered one of the therapeutic compositions disclosed herein.

[0135] In some embodiments, the administered composition confers an enhanced effector function of the recombinant cells, e.g., engineered T cells. Examples of effector functions that are enhanced in the recombinant cells, e.g., engineered T cells include, but are not limited to growth rate (proliferation), cytokine production, target cell inhibition (e.g., anti-cancer cytotoxicity), macrophage activation, maintenance of enhanced effector cell function, NK cell activation, exhaustion resistance, and in vivo persistence (e.g, survival). In some embodiments, an effector function of the engineered T cells included in the composition of the disclosure is enhanced at levels that are at least 10% higher, such as at least 10% higher than about 10%, at least higher than about 20%, at least higher than about 30%, at least higher than about 40%, at least higher than about 50%, at least higher than about 60%, at least higher than about 70%, at least higher than about 80%, at least higher than about 90%, at least higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times, higher than about 50 times, higher than about 100 times, or higher than about 200 times compared to a reference T cell. In some embodiments, the reference T cell does not include a composition of the disclosure.

[0136] In some embodiments, the enhanced effector function includes increased production of one or more cytokines in the recombinant cell, e.g., engineered T cell. In some embodiments, the one or more cytokines includes interferon gamma (IFNy), tumor-necrosis factor a (TNFa), and / or interleukin-2 (IL-2). In some embodiments, the production of one or more cytokines in the recombinant cell, e.g., engineered T cell is increased at levels that are at least 10% higher, such as at least 10% higher than about 10%, at least higher than about 20%, at least higher than about 30%, at least higher than about 40%, at least higher than about 50%, at least higher than about 60%, at least higher than about 70%, at least higher than about 80%, at least higher than about 90%, at least higher than about 2 times, higher than about three times, higher than about four time, higher than about five times, higher than about six times, higher than about seven times, higher than about eight times, higher than about nine times, higher than about 20 times,Attorney Docket No.: 078430-546001 WOhigher than about 50 times, higher than about 100 times, or higher than about 200 times, when compared to a reference T cell. In some embodiments, the reference T cell does not include a composition of the disclosure.

[0137] An effective amount of the compositions described herein, e.g., engineered T cells, recombinant nucleic acids, and / or pharmaceutical compositions, can be determined based on the intended goal, for example cancer regression. For example, where existing cancer is being treated, the amount of a composition disclosed herein to be administered can be greater than where administration of the composition is for prevention of cancer. One of ordinary skill in the art would be able to determine the amount of a composition to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each subject. For example, frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.

[0138] Determination of the amount of compositions to be administered will be made by one of skill in the art, and will in part be dependent on the extent and severity of cancer, and whether the recombinant cells, e.g., engineered T cells, are being administered for treatment of existing cancer or prevention of cancer. For example, longer intervals between administration and lower amounts of compositions can be employed where the goal is prevention. For instance, amounts of compositions administered per dose can be 50% of the dose administered in treatment of active disease, and administration can be at weekly intervals. One of ordinary skill in the art, in light of this disclosure, would be able to determine an effective amount of compositions and frequency of administration. This determination would, in part, be dependent on the particular clinical circumstances that are present (e.g., type of cancer, severity of cancer).

[0139] In some embodiments, it can be desirable to provide a continuous supply of a composition disclosed herein to the subject to be treated, e.g., a patient. In some embodiments, continuous perfusion of the region of interest (such as a tumor) can be suitable. The time period for perfusion would be selected by the clinician for the particular subject and situation, but times could range from about 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2 weeks or longer. Generally, the dose of the composition viaAttorney Docket No.: 078430-546001 WOcontinuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.

[0140] In some embodiments, administration is by intravenous infusion. In some embodiments, administration is by intracranial infusion. An effective amount of the recombinant cells (e.g., engineered T cells), recombinant nucleic acids, and / or pharmaceutical compositions disclosed herein can be determined based on the intended goal, for example tumor regression. For example, where existing cancer is being treated, the number of cells to be administered can be greater than where administration of the recombinant cells (e.g. , engineered T cells) disclosed herein is for prevention of cancer. One of ordinary skill in the art would be able to determine the number of cells to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the individual to be treated, the state of the individual, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.Administration of recombinant cells to a subject

[0141] In some embodiments, the methods of the disclosure involve administering an effective amount or number of the recombinant cells, e.g., engineered T cells, provided herein to a subject in need thereof. This administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells, e.g., engineered T cells, can be infused directly in the subject’s bloodstream or otherwise administered to the subject.

[0142] In some embodiments, the methods disclosed herein include administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” recombinant cells, e.g., engineered T cells, into an individual, by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant cells, e.g., engineered T cells, or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remainAttorney Docket No.: 078430-546001 WOviable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the individual, e.g., long-term engraftment.

[0143] When provided prophylactically, the recombinant cells, e.g. , engineered T cells, described herein can be administered to a subject in advance of any symptom of a disease or health condition to be treated. Accordingly, in some embodiments the prophylactic administration of an engineered T cell population prevents the occurrence of symptoms of the disease or health condition.

[0144] When provided therapeutically in some embodiments, recombinant cells are provided at (or after) the onset of a symptom or indication of a disease or health condition, e.g., upon the onset of disease or health condition.

[0145] For use in the various embodiments described herein, an effective amount of recombinant cells, e.g., engineered T cells, as disclosed herein, can be at least 102cells, at least 5 x 102cells, at least 103cells, at least 5 x 103cells, at least 104cells, at least 5x104cells, at least 105cells, at least 2 x 105cells, at least 3x105cells, at least 4 x 105cells, at least 5x105cells, at least 6 x 105cells, at least 7x105cells, at least 8x105cells, at least 9x105cells, at least 1 x 106cells, at least 2 x 106cells, at least 3x106cells, at least 4 x 106cells, at least 5x106cells, at least 6 x 106cells, at least 7x106cells, at least 8x106cells, at least 9x106cells, or multiples thereof.

[0146] In some embodiments, the recombinant cells, e.g., T cells, are non-autologous to the subject in need of treatment. In some embodiments, the adoptive cell therapy is an allogeneic adoptive cell therapy. For example, in some embodiments, the recombinant cells, e.g., engineered T cells, are allogeneic to the subject in need of treatment. In an allogeneic adoptive cell therapy, the recombinant cells, e.g., engineered T cells, are not derived from the individual receiving the adoptive cell therapy. Allogeneic cell therapy generally refers to a therapy whereby the individual (donor) who provides the immune cells is a different individual (of the same species) than the individual receiving the cell therapy. For example, a population of engineered cells being administered to an individual is derived from one more unrelated donors, or from one or more non-identical siblings. Accordingly, the engineered cells can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the engineered cells are expanded in culture prior to administration to a subject in need thereof.Attorney Docket No.: 078430-546001 WO

[0147] In some embodiments, the delivery of a cell composition (e.g., a composition including a plurality of recombinant cells, e.g., engineered T cells, according to any of the cells described herein) into a subject by a method or route results in at least partial localization of the cell composition at a desired site. A composition including recombinant cells, e.g., engineered T cells, can be administered by any appropriate route that results in effective treatment in the subject, e.g., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, e.g., at least 1 x 104cells, is delivered to the desired site for a period of time. Exemplary modes of suitable administration include injection, infusion, and instillation. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, delivery by injection or infusion is often considered a standard mode of administration.

[0148] In some embodiments, the recombinant cells, e.g., engineered T cells, are administered systemically, e.g., via infusion or injection. For example, a population of recombinant cells, e.g., engineered T cells, as described herein are administered other than directly into a target site, tissue, or organ, such that it enters, the subject’s circulatory system and, thus, is subject to metabolism and other similar biological processes.

[0149] The efficacy of a treatment including any of the compositions provided herein for the prevention or treatment of a disease or health condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a prevention or treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions (e.g. , progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g. , causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.Attorney Docket No.: 078430-546001 WO

[0150] Measurement of the degree of efficacy is based on parameters selected with regard to the disease being treated and the symptoms experienced. In general, a parameter is selected that is known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community. For example, in the treatment of a solid cancer, suitable parameters can include reduction in the number and / or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof. It will be understood that the effective dose and the degree of efficacy will generally be determined with relation to a single subject and / or a group or population of subjects. Therapeutic methods of the disclosure reduce symptoms and / or disease severity and / or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%.

[0151] As discussed above, a therapeutically effective amount of a pharmaceutical composition can be an amount of the pharmaceutical composition that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a disease or health condition. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease or health condition, alter the course of a symptom of the disease or health condition (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease or health condition. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.Additional therapies

[0152] As discussed above, any one of the compositions as disclosed herein, e.g., antigenbinding molecules, CARs, recombinant cells (e.g., engineered T cells), recombinant nucleic acids, and / or pharmaceutical compositions, can be administered to a subject in need thereof as a single therapy (e.g. , monotherapy). In addition or alternatively, in some embodiments of the disclosure, one or more of the antigen-binding molecules, CARs, recombinant cells (e.g., engineered T cells), recombinant nucleic acids, and / or pharmaceutical compositions described herein can be administered to the subject in combination with one or more additional (e.g. , supplementary) therapies, e.g., at least one, two, three, four, or five additional therapies.Attorney Docket No.: 078430-546001 WO

[0153] Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include therapeutic agents such as chemotherapeutics, anti-cancer agents, and anti-cancer therapies.

[0154] Accordingly, in some embodiments, a composition according to the present disclosure is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies (e.g. , second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.KITS

[0155] Also provided herein are kits for the practice of a method described herein. A kit can include one or more of the antigen-binding molecules, CARs, recombinant cells (e.g., engineered T cells), recombinant nucleic acids encoding the antigen-binding molecules and / or CARs, and pharmaceutical compositions as described and provided herein. For examples, provided herein, in some embodiments, are kits that include one or more engineered T cells of the disclosure. In some embodiments, provided herein are kits that include one or more pharmaceutical compositions of the disclosure. In some embodiments, the kits of disclosure further include written instructions for making the antigen-binding molecules, CARs, recombinant cells (e.g,Attorney Docket No.: 078430-546001 WOengineered T cells), recombinant nucleic acids, and / or pharmaceutical compositions of the disclosure and using the same.

[0156] In some embodiments, the kits of the disclosure further include one or more means useful for the administration of any one of the provided antigen-binding molecules, CARs, recombinant cells, recombinant nucleic acids, and pharmaceutical compositions to an individual. For example, in some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer one any of the provided antigen-binding molecules, CARs, recombinant cells, recombinant nucleic acids, and pharmaceutical compositions to a subject in need thereof. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g. , for modulating an activity of a cell, inhibiting a target cancer cell, diagnosing, preventing, or treating a health condition in a subject in need thereof.

[0157] For example, any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control T-cell populations, positive control T-cell populations, reagents for ex vivo production of the T-cell populations.

[0158] In some other examples, any of the above-described kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents suitable for in vitro production of the antigen-binding molecules and / or CARs.

[0159] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. For example, in some embodiments of the disclosure, the kit includes one or more of the provided antigenbinding molecules, CARs, recombinant cells, recombinant nucleic acids, and pharmaceutical compositions as described herein in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g. , in a sterile glass or plastic vial).

[0160] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. For example, the kit can include a packageAttorney Docket No.: 078430-546001 WOinsert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure can be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and intellectual property information.

[0161] In some embodiments, a kit can include further instructions for using the components of the kit to practice the methods disclosed herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0162] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0163] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0164] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patentAttorney Docket No.: 078430-546001 WOdocuments, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0165] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.EXAMPLES

[0166] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY : Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY : Cold Spring Harbor Laboratory Press;Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0167] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.Attorney Docket No.: 078430-546001 WOEXAMPLE 1Construction of library of 376.96 scFv variants

[0168] This Example describes experiments performed to assess the affinity of yeast-surface displayed 376.96 scFv for human and mouse B7-H3 and the generation of a library of mutant 376.96 variants. 376.96 was cloned into the pCTCON2 vector and displayed on the surface of yeast as an Aga2p fusion with a C-terminal c-myc tag to detect protein expression (see, e.g., FIGS. 1A-1B). While yeast-displayed 376.96 demonstrated high affinity to human B7-H3 (~9 nM), binding to mouse B7-H3 was largely undetectable (see, e.g., FIG.2A). However, very weak binding to 376.96 was observed at high concentrations of mB7-H3 when compared to an scFv that was known to not bind mB7-H3 (Macrogenics), indicating that 376.96 was capable of interacting with mB7-H3, but with very low affinity (see, e.g., FIGS.2B-2C). To improve the affinity of 376.96 towards mB7-H3, a library of mutant 376.96 variants was generated using error-prone PCR. This library was then transformed into EBY 100 yeast, and sorted over iterative rounds to enrich for specific binders to mB7-H3. Yeast plasmids were sequenced after each round of sorting to identify consensus mutations that might lead to superior binding properties (see, e.g., FIG. 3). These results supported that the affinity of 376.96 for mB7-H3 could potentially be improved through directed evolution and affinity maturation, given that 376.96 possessed a weak, but measurable binding interaction with mB7-H3.EXAMPLE 2Sorting strategy for identification of 376.96 scFv variants with higher affinity to mouse B7-H3

[0169] This Example describes experiments performed to identify variants of the 376.96 scFv with improved affinity for mB7-H3.

[0170] The 376.96 library generated as described in Example 1 was sorted over four increasingly stringent rounds using magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to select for variants with improved binding to mB7-H3 (see, e.g., FIGS. 4A-4B). After four sorting rounds, three notable consensus mutations were identified that greatly improved the affinity of 376.96 scFv variants for mB7-H3, while maintaining high affinity for hB7-H3: A173T, Y180S, and T181I (see, e.g., FIGS. 5A-5B through 8A-8C). All three mutations were located within the HCDR2 of the scFv, indicating a critical interaction of this loop with mB7-H3.Attorney Docket No.: 078430-546001 WO

[0171] Subsequently various combinations of these mutations were profiled as yeast-displayed scFv. It was found that all of these mutations possessed >100 fold improvements in affinity for mB7-H3 compared to the wild-type (WT) 376.96 parent scFv sequence (see, e.g., FIGS. 7A-7C). Conversely, very little variation in the affinities of scFv mutants versus wild-type was observed in binding to hB7-H3 (see, e.g., FIGS. 8A-8C). Minimal off-target binding was observed for any of these mutant variants, indicating a retained specificity for B7-H3 binding (see, e.g., FIGS.9A-9B). Notably, the highest affinity variant was the single point mutant Y180S, with a measured affinity of -180 pM to mB7-H3 and -110 pM to hB7-H3, with minimal off-target binding (FIGS. 7A-7C through 9A-9B). Thus, these experiments demonstrated the identification of numerous anti-B7-H3 scFv variants with robustly enhanced affinity for mB7-H3, while maintaining affinity for hB7-H3 and specificity for B7-H3 binding.EXAMPLE 3Expression and antitumor efficacy of CARs containing engineered scFv variants on primary human T cells

[0172] This Example describes experiments performed to demonstrate functionality of engineered scFv variants as antigen sensing domains incorporated into full-length human CAR sequences capable of sensing and responding to human and murine B7-H3.

[0173] Additional experiments were performed to demonstrate that these engineered scFv variants could be expressed as full-length human CARs on human T cells, containing either 4-1BB (see, e.g., FIGS. 10A-10C) or CD28 (see, e.g., FIG. 15) co-stimulatory domains, fused to a CD3z tail. It was observed that these CARs bound mB7-H3 with higher affinity than the WT CAR (see, e.g., FIGS. 11A-11B) and were able to kill mB7-H3+ tumor cells in vitro in an Incucyte assay, while the WT CAR was ineffective and performed more similarly to Mock, untransduced T cells (see, e.g., FIGS. 13 and 14A-14C). Notably, these CARs presented ideal therapeutic development properties, with minimal antigen independent tonic signal at rest, as observed through low levels of activation markers (e.g. CD25, CD69) and exhaustion markers (e.g. CD39, LAG3, PD1), but potent induction of activation markers (e.g. CD25, CD69, PD1, LAG3) upon stimulation with both murine and human B7-H3 expressing tumors (FIGS. 12A-12B and 16A-16B, respectively). Additional experiments were performed to demonstrate that identical antitumor efficacy of these engineered CARs vs the WT CAR in targeting hB7-H3+ Nalm6 tumors (see, e.g., FIGS. 17A-17C), as would be expected based on the maintenance ofAttorney Docket No.: 078430-546001 WOhB7-H3 affinity after engineering. Thus, these new CARs possessed the same ability to kill human tumors but had gained the functionality to also kill murine tumors through the expression of the same binder.

[0174] These results demonstrated that engineered CAR variants expressed on primary human T cells were capable of activating T cells and initiating an antigen dependent killing signal against both human and mouse B7-H3+ tumor lines with similar potency, thus establishing these variants as truly species cross-reactive agents. Conversely, while the parent wild-type 376.96 CAR generated a potent activation signal in response to hB7-H3+ tumors, this CAR was unable to create a similarly strong activation signal against mB7-H3+ tumors, limiting its utility as a cross-reactive agent.EXAMPLE 4Therapeutic efficacy of CARs containing engineered scFv variants in murine T cells in immunocompetent models

[0175] This Example describes experiments performed to explore the efficacy of engineered CAR variants in immunocompetent mouse models against tumor expressing mB7-H3.

[0176] Subsequently, the superior therapeutic properties of these engineered CARs against mouse B7-H3+ tumors in vitro was used to explore the efficacy of the high affinity Y180S CAR (MUT CAR) in murine T cells in immunocompetent models. Experiments were performed to demonstrate that these CARs express well as fully murine CD28z CARs (see, e.g., FIG. 18) and retain low tonic signal at rest in the fully murine setting, as indicated by low levels of the activation marker CD69 and exhaustion markers PD1, CD39, and TfM3 (see, e.g., FIGS. 19A-19B). Murine MUT CARs were activated by osteosarcoma tumor lines expressing low levels of mB7-H3, as indicated by production of fL-2 and IFNy after tumor exposure (see, e.g., FIGS. 20A-20B and 21A-21B). In contrast, the WT CAR produced much lower levels of cytokine in response to mB7-H3+ tumors, indicating inferior activation, in keeping with in vitro data in the human setting. Consistent with cytokine secretion, the MUT CAR was able to control the growth of multiple tumor lines in vitro with varying expression of mB7-H3, while the WT CAR largely struggled to elicit an antitumor response (see, e.g., FIG. 22A-22C). In vivo, MUT CAR T cells expanded and persisted to a superior level compared to WT CAR T cells (see, e.g., FIGS. 23A-23C) and demonstrated superior efficacy in controlling osteosarcoma (see, e.g., FIGS.24A-24B and 25B) and medulloblastoma (see, e.g., FIG. 26) murine tumor models. Notably, it was alsoAttorney Docket No.: 078430-546001 WOobserved that there was no toxicity upon a high dose of MUT CAR T cells and prolonged treatment (see, e.g. , FIGS.25A), demonstrating a favorable developability profile for clinical testing, where other B7-H3 CARs have produced some adverse events.

[0177] Experimental studies described herein demonstrated that the newly engineered CARs are efficacious in controlling both murine and human B7-H3+ tumor growth in vitro and in vivo. This efficacy has been further demonstrated with limited toxicity in vivo against multiple mB7-H3+ tumors.EXAMPLE 5General methods and materialsYeast surface display vectors

[0178] A DNA sequence encoding the 376.96 scFv was cloned into the pCTCON2 yeastsurface display vector (Addgene) using the Nhel and BamHI sites.Yeast surface display binding assays

[0179] EBY 100 yeast were transformed with pCTCON2 plasmids and selected on SD-CAA-Agar plates. Yeast (-100,000 per sample) were grown and induced in SG-CAA, and binding set up over a range of soluble ligand or receptor concentrations in phosphate -buffered saline (PBS) containing 1 mg ml-1bovine serum albumin (BSA; BPBS), taking into account ligand depletion and equilibrium time. After incubation with binding partner, yeast cells were washed once with BPBS, then incubated with a 1 :5,000 dilution of chicken anti-c-myc antibody (A21281, fnvitrogen) and incubated for 30 min at 4°C in the dark. After primary addition, samples were washed once with BPBS, and secondary antibodies were added. Expression was detected with a 1:500 dilution of goat anti-chicken Alexa Fluor 488 or Alexa Fluor 647 (fnvitrogen). Binding of B7-H3-Fc was detected using a 1:500 dilution of goat anti-human Alexa Fluor 488 or Alexa Fluor 647 (fnvitrogen). Secondary antibodies were incubated for 15 min at 4°C in the dark. After secondary incubation, samples were washed once with BPBS, pelleted, and left pelleted on ice until analysis. Samples were analyzed by resuspending them in 50 pL of BPBS and running flow cytometry using a BD Accuri C6 (BD Biosciences). Samples were gated for bulk yeast cells (forward scatter (FSC) vs. side scatter (SSC)) and then for single cells (FSC-Height vs. FSC-Area). Expressing yeast were determined and gated via C-terminal c-myc tag or GFP detection. The geometric mean of the binding fluorescence signal was quantified from the expressingAttorney Docket No.: 078430-546001 WOpopulation and used as a raw binding value. When comparing binding signals, the average fluorescence expression signal was quantified for different protein variants and used to normalize binding signal. To determine “fraction bound,” (i.e. “normalized fluorescence”) binding signals were divided by the signal derived from the highest signal observed for each variant. To calculate KD values, data were analyzed in GraphPad Prism (v9.3.1) using non-linear regression curve fit.Yeast surface display library generation, sorting, and sequencing

[0180] The 376.96 scFv was expressed in Saccharomyces cerevisiae (strain: EBY100; ATCC MYA-4941) as a genetic fusion to the agglutinin mating protein Aga2p. An error-prone PCR library was created using the 376.96 scFv as a template and mutations were introduced with a Gene Morph 11 random mutagenesis kit (Agilent), following the manufacturer’s instructions. Separate PCRs were performed using various concentrations of Mutazyme II enzyme. Products from these reactions were purified via gel electrophoresis, pooled, and amplified with standard PCR using Phusion polymerase (New England BioLabs). Purified mutant DNA and linearized plasmid were electroporated into EBY 100 yeast, where they were assembled in vivo through homologous recombination. A diversity of 1.28x108variants were estimated for the library, determined by dilution plating and colony counting. Yeast were grown in SD-CAA media and induced for scFv protein expression by growth in media containing 90% SG-CAA and 10% SD-CAA overnight. Yeast displaying 376.96 scFv variants were isolated via magnetic-activated cell sorting and fluorescence-activated cell sorting (FACS) using a SONY SH800S cell sorter (SONY) and analyzed with a BD Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (BD Biosciences). Screens were carried out using equilibrium binding conditions where yeast were incubated at room temperature in BPBS with the following concentrations of mB7-H3-hFc or Isotype control (Obinutuzumab) for two hours. For positive sorts to mB7-H3, the binding populations of yeast were collected. For negative sorts to isotype control, the non-binding populations of yeast were collected. Sort 1.1, positive sort via MACS, 1 uM mB7-H3-hFc; Sort 1.2, negative sort via MACS, 1 uM isotype control; Sort 2.1, positive sort via MACS, 1 uM mB7-H3-hFc; Sort 2.2, negative sort via MACS, 1 uM isotype control; Sort 3, positive sort via FACS, 30 nM mB7-H3-hFc; Sort 4, positive sort via FACS, 300 pM mB7-H3-hFc. After incubation with mB7-H3 or isotype control, yeast were pelleted, washed, and labeled with antibodies as described above prior to sorting. Sorted yeast clones were propagated, inducedAttorney Docket No.: 078430-546001 WOfor scFv expression, and subjected to iterative rounds of MACS or FACS as described above. After each round of screening, plasmid DNA was recovered using a Zymoprep yeast plasmid miniprep I kit (Zymo Research Corp), transformed into DH10B electrocompetent cells (Thermo Fisher), and isolated using a GeneJET plasmid miniprep kit (Thermo Fisher). Sequencing was performed by ELIM Biopharmaceuticals, Inc. (Hayward, CA).Cell lines

[0181] The Nalm6 B-ALL cell line was provided by David Barrett (Children’s Hospital of Philadelphia) and retrovirally transduced to express GFP, firefly luciferase, and hB7-H3. The 293 GP retroviral packaging line was provided by the Surgery Branch (National Cancer Institute, National Institutes of Health). Nalm6-B7-H3 were cultured in RPMI-1640 (Gibco). 293 GP were cultured in DMEM (Gibco). Cell line culture media was supplemented with 10% FBS, lOmM HEPES, 2mM L-glutamine, 100 U / mL penicillin, and I OOpg / mL streptomycin (Gibco). STR DNA profiling of all cell lines was conducted once per year (Genetica Cell Line testing). All cell lines were routinely tested for mycoplasma.

[0182] Mouse group 3 medulloblastoma cells were provided by Martine Roussel (St. Jude Children's Research Hospital) and lentivirally transduced to overexpress mB7-H3 followed by puromycin selection. Mouse G3MB-B7H3 were culture in Neurobasal (Gibco) supported with B-27 (Gibco) and N2 supplements (Gibco), EGF (20ng / ml) and FGF-2 (20ng / ml) (lOmM HEPES, 100 U / mL penicillin, and 1 OOpg / mL streptomycin (Gibco).

[0183] K7M2 cell line was obtained from ATCC (CRL-2836). F331 and F420 were provided by Alejandro Sweet-Cordero (UCSF). K7M2, F331, and F420 were cultured in DMEM (Gibco) supplemented with 10% FBS, lOmM HEPES, 2mM L-glutamine, 100 U / mL penicillin, and lOOpg / mL streptomycin (Gibco).

[0184] All cell lines were cultured at 37°C in a 5% CO2 environment.Source of primary human and murine T cells

[0185] Buffy coats from healthy donors were purchased from the Stanford Blood Center under an IRB-exempt-protocol. Leukopaks from healthy donors were purchased from STEMCELL Technologies. Primary human T cells were purified by negative selection using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) and SepMate-50 tubes. T cells were cryopreserved at 2* 107cells per mL in CryoStor CS10 cryopreservation media (Stem Cell Technologies) until use.Attomey Docket No.: 078430-546001 WO

[0186] Mouse T cells were isolated from spleens of 8 weeks old B6. SJL -Ptprca Pepcb / BoyJ mice (The Jackson Laboratory). Spleens were harvested and mechanically dissociated by passing through a 70um cell strainer twice. Mouse primary T cells were purified by using the mouse pan T cell isolation kit (Miltenyi Biotech, CA) following the manufacturer instructions.Viral vector construction

[0187] All retroviral constructs were cloned into the MSGV1 retroviral vector. B7-H3.BBz variants were generated by fusing, from N to C terminus, a human GM-CSFR leader sequence, scFv variants derived from 376.96 in the VL-(G4S)3 linker- VH orientation, CD8a hinge and transmembrane sequence, and human 4- IBB and CD3z intracellular signaling domains. Human B7-H3.28z variants were generated by fusing, from N to C terminus, a human GM-CSFR leader sequence, scFv variants derived from 376.96 in the VL-(G4S)3 linker- VH orientation, CD28 hinge and transmembrane sequence, and human CD28 and CD3z intracellular signaling domains. Murine B7-H3.28z variants were generated by fusing, from N to C terminus, GM-CSFR leader sequence, scFv variants derived from 376.96 in the VL-(G4S)3 linker- VH orientation, murine CD28 hinge and transmembrane sequence, and murine CD28 and CD3z intracellular signaling domains. Macrogenics (MGA) control B7-H3.BBz and B7-H3.28z control CAR constructs were generated by replacing the 376.96 scFv with an scFv derived from MGA271 in the VH-(G4S)3linker- VL orientation. In some instances, CAR transduction was detected through inclusion of a porcine teschovirus- 1 2A (P2A) ribosomal skipping sequence following the CD3z sequence, followed by the cell-surface marker human truncated NGFR which lacked the intracellular domain.Virus production

[0188] Retroviral supernatant was packaged using 293 GP cells, the RD114 envelope plasmid for human B7-H3 CAR variants and the pCL-Eco vector for murine B7-H3 CAR variants. In brief, for production of human B7-H3 CAR virus, 11 pg RD114 and 22 pg of the corresponding MSGV 1 transfer plasmid were delivered to 293GP cells grown on 150mm poly-D-lysine dishes (Coming) to 80% confluency by transient transfection with Lipofectamine 2000 (Thermo Fisher). For production of murine B7-H3 CAR virus, 1 I g pCL-Eco and 22pg of the corresponding MSGV1 transfer plasmid were delivered to 293GP cells grown on 150mm poly-D-lysine dishes (Coming) to 80% confluency by transient transfection with Lipofectamine 2000 (Thermo Fisher). Media was replenished every 24 hours. Vims production was performed side-Attorney Docket No.: 078430-546001 WOby-side for comparable CAR constructs. Retroviral supernatant was harvested 48 and 72-hour post transfection. Supernatant from replicate dishes were pooled, centrifuged to deplete cell debris, and stored at -80 °C until use.CAR T manufacturing

[0189] Human T cell production: At Day 0, primary human T cells were thawed and activated with anti-CD3 / CD28 Human T-Expander Dynabeads (Thermo Fisher) at a 1:1 bead to cell ratio. On Day 2 virus coated culture plates were prepared on non-TC-treated 12-well plates that had been pre-coated with RetroNectin (Takara Bio) according to the manufacturer’s instructions, by incubating with ImL of retroviral supernatant (2x 107-5x107TU / mL) and centrifugation at 3200 RPM, 32 °C for two hours. The supernatant was subsequently aspirated off of the wells and 0.5* 106T cells were added in ImL of T cell media comprised of: AIM V (Thermo Fisher), 5% fetal bovine serum (FBS), 100 U / mL penicillin (Gibco), 100 mg / mL streptomycin (Gibco), 2 mM L-glutamine (Gibco), 10 mM HEPES (Gibco), and 40 U / mL rhfL-2 (Peprotech). After addition of the T cells, the plates were gently spun down at 1000 RPM for 2 min then incubated for 24hrs at 37°C 5% CO2. This transduction process was repeated on Day 3. Dynabeads were removed on Day 4 by magnetic separation. Cells were maintained between 0.4 - 2x 106cells / mL and expanded until Day 10 - 12.

[0190] Murine T cell production: At Day 0, primary murine T cells were thawed and activated with anti-CD3 / CD28 Mouse T-Expander Dynabeads (Thermo Fisher) at a 1 :2 bead to cell ratio. On Day 1 virus coated culture plates were prepared on non-TC-treated 6-well plates that had been pre-coated with RetroNectin (Takara Bio) according to the manufacturer’s instructions, by incubating with ImL ofretroviral supernatant (6* 107- 1.5* 108TU / mL) and centrifugation at 3200 RPM, 32 °C for two hours. 1.5 ml of supernatant was subsequently aspirated off of the wells and 1 x 1 o6T cells were added in 1.5mL of T cell media comprised of: RPMf (Thermo Fisher), 10% fetal bovine serum (FBS), 100 U / mL penicillin (Gibco), 50 pM 2-BME (Sigma-Aldrich), 100 mg / mL streptomycin (Gibco), 2 mM L-glutamine (Gibco), 10 mM HEPES (Gibco), 40 U / mL rhfL-2 (Peprotech), lOng / ml recombinant mouse IL-7 (BioLegend) and hfl-15 lOng / ml (Peprotech). After addition of the T cells, the plates were gently spun down at 1000 RPM for 2 min then incubated for 24hrs at 37°C 5% CO2. This transduction process was repeated on Day 2. Dynabeads were removed on Day 3 by magnetic separation. Cells were maintained between 0.4 - 2x 106cells / mL and expanded until Day 4-6.Attorney Docket No.: 078430-546001 WOFlow cytometry

[0191] Recombinant hB-H3-hFc (R&D systems) and mB7-H3-hFc (Sino Biological) were used to detection B7-H3 surface CAR. CAR detection reagents were fluorescently labeled with the DyLight 650 Microscale Antibody Labeling Kit (Thermo Fisher). The following antibodies were used for detection of cell-surface proteins: human CD4 (clone SK3; BD); human CD8 (clone SKI; BD); human CD69 (clone FN50; BioLegend); human CD39 (clone Al; BioLegend); human LAG3 (clone 3DS223H; Invitrogen); human PD1 (clone JI 05; Invitrogen); human NGFR (clone ME20.4; BioLegend); mouse CD45.1 (clone A20; BioLegend); mouse CD69 (clone H1.1F3; BioLegend); mouse PD1 (clone 29F.1A12; BioLegend); mouse CD39 (clone Duha59; BioLegend); mouse TIM3 (clone RMT3-23; BioLegend); mouse B7-H3 (clone MIH32; BD); mouse CD4 (clone GK1.5; BD); mouse CD8 (clone 53-6.7; BioLegend). Surface protein was stained by incubation with 3 pg / mL of detection reagents (or at the concentrations indicated in the figures) for 30 min at 4 °C. Flow cytometry was performed on BD Fortessa and BD Accuri instruments.Incucyte tumor killing assays, cytokine analysis, and T cell activation marker detection 5X104GFP-labeled tumor cells were cocultured with CAR T cells in 200pL RPMI supplemented with 10% FBS, lOmM HEPES, 2mM L-glutamine, 100 U / mL penicillin, and lOOpg / mL streptomycin. For various E : T ratios, the following number of CAR T cells were added to each well containing 5X104tumor cells: 2:1 E:T - 10X104T cells; 1:1 E:T - 5X104T cells; 1:2 E:T - 2.5X104T cells; 1:4 E:T - 1.25X104T cells; 1:8 E:T - 6.25X103T cells; 1:16 E:T - 3.13X103T cells; 1:32 E:T - 1.57X103T cells; 1:64 E:T - 7.83X102T cells. Triplicate wells were plated in 96-well flat-bottom plates for each condition. Tumor fluorescence was monitored every 2-3 hours with a lOx objective using the Incucyte Zoom system (Sartorius), housed in a cell culture incubator at 37°C and 5% CO2, set to take 4 images per well at each time point. Total integrated GFP intensity was quantified using the Zoom software (Sartorius). Data were normalized to the first timepoint and plotted as fold change in tumor fluorescence over time. For cytokine secretion and T cell marker analysis, cocultures were set up as above except in 96-well round bottom plates. After approximately 24 hours, plates were spun down to pellet cells and 150pL of supernatant was harvested and stored at -80 °C until analysis, while cell pellets were immediately processed for flow cytometry. IFNy and IL-2 levels in coculture supernatants were quantified by ELISA (BioLegend) according to the manufacturer’sAttorney Docket No.: 078430-546001 WOinstructions. Negative cytokine values were set to 0. For analysis of T cell markers after activation by tumor cells, pellets from spun plates were pooled together for triplicate wells, stained for live cells, hCD4, hCD8, hCD69, hCD25, hPDl, hCD39, hLAG3, and hNGFR and analyzed by flow cytometry. Coculture experiments were setup using day 10 T cells.Animal models

[0192] C57B16 / J mice were purchased from the Jackson Laboratory and cared for under Stanford University APLAC-approved protocols. Healthy male and female mice were used for in vivo experiments between 6 and 10 weeks old at tumor engraftment and were drug naive, and not involved in previous procedures. Mice were housed in sterile cages in a barrier facility at Stanford University with a 12-hour light / dark cycle. Veterinary Services Center (VSC) staff at Stanford University monitored the mice daily and were euthanized when mice manifested persistent hunched posture, persistent scruffy coat, paralysis, impaired mobility, greater than 20% weight loss, if tumors significantly interfered with normal bodily functions, or if they exceeded limits designated in APLAC-approved protocols. Per recommendation by VSC staff, mice with morbidities were supported with 500pL subcutaneous saline, diet gel (DietGel® 76A, ClearH2O), and wet chow.Quantification of T cells from blood

[0193] Mouse blood was collected from the retro-orbital sinus into Microvette blood collection tubes with EDTA (Fisher Scientific). Red blood cells were depleted by ACK lysis (Quality Biological Inc.), followed by two washes with FACS buffer (PBS + 2% FBS). Samples were stained with anti-mCD45.1, anti-mCD4, and anti-mCD8 reagents. Samples were mixed with CountBright Absolute Counting beads (Thermo Fisher) before flow cytometry analysis.Osteosarcoma tumor models

[0194] Mice were treated with one dose of 50 mg / kg cyclophosphamide and 100 mg / kg fludarabine for lymphodepletion four days before tumor engraftment. 1.5X106F331 cells in lOOpL DPBS were injected into the tibia periosteum of six- to ten-week-old C57B16 / J male or female mice. Four days after tumor implantation and after visual confirmation of tumor formation, mice were treated with 10X106murine B7-H3.28z-CAR T cells (either containing the parent 376.96 [wt] sequence or the 376.96 Y180S mutant sequence [mut]). Tumor progressionAttorney Docket No.: 078430-546001 WOwas monitored by caliper measurement. Mice were monitored for weight change and euthanized according to the criteria described in the Animal Models section.Medulloblastoma tumor models

[0195] Mice were anaesthetized with 3% isofluorane in an induction chamber, mice were maintained under 1.5% isofluorane anesthesia during brain surgery. 1.5X106mouse G3 MB cells in 3pL DPBS were injected into the cerebellum (2 mm posterior to lambda and 2 mm deep) utilizing a digital mouse stereotactic frame (Stoelting Instruments) with an automatic microinjector (at an infusion rate of 0.8uL / min) of six- to ten-week-old C57B16 / J male or female mice. Four days after tumor implantation mice were infused with 1X106murine B7-H3.28z-CAR T cells into the posterior fossa (either containing the parent 376.96 [wt] sequence or the 376.96 Y180S mutant sequence [mut]). Mice were monitored for tumor symptoms, as domed head, abnormal gait, hydrocephaly or hunched back and euthanized according to the criteria described in the Animal Models section.

[0196] As discussed above, the compositions and methods disclose herein have several advantages and improvements over existing methods. For example, it is believed the crossspecies B7-H3 -binding molecules disclosed herein have significant advantages compared to other B7-H3 targeting CARs in existence which position these new B7-H3-binding molecules for clinical success. In particular, the Y180S-based CAR disclosed herein possesses near identical affinity for mB7-H3 and hB7-H3, an ideal property that allows for equitable comparisons in both human and murine tumor models. Furthermore, the engineered CARs disclosed herein display very favorable signaling properties, with minimal tonic signal and potent induction of T cell activation upon antigen stimulation. This is in contrast to other B7-H3 -targeting CARs, and notably the MGA B7-H3.BBz CAR previously developed. This B7-H3 CAR produces a high level of tonic signal and is difficult to pair with other CARs in a single cell, limiting its potential utility and further underscoring the importance of the newly engineered B7-H3-targeting CARs disclosed herein.

[0197] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

Claims

Attorney Docket No.: 078430-546001 WOCLAIMS WHAT IS CLAIMED IS:

1. An antigen-binding molecule having a binding affinity to a human B7-H3 (hB7-H3) and a murine B7-H3 (mB7-H3), the antigen-binding molecule comprising:(a) a variable light chain (VL) domain comprising complementarity determining regions LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 21, 22, and 23, respectively; and(b) a variable heavy chain (VH) domain comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 17, 18, and 19, respectively; and further comprising one or more amino acid substitutions at a position selected from the group consisting of Al 73, Y180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11.

2. The antigen-binding molecule of claim 1, the VL domain comprises the amino acid sequence of SEQ ID NO: 1.

3. The antigen-binding molecule of claim 1 or 2, the VH domain comprises the amino acid sequence of SEQ ID NO: 2.

4. The antigen-binding molecule of any one of claims 1-3, wherein the antigen-binding molecule is a full-length antibody or an antigen-binding fragment.

5. The antigen-binding molecule of claim 4, wherein the antigen-binding fragment is a single-chain antibody fragment (scFv), a F(ab), a F(ab'), a Fab'-SH, a F(ab')2, or a Fv fragment.

6. The antigen-binding molecule of claim 5, wherein the antigen-binding fragment is or includes a scFv.

7. The antigen-binding molecule of claim 5, wherein the scFv is or includes an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-16.Attorney Docket No.: 078430-546001 WO8. The antigen-binding molecule of any one of claims 6-7, wherein the VL and VH domains of the scFv are operably linked to one another via a polypeptide linker.

9. The chimeric antigen receptor of claim 8, wherein the polypeptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

10. The antigen-binding molecule of any one of claims 1-9, wherein the substitution at position Al 73 is an Alanine-to-Threonine (A173T) substitution.

11. The antigen-binding molecule of any one of claims 1-10, wherein the substitution at position Y50 is a Tyrosine-to-Serine (Y180S) substitution.

12. The antigen-binding molecule of any one of claims 1-11, wherein the substitution at position T181 is a Threonine -to-Isoleucine (T181I) substitution.

13. The antigen-binding molecule of any one of claims 1-12, wherein the antigen-binding molecule comprises the following substitutions: A173T and Y180S.

14. The antigen-binding molecule of any one of claims 1-13, wherein the antigen-binding molecule comprises the following substitutions: A173T and T181I.

15. The antigen-binding molecule of any one of claims 1-14, wherein the antigen-binding molecule comprises the following substitutions: A173T, Y180S, and T181I.

16. The antigen-binding molecule of any one of claims 1-15, wherein the antigen-binding molecule has a higher affinity for mB7-H3 as compared to 376.96 scFv, while maintaining high affinity for hB7-H3.

17. The antigen-binding molecule of any one of claims 1-16, wherein the antigen-binding molecule is capable of binding mB7-H3 with an equilibrium dissociation constant (KD) value of less than 2.34 pM.

18. The antigen-binding molecule of any one of claims 1-17, wherein the antigen-binding molecule is capable of binding mB7-H3 with a KD value of less than 8.2 nM, less than 1.3 nM, less than 840 pM, less than 810 pM, or less than 190 pM.Attorney Docket No.: 078430-546001 WO19. The antigen-binding molecule of any one of claims 1-18, wherein the antigen-binding molecule is capable ofbinding hB7-H3 with a KD value of less than 180 pM, less than 150 pM, or less than 110 pM.

20. A chimeric antigen receptor (CAR) comprising an antigen-binding moiety derived from an antigen-binding molecule, or an antigen-binding fragment thereof, of any one of claims 1-19.

21. The chimeric antigen receptor of claim 20, wherein the antigen-binding moiety comprises a scFv comprising:(a) a variable light chain (VL) domain comprising the amino acid sequence of SEQ ID NO: 1; and(b) a variable heavy chain (VH) domain comprising the amino acid sequence of SEQ ID NO: 2, and further comprising one or more amino acid substitutions at a position selected from the group consisting of Al 73, Y 180, and T 181 , wherein the substitutions are numbered in accordance with the amino acid sequence of SEQ ID NO: 11.

22. The chimeric antigen receptor of claim 21, further comprising one or more of the following domains: (a) a signal peptide; (b) polypeptide linker inserted between the VH and VL domains; (a) a transmembrane domain (TMD); (d) a hinge domain; (e) a costimulatory domain; and (f) a CD3^ chain.

23. The chimeric antigen receptor of claim 22, wherein the signal peptide is from a human GM-CSF receptor (GMCSR), CD45, CD8ct, Ig-Kappa, or IL-2.

24. The chimeric antigen receptor of claim 23, wherein the signal peptide is from GM-CSF receptor ct (GMCSR2).

25. The chimeric antigen receptor of any one of claims 22-24, wherein the TMD is derived from a protein selected from a T-cell receptor (TCR) alpha chain, a TCR beta chain, a TCR zeta chain, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD27 (TNFRSF19), CD28, CD33, CD45, CD80, CD83, CD86, CD134, CD137, CD152 (CTLA4), CD154, CD279, and PD-1.Attorney Docket No.: 078430-546001 WO26. The chimeric antigen receptor of any one of claims 22-25, wherein the TMD is a CD8 TMD or a CD28 TMD.

27. The chimeric antigen receptor of any one of claims 21-26, wherein the hinge domain is derived from a protein selected from the group consisting of LFA-1 (CD1 la / CD18), LFA-2 (CD2), CD4, CD5, CD8, CD27 (TNFRSF7), CD28, CD70, 4-1BB, 0X40 (CD134), CD152 (CTLA4), ICOS (CD278), IgGl Fc region, and IgG4 Fc region.

28. The chimeric antigen receptor of any one of claims 22-27, wherein the hinge domain is derived from CD 8 or CD28.

29. The chimeric antigen receptor of any one of claims 22-28, wherein the costimulatory domain is derived from a protein selected from the group consisting of 4-1BB (CD137), CD27 (TNFRSF7), CD28, 0X40 (CD134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CD1 la / CD18), DAP10, DAP12, and a co-stimulatory inducible T-cell costimulatory (ICOS) protein.

30. The chimeric antigen receptor of any one of claims 22-29, wherein the costimulatory domain is a CD28 costimulatory domain or a 4- IBB costimulatory domain.

31. The chimeric antigen receptor of any one of claims 21-30, wherein the VL and VH domains of the scFv are operably linked to one another via a polypeptide linker.

32. The chimeric antigen receptor of claim 31 , wherein the polypeptide linker comprises or consists of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 10).

33. The chimeric antigen receptor of any one of claims 21-32, wherein the scFv is operably linked to the hinge domain via a polypeptide connector.

34. The chimeric antigen receptor of claim 33, wherein the polypeptide connector comprises or consists of the amino acid sequence Ala-Ala-Ala.

35. The chimeric antigen receptor of any one of claims 21-34, wherein the substitution at position Al 73 is an Alanine-to-Threonine (A173T) substitution.Attorney Docket No.: 078430-546001 WO36. The chimeric antigen receptor of any one of claims 21-35, wherein the substitution at position Y50 is a Tyrosine-to-Serine (Y180S) substitution.

37. The chimeric antigen receptor of any one of claims 21-36, wherein the substitution at position T181 is a Threonine -to-Isoleucine (T181I) substitution.

38. The chimeric antigen receptor of any one of claims 21-37, wherein the antigen-binding molecule comprises the following substitutions: A173T and Y180S.

39. The chimeric antigen receptor of any one of claims 21-38, wherein the antigen-binding molecule comprises the following substitutions: A173T and T181I.

40. The chimeric antigen receptor of any one of claims 21-39, wherein the antigen-binding molecule comprises the following substitutions: A173T, Y180S, and T181I.

41. The chimeric antigen receptor of any one of claims 20 40, wherein the chimeric antigen receptor comprises, in N-terminus to C-terminus direction:(a) GMCSFR signal sequence(b) an antigen-binding moiety derived from an antigen-binding molecule, or an antigenbinding fragment thereof, of any one of claims 1-19;(c) a CD8 hinge domain;(d) a CD8 TMD;(e) a 4- 1 BB costimulatory domain; and(f) a CD3^.

42. The chimeric antigen receptor of any one of claims 20 40, wherein the chimeric antigen receptor comprises, in N-terminus to C-terminus direction:(a) GMFSFR signal sequence;(b) an antigen-binding moiety derived from an antigen-binding molecule, or an antigenbinding fragment thereof, of any one of claims 1-19;(c) a CD28 hinge domain;(d) a CD28 TMD;(e) a CD28 costimulatory domain; and(f) a CD3^.Attorney Docket No.: 078430-546001 WO43. The chimeric antigen receptor of any one of claims 20 42, wherein the antigen-binding molecule is capable of binding mB7-H3 with a KD value of less than 2.34 pM.

44. The antigen-binding molecule of any one of claims 20 43 , wherein the antigen-binding molecule is capable of binding mB7-H3 with a KD value of less than 8.2 nM, less than 1.3 nM, less than 840 pM, less than 810 pM, or less than 190 pM.

45. The antigen-binding molecule of any one of claims 20 44, wherein the antigen-binding molecule is capable ofbinding hB7-H3 with a KD value of less than 180 pM, less than 150 pM, or less than 110 pM.

46. The chimeric antigen receptor of any one of claims 43 45, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9.

47. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding:(a) an antigen-binding molecule according to any one of claims 1-19; and / or(b) a CAR according to any one of claims 20 46.

48. The recombinant nucleic acid molecule of claim 43, wherein the recombinant nucleic acid molecule is operably linked to one or more heterologous nucleic acid sequences.

49. The recombinant nucleic acid molecule any one of claims 47-48, wherein the one or more heterologous nucleic acid sequences comprises a promoter sequence.

50. The recombinant nucleic acid molecule of any one of claims 47 48, wherein the recombinant nucleic acid molecule is incorporated into an expression cassette or a vector.

51. A recombinant cell comprising:(a) an antigen-binding molecule according to any one of claims 1-19;(b) a CAR according to any one of claims 20 46; and / or(c) a recombinant nucleic acid according to any one of claims 47-50.

52. The recombinant cell of claim 51 , wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell.Attorney Docket No.: 078430-546001 WO53. The recombinant cell of claim 52, wherein the eukaryotic cell is a mammalian cell.

54. The recombinant cell of claim 53, wherein the mammalian cell is a human cell.

55. The recombinant cell of any one of claims 53-54, wherein the mammalian cell is a T lymphocyte.

56. The recombinant cell of claim 54, wherein the T cell is a CD8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell.

57. The recombinant cell of any one of claims 55-56, wherein the T cell was obtained leukapheresis of a sample obtained from a subject.

58. A cell culture comprising at least one recombinant cell according to any one of claims 51-57, and a culture medium.

59. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more of the following:(a) an antigen-binding molecule according to any one of claims 1-19;(b) a CAR according to any one of claims 20 46;(c) a recombinant nucleic acid according to any one of claims 47-50; and(d) a recombinant T cell according to any one of claims 55-57.

60. The pharmaceutical composition of claim 59, wherein the composition comprises a recombinant T cell according to any one of claims 55-57, and a pharmaceutically acceptable carrier.

61. A method for diagnosing, preventing, and / or treating a health condition in a subject in need thereof, comprising administering to the subject a composition comprising one or more of the following:(a) an antigen-binding molecule according to any one of claims 1-19;(b) a CAR according to any one of claims 20 46;(c) a recombinant nucleic acid according to any one of claims 47-50;(d) a recombinant T cell according to any one of claims 55-57; and(e) a pharmaceutical composition according to any one of claims 59-60.Attorney Docket No.: 078430-546001 WO62. The method of claim 61, wherein the recombinant cells are allogeneic relative to the subject.

63. The method of claim 61, wherein the recombinant cells are autologous relative to the subject.

64. The method of any one of claims 61-63, wherein the health condition is a proliferative disorder.

65. The method of claim 64, wherein the proliferative disorder is a cancer.

66. The method of claim 65, wherein the cancer is positive for B7-H3 expression.

67. The method of claim 66, wherein the B7-H3 -positive cancer is selected from the group consisting of nervous system cancer, cervical cancer, sarcoma, neuroblastoma, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, and hepatocellular carcinoma.

68. The method of claim 66, wherein the B7-H3 -positive cancer is an adult malignancy or a pediatric cancer.

69. The method of claim 68, wherein the pediatric cancer is osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, atypical teratoid rhabdoid tumor, medulloblastoma, or neuroblastoma.

70. The method of any one of claims 61-69, wherein the T cells are obtained from tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs).

71. The method of any one of claims 61-70, wherein the administered composition confers an enhanced effector function of the recombinant T cells.

72. The method of claim 71, wherein the enhanced effector function of the recombinant T cells is selected from the group consisting of growth rate (proliferation), cytokine production, target cell inhibition (e.g., anti-cancer cytotoxicity), macrophage activation, maintenance of enhanced effector cell function, NK cell activation, exhaustion resistance, and in vivo persistence (e.g. , survival).Attorney Docket No.: 078430-546001 WO73. The method of any one of claims 71-72, wherein the enhanced effector function comprises increased production of one or more cytokines.

74. The method of claim 73, wherein the one or more cytokines comprises interferon gamma (IFNy), tumor-necrosis factor a (TNFa), and / or interleukin-2 (IL-2).

75. The method of any one of claims 61-74, wherein the composition is administered to the subject individually (monotherapy) or in combination with a second therapy.

76. The method of claim 75, wherein the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, or surgery.

77. A kit for the diagnosis, prevention, and / or treatment of a health condition in a subject in need thereof, the kit comprising one or more of the following:(a) an antigen-binding molecule according to any one of claims 1-19;(b) a CAR according to any one of claims 20 46;(c) a recombinant nucleic acid according to any one of claims 47-50;(d) a recombinant T cell according to any one of claims 55-57; and(e) a pharmaceutical composition according to any one of claims 59-60.

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