SEMA4a-binding chimeric antigen receptors

Abstract

Disclosed are compositions and methods for targeted treatment of semaphorin 4A (SEMA4A)-expressing cancers. In particular, chimeric antigen receptors (CAR) are provided that are capable of targeting SEMA4A-expressing malignant cells. Also disclosed are isolated nucleic acid sequences encoding the disclosed CAR polypeptides, vectors comprising these isolated nucleic acids, and cells containing these vectors. Also disclosed is a method of providing an anti-tumor immunity in a subject with a SEMA4A-expressing cancer that involves administering to the subject an effective amount of an immune effector cell genetically modified with a disclosed SEMA4Aspecific CAR.

Description

[0001] Attorney Docket No. MOF 24MB032 PCT

[0002] SEMA4A-BINDING CHIMERIC ANTIGEN RECEPTORS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims benefit of U.S. Provisional Application No. 63 / 715,812, filed November 4, 2024, which is hereby incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT INTEREST

[0005] This invention was made with Government Support under Grant No. CA276939 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application contains a sequence listing filed in ST.26 format entitled “MOF 24MB032 PCT Sequence Listing” created on November 3, 2025, and having 18,258 bytes. The content of the sequence listing is incoiporated herein in its entirety.

[0008] BACKGROUND OF THE INVENTION

[0009] Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Two B cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapies - idecabtagene vicleucel (ide-cel), and ciltacabtagene autoleucel (cilta-cel) have been approved by the US Food and Drug Administration (FDA) for the treatment of adult patients with relapsed or refractory multiple myeloma (RRMM). These products have shown unprecedented activity in RRMM but, most patients have short disease remissions and commonly relapse (Kumar, S.K., et al. Nat Rev Dis Primers 2017 3:17046; Costa, L.J., ct al. Blood Adv 2017 1:282-287).

[0010] Downregulation of target antigen density on tumor cells is emerging as an important factor contributing to relapse after CAR T cell therapy (Brudno, J.N., et al. J Clin Oncol 2018 36:2267- 2280; Da Via, M.C., et al. Nat Med 2021 27:616-619). A few thousand molecules are required to trigger CD19-directed CAR T cell activation upon antigen recognition (Dong, R., et al. EMBO J 2020 39:el04730). The efficient cytotoxicity threshold for 4-lBB-based CD19-CARs is around 1x103 target molecules per cell (Majzner, R.G., et al. Cancer Discov 2020 10:702-723; Hamieh, M., et al. Nature 2019 568: 112-116; Watanabe, K., et al. J Immunol 2015 194:911-920), and decreased antigen density upon CD19-CAR T cell therapy represents a mechanism of relapse with antigen-low variants (Cohen, A.D., ct al. J Clin Invest 2019 129:2210-2221).

[0011] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0012] 45791084.1 1 Attorney Docket No. MOF 24MB032 PCT

[0013] SUMMARY OF THE INVENTION

[0014] Disclosed herein are semaphorin 4A (SEMA4A) antibodies and chimeric antigen receptor (CAR) polypeptides that can be used to target and kill cancers. The disclosed CAR polypeptides contain an ectodomain that contains an anti-SEMA4A binding domain, a hinge domain, a transmembrane (TM) domain, and an endodomain that contains a signaling region.

[0015] Also disclosed are isolated nucleic acid sequences encoding the disclosed CAR polypeptides, vectors comprising these isolated nucleic acids, and cells containing these vectors. For example, the cell can be an immune effector cell selected from the group consisting of an alphabeta T cells, a gamma-delta T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, and a regulatory T cell.

[0016] In some embodiments, the cell exhibits an anti-tumor immunity when the antigen binding domain of the CAR binds to a SEMA4A on a tumor.

[0017] Also disclosed is a method of providing an anti-tumor immunity in a subject with a SEMA4A-expressing cancer that involves administering to the subject an effective amount of an immune effector cell genetically modified with a disclosed SEMA4A-specific CAR.

[0018] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0019] BRIEF DESCRIPTION OF FIGURES

[0020] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0021] FIGs. 1 A to IK show SEMA4A is frequently and highly expressed in RRMM patients. FIG. 1A contains representative FACS plots showing downregulation of BCMA expression in clinical samples collected before and after BCMA CAR T cell therapy from the same patient. FIG. IB shows number of BCMA molecules per cell in bone marrow samples collected before and after BCMA CAR T cell therapy from the same patient (n=6 patients), determined by QuantiBRITE assay. ** indicates p < 0.01. The blue box highlights the median (1191) BCMA density greater than 1000 molecules per cell in patients before BCMA CAR T cell therapy. The red box highlights the 45791084.1 2 Attorney Docket No. MOF 24MB032 PCT median (725) BCMA density less than 1000 molecules per cell in patients relapsed after BCMA CAR T cell therapy. FIG. 1C shows absolute BCMA antigen density in 41 samples from MM patients relapsed after BCMA CAR T cell therapy by QuantiBRITE assay. FIG. ID shows distribution of MM-associated targets in 829 MM patients. SEMA4A and BCMA (TNFRS17) as well as the targets currently used in pre-clinical and clinical developments are shown. RNA expression levels are calculated by RNA-Seq by Expectation Maximization (RSEM) from the aligned BAM files in terms of Transcript Per Million mapped reads (TPM). The log2-transformed TPM values (log2TPM) with a pseudo count of 1 is used for the statistical analysis. Distribution of these MM-associated targets in the MMRF database of MM patients is shown in Fig. 6A. FIG. IE shows SEMA4A density in 41 bone marrow samples from MM patient relapsed after BCMA CAR T cell therapy analyzed by flow cytometry using QuantiBRITE assay. *** indicates p < 0.001 comparing SEMA4A to BCMA values. FIG. IF shows percentage of cells positive for SEMA4A or BCMA in 41 samples from MM patients relapsed after BCMA CAR T cell therapy by flow cytometric analysis * indicates p < 0.05. Flow plots from each patient analyzed are shown in Fig. 6B. FIG. 1G shows number of SEMA4A molecules per cell in bone marrow samples collected before and after BCMA CAR T cell therapy from the same patient (n=6 patients), determined by QuantiBRITE assay. ** indicates p < 0.01. Fig. 1H shows Pearson’s correlation showing lack of linear correlation between SEMA4A and BCMA expression based on the Moffitt database of MM patient data (n=829). Python ‘scipy’ and ‘seaborn’ packages are used to perform the statistical analysis and visualization. FIG. II shows Pearson’s correlation shows lack of linear correlation between SEMA4A and BCMA expression based on the MMRF database of MM patient data (n=832). Python ‘scipy’ and ‘seaborn’ packages are used to perform the statistical analysis and visualization. FIG. 1 J shows statistically significant difference in MM patient overall survival based on SEMA4A expression. Log-rank p<le16. FIG. IK shows differential expression between TP53 mutated (N=38) and TP53 wild type patients (N=794). Patients with TP53 at least one coding region affecting mutation (missense, nonsense, in-frame and frame-shift indels and splice site changes) are categorized as TP53 Mutant. Box plot data represent median and IQR.

[0022] FIGs. 2A to 2F show SEMA4A expression in normal tissues. FIG. 2A is a schematic representation of the normal Tissue Micro Array we build. FIG. 2B shows IHC characterization of skin, medulla oblongata, aorta, puberal testis, adult testis, parietal cortex, bladder, prostate, colon, adrenal, ovary, pons, endometrium, hippocampus, thyroid, lung, thymus, and myocardium in a tissue microarray from 36 samples. (Magnification 200X). Control staining per tissue is shown in Fig. 7 A. FIG. 2C shows IHC characterization of thyroid, lung and thymus (upper panel) and relative magnification to highlight positive macrophages (arrows in the lower panel). FIG. 2D

[0023] 45791084.1 3 Attorney Docket No. MOF 24MB032 PCT shows IHC images for liver, kidney, and myocardium (upper panel). FACS plots of THLE2 liver epithelial cells, serving as a model for the normal liver, RPTEC cells derived from the Renal Proximal Tubule Epithelium as a model for the normal kidney and AGIO cells, derived from human cardiomyocytes, as a model for the normal myocardium. FIG. 2E shows single-cell RNA-seq analyses from >30,781 bone marrow cells and 5 healthy donors to map SEMA4A expression in the hematopoietic lineage. Marker per cell type is also shown. The color of each dot encodes the average expression level after scaling, while the size reflects the proportion of positive cells. FIG. 2F shows two-dimensional UMAP visualization by major cell types (left) and normalized gene expression of SEMA4A (right).

[0024] FIGs. 3A to 3H shows genetic targeting of SEMA4A impacts MM cell proliferation and migration in vitro and in vivo. FIG. 3A shows MM cell proliferation assessed in four MM cell lines targeted with two sgRNAs per SEMA4A. Cells are seeded at the same concentration on day 0 and counted manually by trypan blue 24-48-72-96-120 hours after seeding. Knock-out validation in Fig. 8A. Additional MTS proliferation assays are shown in Fig. 8B. FIG. 3B shows MM cell proliferation assessed in four MM cell lines targeted with two sgRNAs per SEMA4A. Cells are seeded at the same concentration on day 0 and counted manually by trypan blue 120 hours after seeding. ** indicates p < 0.01, * indicates p < 0.05. Cell cycle analysis is shown in Figs. 8C-8D. Data on BCMA-KO MM cells are shown in Fig. 8F. FIG. 3C shows KO and control cells are seeded in hollow plastic chambers sealed at one end with a porous membrane and filled with serum free media, as previously described. This chamber was suspended over a larger well containing media supplemented with 10% FBS. The percentage of cells counted in the lower chambers at 48 hours over cells seeded in the upper chambers at the time of input is shown. MTS assays are shown in Fig. 8E. ** indicates p < 0.01. FIG. 3D shows overall survival of tumor-bearing mice injected with SEMA4AKO or control (expressing an empty vector - EV) MM cell lines. Surviving mice are sacrificed 221 days after cell injection. * indicates p < 0.05. FIG. 3E shows human kappa (KMS 11) chain concentrations are measured in the mice serum at multiple time points. ** indicates p < 0.01 . FIG. 3F shows human lambda (U266) chain concentrations are measured in the mice serum at multiple time points. *** indicates p < 0.001. FIG. 3G shows bone marrow tumor infiltration in mice is assessed by IHC. Bone sections (femur) from mice injected with SEMA4AKO or control (EV) KMS11 (left) or U266 (right) MM cell lines are stained with hematoxylin eosin (HE), tartrateresistant acid phosphatase (TRAP), CD 138, and control IgG. TRAP serves to stain osteoclasts which are associated to increased bone destruction, a typical effect of myeloma infiltration. Myeloma cells are stained with CD 138. FIG. 3H shows H-score calculated based on the intensity of CD138 staining. H-score is a digital image analysis-based metric quantifying membrane biomarker

[0025] 45791084.1 4 Attorney Docket No. MOF 24MB032 PCT expression from immunohistochemistry images. * indicates p < 0.05 I) TRAP-positive osteoclast number over bone surface (number of osteoclast / mm2) is manually counted. * indicates p < 0.05, ** indicates p < 0.01. All data are from three independent experiments. All data are represented as mean ± SEM.

[0026] FIGs. 4A to 41 shows generation and assessment of the efficacy of SEMA4A CAR T cells in eliminating myeloma cells. FIG. 4A is a schematic representation of the strategy utilized to generate human antibodies recognizing SEMA4A. NIH / 3T3 cells overexpressing human SEMA4A and recombinant peptides covering the ECD of SEMA4A are used to immunize humanized ATX- GXTM mice. FIG. 4B shows MFI values from flowcytometric analyses of 8 selected antibodies conjugated with secondary antibodies or the SEMA4A commercially available antibody (Cat#148406 Biolegend) in U266 MM cell lines endogenously expressing SEMA4A or knocked- out for SEMA4A. Normal cardiomyocyte (AGIO), Primary Renal Proximal Tubule Epithelial Cells (RPTEC) and liver epithelial cells (THLE-2) are used as negative controls. Experiments are performed in duplicates. Results on an additional cell line (KMS11) are shown in Fig. 9A. FACS plots are shown in Fig. 9B. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. FIG. 4C shows heavy (VH) and light (VL) chain variable fragments of each SEMA4A-binding antibody used to develop single-chain variable fragment (scFv) for CAR T constructs. FIG. 4D shows cytotoxicity assays using a 16h bioluminescence assay, using firefly luciferase expressing KMS11 and U266 as targets cells. (n=3 independent biological experiments from 3 healthy donors). * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001. Immunophenotype of engineered CAR T cells is shown in Figs. 9C-9D. Lack of T cell killing is shown in Fig. 9E. FIG. 4E shows representative cumulative cell counts of CAR T cells upon weekly stimulations with KMS11 MM cells. Arrows indicate stimulation time point. (n=3 independent biological experiments from 3 healthy donors). FIG. 4F shows cytokines produced by CAR T cells after 1 : 1 co-culture with KMS 11 MM cells or alone for 16h, measured by Luminex assay. (n=3 independent biological experiments from 3 healthy donors). FIG. 4G shows imaging of NSG mice injected intravenously with 1x106 KMS11-ffLuc MM cells followed by IxlO6CAR T cells, 7 days later. Tumor burden shown as bioluminescent signal quantified per animal every 2 weeks is shown in Fig. 9F. FIG. 4H shows overall survival data of NSG mice injected intravenously with IxlO6KMS11-ffLuc MM cells followed by IxlO6CAR T cells, 7 days later. FIG. 41 shows screening used to identify lead CAR constructs. Data are represented as mean ± SEM. FIG. 4F shows patient data.

[0027] FIGs. 5 A to 5C show limiting doses of BCMA CAR T cells reproduce myeloma with low BCMA levels. FIG. 5A shows an experimental design. Mice bearing H929 MM cells are treated

[0028] 45791084.1 5 Attorney Docket No. MOF 24MB032 PCT with sub-curative doses of BCMA CAR T cell and at day 30, arc infused with SEMA4A-dircctcd CAR T cells. FIG. 5B shows after the first infusion of BCMA CAR T cells, tumor burden is monitored as the average radiance of bioluminescence (photons s-1 cm -2 sr-1) at day 8,15, 22, and 29 post MM cell injection. FIG. 5C contains histograms showing downregulation of human BCMA in vivo on CD138+ cells at 28 post-injection in the group treated with 0.2xl06CAR T cells compared to progressing cells in the group of untransduced mice. FIG. 5D shows average radiance of bioluminescence (photons s-1 cm -2 sr-1) indicating the tumor burden after the second infusion of CAR T cells. Data are represented as mean ± SD. *** indicates p < 0.001, **** indicates p < 0.0001.

[0029] FIGs. 6A and 6B show BCMA and SEMA4A expression in primary patients. FIG. 6A shows distribution of candidate targets in the MMRF database of MM patient data. FIG. 6B shows FACS plots from 41 MM patient samples.

[0030] FIG. 7 shows SEMA4A expression in normal tissues, i.e. Tissue Micro Array pankeratin controls matching samples shown in Figure 2B.

[0031] FIGs. 8A to 8F shows genetic targeting of SEMA4A. FIG. 8A shows knock-out of SEMA4A shown by flowcytometry in 6 MM cell lines genetically targeted with 2 sgRNAs and CRISPR-Cas9 technology. FIG. 8B shows cell viability determined by MTS assay in MM cell lines targeted with two sgRNAs per molecule. * indicates p < 0.05, ** indicates p < 0.01. FIG. 8C contains representative FACS plots of cell cycle analysis in KMS 11 MM cell line expressing an empty vector or SEMA4A KO. FIG. 8D shows percentages of KMS11 MM cells that are in either the G1 or G2 phase. FIG. 8E shows measured absorbance of KO or control migrated cells after 48 hours by MTS assay. * indicates p < 0.05, ** indicates p < 0.01. FIG. 8F shows cell viability determined by manual counting MM cell lines targeted with sgRNAs for BCMA or SEMA4A. *** indicates p < 0.001, **** indicates p < 0.0001. All data are from three independent experiments. All data are represented as mean ± SEM.

[0032] FIGs. 9A to 9F show SEMA4A CAR T cells. FIG. 9A shows MFI values from flowcytometric analyses of 8 selected antibodies conjugated with secondary antibodies or the SEMA4A commercially available antibody in KMS 11 MM cell lines endogenously expressing SEMA4A or knocked-out for SEMA4A. Experiments are performed in duplicates and results on MM cells compared to KO cells ** indicates p < 0.01, *** indicates p < 0.001. FIG. 9B shows flowcytometric analysis of 8 selected antibodies conjugated with secondary antibodies in U266 and KMS 11 MM cell lines expressing SEMA4A or knocked-out for SEMA4A. FIG. 9C shows immunophenotype of untransduced T cells compared to T cells lentivirally expressing different CAR constructs differing on the scFv recognizing SEMA4A or BCMA. Plots indicate the

[0033] 45791084.1 6 Attorney Docket No. MOF 24MB032 PCT phenotypes of the CAR-positive T cells measured by flow cytometry analysis of CD62L and CD45RA expression. T cells were freshly purified, activated (day 0) and infected to lentivirally express different CAR constructs and analyzed at day 5. FIG. 9D contains representative FACS plots. FIG. 9E shows cell killing of T cells induced by CAR T cells integrating the indicated scFv after 16h of co-culture. Dead cells are measured by CytoTox-Glo™ Cytotoxicity Assay. FIG. 9F shows NSG mice injected intravenously with IxlO6KMSl l-ffLuc MM cells are treated with IxlO6CAR T cells, 7 days after MM cell injection. Tumor burden shown as bioluminescent signal quantified per animal every 2 weeks. All data are from three independent experiments. All data are represented as mean ± SEM.

[0034] DETAILED DESCRIPTION

[0035] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0036] 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.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0038] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0039] 45791084.1 7 Attorney Docket No. MOF 24MB032 PCT

[0040] Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0041] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0042] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0043] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0044] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0045] Definitions

[0046] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0047] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of

[0048] 45791084.1 8 Attorney Docket No. MOF 24MB032 PCT those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.

[0049] The term “aptamer” refers to oligonucleic acid or peptide molecules that bind to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity. A “nucleic acid aptamer” is a DNA or RNA oligonucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of such molecule. A nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof. A “peptide aptamer” is a combinatorial protein molecule with a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast dihybrid conditions, which enhances the probability for the selected peptide aptamers to be stably expressed and correctly folded in an intracellular context.

[0050] The term “carrier” means a compound, composition, substance, or slruclure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0051] The term “chimeric molecule” refers to a single molecule created by joining two or more molecules that exist separately in their native state. The single, chimeric molecule has the desired functionality of all of its constituent molecules. One type of chimeric molecules is a fusion protein.

[0052] The term “fusion protein” refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

[0053] The term “identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching

[0054] 45791084.1 9 Attorney Docket No. MOF 24MB032 PCT nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0055] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0056] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0057] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0058] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity,

[0059] 45791084.1 10 Attorney Docket No. MOF 24MB032 PCT irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0060] The term “protein domain” refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.

[0061] A “spacer" as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.

[0062] The term “specifically binds", as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody “specifically binds” to its particular “target" (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 105M1(e.g., 106M ’, 107M ', 108M '. 109M ’, IO10M 1011M and 1012M1or more) with that second molecule.

[0063] The term “specifically deliver” as used herein refers to the preferential association of a molecule with a cell or tissue bearing a particular target molecule or marker and not to cells or tissues lacking that target molecule. It is, of course, recognized that a certain degree of non-specific interaction may occur between a molecule and a non- target cell or tissue. Nevertheless, specific delivery, may be distinguished as mediated through specific recognition of the target molecule. Typically specific delivery results in a much stronger association between the delivered molecule and cells bearing the target molecule than between the delivered molecule and cells lacking the target molecule.

[0064] The term “subject" refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0065] 45791084.1 11 Attorney Docket No. MOF 24MB032 PCT

[0066] The term “therapeutically effective’' refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0067] The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.

[0068] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0069] The term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid subsitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0070] The term “vector” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0071] SEMA4A-specific chimeric antigen receptors (CAR)

[0072] CARs generally incorporate an antigen recognition domain from the single-chain variable fragments (scFv) of a monoclonal antibody (mAb) with transmembrane signaling motifs involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 2003 3:35-45). Disclosed herein is a SEMA4A-specific chimeric antigen receptor (CAR) that can be that can be expressed in immune effector cells to enhance antitumor activity against SEMA4A-specific CARs.

[0073] The disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain comprises the SEMA4A-binding region and is

[0074] 45791084.1 12 Attorney Docket No. MOF 24MB032 PCT responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell. The transmembrane domain (TD), is as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell. The endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition. For example, the endodomain can contain an intracellular signaling domain (ISD) and optionally a co-stimulatory signaling region (CSR). In some embodimenst the disclosed CARs contain in the endodomain the intracellular domain of NKG2D.

[0075] A “signaling domain (SD)” generally contains immunoreceptor tyrosine-based activation motifs (IT AMs) that activate a signaling cascade when the IT AM is phosphorylated. The term “costimulatory signaling region (CSR)’’ refers to intracellular signaling domains from costimulatory protein receptors, such as CD28, 41 BB, and ICOS, that are able to enhance T-cell activation by T- cell receptors.

[0076] In some embodiments, the endodomain contains an SD or a CSR, but not both. In these embodiments, an immune effector cell containing the disclosed CAR is only activated if another CAR (or a T-cell receptor) containing the missing domain also binds its respective antigen.

[0077] In some embodiments, the disclosed CAR is defined by the formula: SP- BD -HG-TM-CSR-SD; or

[0078] SP BD HG TM SD CSR; wherein “SP” represents an optional signal peptide, wherein “BD” represents a SEMA4A-binding region, wherein “HG” represents an optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CSR” represents one or more co-stimulatory signaling regions, wherein “SD” represents a signaling domain, and wherein represents a peptide bond or linker.

[0079] In some embodiments, the disclosed CAR is defined by the formula: SP-BD-HG-TM-NKRIC; wherein “SP” represents an optional signal peptide, wherein “BD” represents a SEMA4A target binding domain,

[0080] 45791084.1 13 Attorney Docket No. MOF 24MB032 PCT wherein “HG” represents an optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “NKRIC” represents an NK cell receptor intracellular domain, and wherein represents a peptide bond or linker.

[0081] Additional CAR constructs are described, for example, in Fresnak AD, et al. Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23; 16(9):566-81 , which is incorporated by reference in its entirety for the teaching of these CAR models.

[0082] For example, the CAR can be a TRUCK, Universal CAR, Self-driving CAR, Armored CAR, Self-destruct CAR, Conditional CAR, Marked CAR, TenCAR, Dual CAR, or sCAR.

[0083] TRUCKS (T cells redirected for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an antitumor cytokine. Cytokine expression may be constitutive or induced by T cell activation. Targeted by CAR specificity, localized production of pro-inflammatory cytokines recruits endogenous immune cells to tumor sites and may potentiate an antitumor response.

[0084] Universal, allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft- versus-host disease (GVHD) or rejection, respectively.

[0085] Self-driving CARs co-express a CAR and a chemokine receptor, which binds to a tumor ligand, thereby enhancing tumor homing.

[0086] CAR T cells engineered to be resistant to immunosuppression (Armored CARs) may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)), with an immune checkpoint switch receptor, or may be administered with a monoclonal antibody that blocks immune checkpoint signaling.

[0087] A self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of activation of human caspase 9 by a small-molecule dimerizer.

[0088] A conditional CAR T cell is by default unresponsive, or switched ‘off’, until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell. Alternatively, T cells may be engineered to express an adaptor-

[0089] 45791084.1 14 Attorney Docket No. MOF 24MB032 PCT specific receptor with affinity for subsequently administered secondary antibodies directed at target antigen.

[0090] Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects.

[0091] A tandem CAR (TanCAR) T cell expresses a single CAR consisting of two linked singlechain variable fragments (scFvs) that have different affinities fused to intracellular co-stimulatory domain(s) and a CD3 domain. TanCAR T cell activation is achieved only when target cells coexpress both targets.

[0092] A dual CAR T cell expresses two separate CARs with different ligand binding targets: one CAR includes only the CD3^ domain and the other CAR includes only the co-stimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets on the tumor.

[0093] A safety CAR (sCAR) consists of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.

[0094] The antigen recognition domain of the disclosed CAR is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen recognition region.

[0095] The endodomain is the business end of the CAR that after antigen recognition transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Therefore, the endodomain may comprise the “intracellular signaling domain” of a T cell receptor (TCR) and optional co-receptors. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.

[0096] Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs). Examples of IT AM containing cytoplasmic signaling

[0097] 45791084.1 15 Attorney Docket No. MOF 24MB032 PCT sequences include those derived from CD8, CD3 , CD38, CD3y, CD3E, CD32 (Fc gamma Rlla), DAP10, DAP12, CD79a, CD79b, FcyRIy, FcyRIIIy, FceRip (FCERIB), and FceRIy (FCERIG).

[0098] In particular embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3Q (TCR zeta, GenBank aceno. BAG36664.1). T-cell surface glycoprotein CD3 zeta (CD3Q chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene.

[0099] First-generation CARs typically had the intracellular domain from the CD3^ chain, which is the primary transmitter of signals from endogenous TCRs. Second-generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, third-generation CARs combine multiple signaling domains to further augment potency. T cells grafted with these CARs have demonstrated improved expansion, activation, persistence, and tumor-eradicating efficiency independent of costimulatory receptor / ligand interaction (Imai C, et al. Leukemia 2004 18:676-84; Maher J, et al. Nat Biotechnol 2002 20:70-5).

[0100] For example, the endodomain of the CAR can be designed to comprise the CD3^ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAPI2, MyD88, BTNL3, and NKG2D. Thus, while the CAR is exemplified primarily with CD28 as the co- stimulatory signaling element, other costimulatory elements can be used alone or in combination with other co-stimulatory signaling elements.

[0101] In some embodiments, the CAR comprises a hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety (e.g., anti-SEMA4A scFv) and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some

[0102] 45791084.1 16 Attorney Docket No. MOF 24MB032 PCT embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0103] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, or CD 154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld. ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRTAM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CD100 (SEMA4D) , SLAMF6 (NTB-A, Lyl08) , SLAM (SLAMF1, CD 150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD 162) , LTBR, and PAG / Cbp. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.

[0104] In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.

[0105] In some embodiments, the CAR is a multi-chain CAR, as described in WO2015 / 039523, which is incorporated by reference for this teaching. A multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction. For example, the multi-chain CAR can comprise a part of an FCERI alpha chain and a part of an FCERI beta chain such that the FCERI chains spontaneously dimerize together to form a CAR.

[0106] In some embodiments, the anti-SEMA4A binding agent is single chain variable fragment (scFv) antibody. The affinity / specificity of an anti-SEMA4A scFv is driven in large part by specific sequences within complementarity determining regions (CDRs) in the heavy (VH) and light (VL) chain. Each VH and VL sequence will have three CDRs (CDR1, CDR2, CDR3).

[0107] 45791084.1 17 Attorney Docket No. MOF 24MB032 PCT

[0108] In some embodiments, the anti-SEMA4A binding agent is derived from natural antibodies, such as monoclonal antibodies. In some cases, the antibody is human. In some cases, the antibody has undergone an alteration to render it less immunogenic when administered to humans. For example, the alteration comprises one or more techniques selected from the group consisting of chimerization, humanization, CDR-grafting, deimmunization, and mutation of framework amino acids to correspond to the closest human germline sequence.

[0109] Also disclosed are bi-specific CARs that target SEMA4A and at least one additional tumor antigen. Also disclosed are CARs designed to work only in conjunction with another CAR that binds a different antigen, such as a tumor antigen. For example, in these embodiments, the endodomain of the disclosed CAR can contain only an signaling domain (SD) or a co-stimulatory signaling region (CSR), but not both. The second CAR (or endogenous T-cell) provides the missing signal if it is activated. For example, if the disclosed CAR contains an SD but not a CSR, then the immune effector cell containing this CAR is only activated if another CAR (or T-cell) containing a CSR binds its respective antigen. Likewise, if the disclosed CAR contains a CSR but not a SD, then the immune effector cell containing this CAR is only activated if another CAR (or T-cell) containing an SD binds its respective antigen.

[0110] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The additional antigen binding domain can be an antibody or a natural ligand of the tumor antigen. The selection of the additional antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-llRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr- abl, HER2, 0-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD 19, TIM3, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM I, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, plysialic acid, PLAC1 , RU1, RU2 (AS), intestinal carboxyl esterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostatecarcinoma tumor antigen- 1 (PCTA-1), ML-IAP, MAGE, MAGE-A1,MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1 , ELF2M, ERG (TMPRSS2 ETS fusion gene), NAU, neutrophil elastase, sarcoma translocation breakpoints, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30,

[0111] 45791084.1 18 Attorney Docket No. MOF 24MB032 PCT

[0112] TIM3, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, R0R1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, TIM3, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof.

[0113] Non-limiting examples of tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm- 23H1, PSA, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F. 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0114] Anti-SEMA4A Binding Domain

[0115] The binding domain is in some embodiments an antibody fragment that specifically binds SEMA4A on a cell. For example, the antigen binding domain can be a Fab or a single-chain variable fragment (scFv) of an antibody that specifically binds the target molecule. The binding domain is in some embodiments an aptamer that specifically binds the tar target molecule. For example, the binding domain can be a peptide aptamer selected from a random sequence pool based on its ability to bind the target molecule. The binding domain can also be a natural ligand of the target molecule, or a variant and / or fragment thereof capable of binding the target molecule.

[0116] In some embodiments, the anti-SEMA4A scFv can comprise a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences.

[0117] In some embodiments, the VH domain comprises the amino acid sequence: EVKLVESGGGLVKPGGSLNLSCAASGFTFNSFTMSWVRQTPAKRLEWVATISSSGYNTYY

[0118] 45791084.1 19 Attorney Docket No. MOF 24MB032 PCT

[0119] PDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGKIYDGYPFAMDFWGQGTSVT VSS (SEQ ID NO:1), and the VL domain comprises the amino acid sequence:

[0120] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYHQKQGKSPQLLVYAATNLADGVPSR

[0121] FSGNGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIK (SEQ ID NO:2).

[0122] In some embodiments, the VH domain comprises the amino acid sequence:

[0123] QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIDPSDSETH

[0124] YNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARHYDGYAVDYWGQGTSVTVSS (SEQ ID NO: 3), and the VL domain comprises the amino acid sequence:

[0125] DIVLTQSPASLPVSLGQRATISCRASQSVTTSRYSYMHWYQQKPGQPPKLLIMYASNLESG VPARFSGSGSGTDFTLNIHPVEEEDIAIYYCQHSWEIPWTFGGGTKLEIK (SEQ ID NO:4).

[0126] In some embodiments, the VH domain comprises the amino acid sequence:

[0127] EVQLHQSGADLVKPGASVKLSCTASGFHIKDTYIHWMKQRPEQGLEWIGRIDPANGNSEY

[0128] DPKFQGKATITADTSSNTAYLHLSSLTSEDTAVYYCAESNYFGSSPYALDYWGQGTSVTVS

[0129] S (SEQ ID NO: 5), and the VL domain comprises the amino acid sequence:

[0130] DIILTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGS GSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLEL (SEQ ID NO:6).

[0131] In some embodiments, the VH domain comprises the amino acid sequence:

[0132] EVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPAKRLEWVATISGSGGNIYY

[0133] PDTVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCTRQEGGLRGFAYWGQGTLVTVSA AKTTPPSVYPL (SEQ ID NO:7), and the VL domain comprises the amino acid sequence:

[0134] DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQLLVYAATNLADGVPSR FSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGIPYTFGGGTKLEIT (SEQ ID NO:8).

[0135] The heavy and light chains are preferably separated by a linker. Suitable linkers for scFv antibodies are known in the art. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:9).

[0136] In some embodiments, the anti-SEMA4A scFv comprises an amino acid sequence:

[0137] EVKLVESGGGLVKPGGSLNLSCAASGFTFNSFTMSWVRQTPAKRLEWVATISSSGYNTYY PDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGKIYDGYPFAMDFWGQGTSVT VSSGGGGSGGGGSGGGGSDIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYHQKQGKS PQLLVYAATNLADGVPSRFSGNGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTK LEIK (SEQ ID NO: 10, VH-linker-VL) or DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYHQKQGKSPQLLVYAATNLADGVPSR FSGNGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIKGGGGSGGGGSGGGG SEVKLVESGGGLVKPGGSLNLSCAASGFTFNSFTMSWVRQTPAKRLEWVATISSSGYNTY

[0138] 45791084.1 20 Attorney Docket No. MOF 24MB032 PCT

[0139] YPDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGKIYDGYPFAMDFWGQGTS

[0140] VTVSS (SEQ ID NO:11, VL-linker-VH).

[0141] In some embodiments, the anti-SEMA4A scFv comprises an amino acid sequence: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIDPSDSETH YNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARHYDGYAVDYWGQGTS VTVSS GGGGSGGGGSGGGGSDIVLTQSPASLPVSLGQRATISCRASQSVTTSRYSYMHWYQQKPG QPPKLLIMYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDIAIYYCQHSWEIPWTFGGGTK LEIK (SEQ ID NO: 12, VH-linker-VL) or DIVLTQSPASLPVSLGQRATISCRASQSVTTSRYSYMHWYQQKPGQPPKLLIMYASNLESG VPARFSGSGSGTDFTLNIHPVEEEDIAIYYCQHSWEIPWTFGGGTKLEIKGGGGSGGGGSGG GGSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIDPSDS ETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARHYDGYAVDYWGQGTSVT VSS (SEQ ID NO: 13, VL-linker-VH).

[0142] In some embodiments, the anti-SEMA4A scFv comprises an amino acid sequence: EVQLHQSGADLVKPGASVKLSCTASGFHIKDTYIHWMKQRPEQGLEWIGRIDPANGNSEY DPKFQGKATITADTSSNTAYLHLSSLTSEDTAVYYCAESNYFGSSPYALDYWGQGTSVTVS SGGGGSGGGGSGGGGSDIILTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRL LIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLEL (SEQ ID NO: 14, VH- linker- VL) or DIILTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGS GSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELGGGGSGGGGSGGGGSEVQL HQSGADLVKPGASVKLSCTASGFHIKDTYIHWMKQRPEQGLEWIGRIDPANGNSEYDPKF QGKATITADTSSNTAYLHLSSLTSEDTAVYYCAESNYFGSSPYALDYWGQGTS VTVSS (SEQ ID NO: 15, VL-linker-VH).

[0143] In some embodiments, the anti-SEMA4A scFv comprises an amino acid sequence: EVKLVESGGGEVKPGGSLKLSCAASGFTFSGYTMSWVRQTPAKREEWVATISGSGGNIYY PDTVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCTRQEGGLRGFAYWGQGTLVTVSA AKTTPPSVYPLGGGGSGGGGSGGGGSDIQMTQSPASLSVSVGETVTITCRASENIYNNLAW YQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGIPY TFGGGTKLEIT (SEQ ID NO: 16, VH-linker-VT.) or DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQLLVYAATNLADGVPSR FSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGIPYTFGGGTKLEITGGGGSGGGGSGGGGSE VKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPAKRLEWVATISGSGGNIYYP

[0144] 45791084.1 21 Attorney Docket No. MOF 24MB032 PCT

[0145] DTVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCTRQEGGLRGFAYWGQGTLVTVSAA

[0146] KTTPPSVYPL (SEQ ID NO: 17, VL-linker-VH).

[0147] Immune effector cells

[0148] Also disclosed are immune effector cells that are engineered to express the disclosed CARs (also referred to herein as “CAR-T cells.” These cells are preferably obtained from the subject to be treated (i.e. are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0149] In some embodiments, the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials. For example, the immune effector cells can comprise lymphocytes, monocytes, macrophages, dentritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof. For example, the immune effector cells can comprise T lymphocytes.

[0150] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.

[0151] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide

[0152] 45791084.1 22 Attorney Docket No. MOF 24MB032 PCT antigens by MHC class II molecules, which arc expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including Tnl, TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response.

[0153] Cytotoxic T cells (Tc cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.

[0154] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4+or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0155] Regulatory T cells (Tregcells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+Tregcells have been described — naturally occurring Tregcells and adaptive Tregcells.

[0156] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD Id.

[0157] In some embodiments, the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T comprise are cytotoxic CD8+T lymphocytes. In some embodiments, the T cells comprise y5 T cells, which possess a distinct T-cell receptor (TCR) having one y chain and one 5 chain instead of a and chains.

[0158] Natural-killer (NK) cells are CD56+CD3 large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8+T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and

[0159] 45791084.1 23 Attorney Docket No. MOF 24MB032 PCT can also eradicate MHC-I-ncgativc cells (Nami-Mancinclli E, ct al. Int Immunol 2011 23:427-431). NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target, off-tumor effects. Although NK cells have a well- known role as killers of cancer cells, and NK cell impairment has been extensively documented as crucial for progression of MM (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676; Fauriat C, et al. Leukemia 2006 20:732-733), the means by which one might enhance NK cell-mediated anti- MM activity has been largely unexplored prior to the disclosed CARs.

[0160] Nucleic Acids and Vectors

[0161] Also disclosed are polynucleotides and polynucleotide vectors encoding the disclosed CAR polypeptides that allow expression of the CAR polypeptides in the cells.

[0162] Nucleic acid sequences encoding the disclosed antibodies, and regions thereof, can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0163] Expression of nucleic acids encoding antibodies is typically achieved by operably linking a nucleic acid encoding the antibody polypeptide to a promoter, and incorporating the const rucl into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0164] The disclosed nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0165] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. In some embodiments, the polynucleotide vectors are lentiviral or retroviral vectors.

[0166] 45791084.1 24 Attorney Docket No. MOF 24MB032 PCT

[0167] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant vims can then be isolated and delivered to cells of the subject either in vivo or ex vivo.

[0168] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor- 1 a (EF- la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian vims 40 (SV40) early promoter, MND (myeloproliferative sarcoma vims) promoter, mouse mammary tumor vims (MMTV), human immunodeficiency vims (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia vims promoter, an Epstein-Barr vims immediate early promoter, a Rous sarcoma vims promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. The promoter can alternatively be an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0169] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.

[0170] In order to assess the expression of an antibody polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes.

[0171] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is

[0172] 45791084.1 25 Attorney Docket No. MOF 24MB032 PCT manifested by some easily detectable property, c.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0173] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0174] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well- known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0175] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.

[0176] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0177] In the case where a non- viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not

[0178] 45791084.1 26 Attorney Docket No. MOF 24MB032 PCT limited to any particular structure in solution. For example, they may be present in a bilaycr structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).

[0179] Pharmaceutical composition

[0180] Also disclosed is a pharmaceutical composition comprising a disclosed molecule in a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. For example, suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21 ed.) ed. PP. Gerbino, Lippincott Williams & Wilkins, Philadelphia, PA. 2005. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0181] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0182] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous

[0183] 45791084.1 27 Attorney Docket No. MOF 24MB032 PCT carriers include water, alcoholic / aqucous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0184] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0185] Methods of Treatment

[0186] Also disclosed is a method for treating a SEMA4A-expressing cancer in a subject by administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition.

[0187] The disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.

[0188] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained.

[0189] The compositions disclosed herein may be administered prophylactically to patients or subjects who are at risk for a SEMA4A-expressing cancer. Thus, the method can further comprise identifying a subject at risk for a SEMA4A-expressing cancer prior to administration of the herein disclosed compositions.

[0190] 45791084.1 28 Attorney Docket No. MOF 24MB032 PCT

[0191] The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A typical daily dosage of the disclosed composition used alone might range from about 1 pg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.

[0192] In some embodiments, the molecule is administered in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about Ipg to about 100 mg per kg of body weight, from about 1 pg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of molecule containing lenalidomide administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 pg, 10 pg, 100 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 1 1 mg, 12 mg, 1 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.

[0193] The cancer of the disclosed methods can be any SEMA4A-expressing cell in a subject undergoing unregulated growth, invasion, or metastasis. Cancers that express SEMA4A include prostate cancer, ovarian cancer, adenocarcinoma of the lung, breast cancer, endometrial cancer, gastric cancer, colon cancer, and pancreatic cancer. SEMA4A has also been found on Jurkat cells. In some aspects, the cancer is a gallbladder cancer, exocrine adenocarcinoma, or apocrine adenocarcinomas.

[0194] 45791084.1 29 Attorney Docket No. MOF 24MB032 PCT

[0195] In some aspects, the cancer can be any neoplasm or tumor for which radiotherapy is currently used. Alternatively, the cancer can be a neoplasm or tumor that is not sufficiently sensitive to radiotherapy using standard methods. Thus, the cancer can be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat include lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, endometrial cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, and pancreatic cancer.

[0196] The disclosed antibodies can be used in combination with any compound, moiety or group which has a cytotoxic or cytostatic effect. Drug moieties include chemotherapeutic agents, which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those which are used for cancer therapy.

[0197] The disclosed antibodies can be used in combination with a checkpoint inhibitor. The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of cosignaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1 , an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS- 936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0198] Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD- 1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies.

[0199] 45791084.1 30 Attorney Docket No. MOF 24MB032 PCT

[0200] Anti-PD-Ll antibodies and uses therefor arc described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-Ll antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0201] In some embodiments, the PDU1 inhibitor comprises an antibody that specifically binds PDU1, such as BMS-936559 (Bristol-Myers Squibb) or MPDU3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds PD1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies to PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-Ul antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-Ul antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0202] The disclosed antibodies can be used in combination with other cancer immunotherapies. There are two distinct types of immunotherapy: passive immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells, without necessarily initiating an immune response in the patient, while active immunotherapy actively triggers an endogenous immune response. Passive strategies include the use of the monoclonal antibodies (mAbs) produced by B cells in response to a specific antigen. The development of hybridoma technology in the 1970s and the identification of tumor-specific antigens permitted the pharmaceutical development of mAbs that could specifically target tumor cells for destruction by the immune system. Thus far, mAbs have been the biggest success story for immunotherapy; the top three best-selling anticancer drugs in 2012 were mAbs. Among them is rituximab (Rituxan, Genentech), which binds to the CD20 protein that is highly expressed on the surface of B cell malignancies such as non-Hodgkin’s lymphoma (NHU). Rituximab is approved by the FDA for the treatment of NHU and chronic lymphocytic leukemia (CUE) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin; Genentech), which revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0203] Generating optimal “killer” CD8 T cell responses also requires T cell receptor activation plus co-stimulation, which can be provided through ligation of tumor necrosis factor receptor family members, including 0X40 (CD134) and 4-1BB (CD137). 0X40 is of particular interest as

[0204] 45791084.1 31 Attorney Docket No. MOF 24MB032 PCT treatment with an activating (agonist) anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors.

[0205] In some embodiments, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5- fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.

[0206] In some embodiments, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin .

[0207] In some embodiments, such an additional therapeutic agent may be selected from an antimitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.

[0208] In some embodiments, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.

[0209] In some embodiments, such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBl (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as a HER2 antibody, e.g. trastuzumab, trastuzumab-DM 1 or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).

[0210] In some embodiments, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0211] Therefore, in some embodiments, a disclosed antibody is used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0212] In some embodiments, a therapeutic agent for use in combination with antibodies as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL- 10, IL- 12, IL- 13, IL- 15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN , GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines may include Glu- Leu-Arg (ELR)- negative chemokines such as IP- 10, MCP-3, MIG, and SDF-la from the human

[0213] 45791084.1 32 Attorney Docket No. MOF 24MB032 PCT

[0214] CXC and C-C chcmokinc families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0215] In some embodiments, a therapeutic agent for use in combination with antibodies as described above may be a cell cycle control / apoptosis regulator (or "regulating agent"). A cell cycle control / apoptosis regulator may include molecules that target and modulate cell cycle control / apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7- hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055) . Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.

[0216] In some embodiments, a therapeutic agent for use in combination with antibodies as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, an antiandrogene (such as flutaminde / eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy- progesterone / provera, megestrol acepate / megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole / arimidex, aminoglutethimide / cytraden, exemestane) or a hormone inhibitor (such as octreotide / sandostatin) .

[0217] In some embodiments, a therapeutic agent for use in combination with antibodies as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0218] Combined administration, as described above, may be simultaneous, separate, or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.

[0219] In some embodiments, the disclosed antibodies are administered in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-i l l.

[0220] 45791084.1 33 Attorney Docket No. MOF 24MB032 PCT

[0221] In some embodiments, the disclosed antibodies arc administered in combination with surgery.

[0222] Embodiments

[0223] Embodiment 1. A chimeric antigen receptor (CAR) polypeptide, comprising a TRP1 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co- stimulatory signaling region, wherein the TRP1 antigen binding domain is a single-chain variable fragment (scFv) of an antibody comprising a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences, and wherein the CDR1 sequence of the VII domain comprises the amino acid sequence GFTFNSFT (SEQ ID NO: 22) the CDR2 sequence of the VH domain comprises the amino acid sequence ISSSGYNT (SEQ ID NO:23), the CDR3 sequence of the VH domain comprises the amino acid sequence ARHGKIYDGYPFAMDF (SEQ ID NO:24), the CDR1 sequence of the VLcomprises the amino acid sequence ENIYSN (SEQ ID NO:25), the CDR2 sequence of the VL domain comprises the amino acid sequence AAT, and the CDR3 sequence of the VL domain comprises the amino acid sequence QHFWGTPWT (SEQ ID NO:26); wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GYTFTSYW (SEQ ID NO:27) the CDR2 sequence of the VH domain comprises the amino acid sequence IDPSDSET (SEQ ID NO:28), the CDR3 sequence of the VH domain comprises the amino acid sequence ARHYDGYAVDY (SEQ ID NO:29), the CDR1 sequence of the VL comprises the amino acid sequence QSVTTSRYSY (SEQ ID NO:30), the CDR2 sequence of the VL domain comprises the amino acid sequence YAS, and the CDR3 sequence of the VL domain comprises the amino acid sequence QHSWEIPWT (SEQ ID NO:31); wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GFHIKDTY (SEQ ID NO:32) the CDR2 sequence of the VH domain comprises the amino acid sequence IDPANGNS (SEQ ID NO:33), the CDR3 sequence of the VH domain comprises the amino acid sequence AESNYFGSSPYAEDY (SEQ ID NO:34), the CDR1 sequence of the VLcomprises the amino acid sequence QSISNN (SEQ ID NO:35), the CDR2 sequence of the VL domain comprises the amino acid sequence YAS, and the CDR3 sequence of the VL domain comprises the amino acid sequence QQSNSWPLT (SEQ ID NO:36); or wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GFTFSGYT (SEQ ID NO:37) the CDR2 sequence of the VH domain comprises the amino acid sequence ISGSGGNI (SEQ ID NO:38), the CDR3 sequence of the VH domain comprises the amino acid sequence TRQEGGLRGFAY (SEQ ID NO:39), the CDR1 sequence of the VL comprises the amino acid sequence ENIYNN (SEQ ID NO:40), the CDR2 sequence of the VL domain comprises

[0224] 45791084.1 34 Attorney Docket No. MOF 24MB032 PCT the amino acid sequence AAT, and the CDR3 sequence of the VL domain comprises the amino acid sequence QHFWGIPYT (SEQ ID NO:41).

[0225] Embodiment 2. The polypeptide of embodiment 1, wherein the VH domain comprises the amino acid sequence:

[0226] EVKLVESGGGLVKPGGSLNLSCAASGFTFNSFTMSWVRQTPAKRLEWVATISSSGYNTYY

[0227] PDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGKIYDGYPFAMDFWGQGTSVT VSS (SEQ ID NO:1), and the VL domain comprises the amino acid sequence:

[0228] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYHQKQGKSPQLLVYAATNLADGVPSR FSGNGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIK (SEQ ID NO:2);

[0229] Embodiment 3. The polypeptide of embodiment 1, wherein the VH domain comprises the amino acid sequence:

[0230] QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIDPSDSETH

[0231] YNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARHYDGYAVDYWGQGTSVTVSS (SEQ ID NO: 3), and the VL domain comprises the amino acid sequence:

[0232] DIVLTQSPASLPVSLGQRATISCRASQSVTTSRYSYMHWYQQKPGQPPKLLIMYASNLESG VPARFSGSGSGTDFTLNIHPVEEEDIAIYYCQHSWEIPWTFGGGTKLEIK (SEQ ID NO:4);

[0233] Embodiment 4. The polypeptide of embodiment 1, wherein the VH domain comprises the amino acid sequence:

[0234] EVQLHQSGADLVKPGASVKLSCTASGFHIKDTYIHWMKQRPEQGLEWIGRIDPANGNSEY

[0235] DPKFQGKATITADTSSNTAYLHLSSLTSEDTAVYYCAESNYFGSSPYALDYWGQGTSVTVS

[0236] S (SEQ ID NO: 5), and the VL domain comprises the amino acid sequence:

[0237] DIILTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGS

[0238] GSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLEL (SEQ ID NO:6); or

[0239] Embodiment 5. The polypeptide of embodiment 1, wherein the VH domain comprises the amino acid sequence:

[0240] EVKLVESGGGEVKPGGSLKLSCAASGFTFSGYTMSWVRQTPAKREEWVATISGSGGNIYY

[0241] PDTVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCTRQEGGLRGFAYWGQGTLVTVSA

[0242] AKTTPPSVYPL (SEQ ID NO:7), and the VL domain comprises the amino acid sequence:

[0243] DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQLLVYAATNLADGVPSR FSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGIPYTFGGGTKLEIT (SEQ ID NO:8).

[0244] Embodiment 6. The polypeptide of embodiment 1, wherein the scFv comprises the amino acid sequence SEQ ID NOTO or 11.

[0245] Embodiment 7. The polypeptide of embodiment 1, wherein the scFv comprises the amino acid sequence SEQ ID NO:12 or 13.

[0246] 45791084.1 35 Attorney Docket No. MOF 24MB032 PCT

[0247] Embodiment 8. The polypeptide of embodiment 1, wherein the scFv comprises the amino acid sequence SEQ ID NO: 14 or 15.

[0248] Embodiment 9. The polypeptide of embodiment 1, wherein the scFv comprises the amino acid sequence SEQ ID NO: 17 or 17.

[0249] Embodiment 10. The polypeptide of any one of embodiments 1 to 9, wherein the costimulatory signaling region comprises the cytoplasmic domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof

[0250] Embodiment 11. The polypeptide of any one of embodiments 1 to 10, wherein the CAR polypeptide is defined by the formula:

[0251] SP-TRP1-HG-TM-CSR-ISD; or

[0252] SP- TRP1-HG-TM-ISD-CSR wherein “SP” represents a signal peptide, wherein “TRP1” represents a TRP1 -binding region, wherein “HG” represents and optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CSR” represents a co-stimulatory signaling region, wherein “ISD” represents an intracellular signaling domain, and wherein represents a bivalent linker.

[0253] Embodiment 12. The polypeptide of any one of embodiments 1 to 11 , wherein the intracellular signaling domain comprises a CD3 zeta (CD3Q signaling domain.

[0254] Embodiment 13. The polypeptide of any one of embodiments 1 to 11, wherein the polypeptide contains only an intracellular signaling domain or a co-stimulatory domain, but not both.

[0255] Embodiment 14. An isolated nucleic acid sequence encoding the recombinant polypeptide of any one of embodiments 1 to 13.

[0256] Embodiment 15. A vector comprising the isolated nucleic acid sequence of embodiment 14.

[0257] Embodiment 16. A cell comprising the vector of embodiment 15.

[0258] Embodiment 17. The cell of embodiment 16, wherein the cell is selected from the group consisting of an af>T cell, y8T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.

[0259] 45791084.1 36 Attorney Docket No. MOF 24MB032 PCT

[0260] Embodiment 18. A method of providing an anti-cancer immunity in a subject with a melanoma, the method comprising administering to the subject an effective amount of an immune effector cell genetically modified to express the CAR polypeptide of any one of claims 1 to 13, thereby providing an anti-tumor immunity in the mammal.

[0261] Embodiment 19. The method of claim 18, wherein the immune effector cell is selected from the group consisting of an aPT cell, y5T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T (Treg) cell, or any combination thereof.

[0262] Embodiment 20. The method of embodiment 18 or 19, further comprising administering to the subject a checkpoint inhibitor.

[0263] Embodiment 21. The method of embodiment 20, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-Ll antibody, anti-CTLA-4 antibody, or a combination thereof.

[0264] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0265] EXAMPLES

[0266] Example 1:

[0267] Introduction

[0268] Here, we quantitatively measure BCMA density (number of molecules per cell) by flowcytometry in a unique cohort of 40+ bone marrow samples from MM patients who relapsed after BCMA CAR T cell therapy, demonstrating that low BCMA density is associated with multiple myeloma relapse after BCMA CAR T cell therapy.

[0269] The identification of alternate targets such as G protein-coupled receptor class C group 5 (GPRC5D) (Smith, E.L., et al. Sci Transl Med 2019 l l(485):eaau7746; Mailankody, S„ et al. N Engl J Med 2022 387:1196-1206; Chari, A., et al. N Engl J Med 2022 387:2232-2244) and Fc receptor-like 5 (FcRH5, also known as IRTA2 / CD307) (Elkins, K., et al. Mol Cancer Ther 2012 11 :2222-2232) is critical to the development of therapeutic options that may rescue patients with BCMA-escape driven RRMM and subvert dismal prognosis and maximize the killing of myeloma cells per patient given the heterogeneity of BCMA expression.

[0270] Previously, we developed a pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing from 900+ MM patients to map the myeloma surface proteome in search of suitable immunotherapeutic targets. Starting from 4,000+ candidates, we identified six promising 45791084.1 37 Attorney Docket No. MOF 24MB032 PCT candidates (SEMA4A, ILT3, CCR1, LRRC8D, FCRL3, IL12RB1) and BCMA (Di Meo, F„ ct al. Cell Rep Med 2023 4:101110).

[0271] A suitable immunotherapeutic target should be highly expressed on the cell surface of most malignant cells per patient and in most patients. As importantly, it should not be expressed on vital normal cells of the whole body to avoid on-target off-tumor effects. Ideally, such target should play a role in cancer biology to avoid downregulation (Perna, F. Mol Ther 2021 29:424-425).

[0272] We demonstrate that SEMA4A meets these strict criteria of an immunotherapeutic target. We first define SEMA4A expression levels in a large cohort of RRMM patients (n=829), as well as in high-risk patient subsets such as in patients bearing p53 mutation (n=38), a major roadblock in myeloma treatment. Further, we precisely quantify SEMA4A density in bone marrow samples from patients who relapsed after BCMA-CAR T cell therapy and downregulate BCMA expression.

[0273] To define target antigen expression in normal tissues, we build normal tissue arrays with a large panel of vital normal tissues and organs and, perform single-cell RNA-sequencing in bone marrow samples from healthy donors.

[0274] To investigate the relevance of SEMA4A, we genetically inactivate SEMA4A in multiple MM cells with the CRISPR-Cas9 technology and report the functional consequences in myeloma cell proliferation and migration in vitro. In xenograft mouse models injected with SEMA4AKO or control MM cells, we measure the tumor burden, tumor tissue infiltration, osteoclast formation, and overall mice survival.

[0275] On the basis of a favorable expression profile and relevant functional data, we run an antibody discovery platform through the immunization of ATX-GXTM humanized mice and select eight novel binders specifically recognizing the extracellular domain of SEMA4A. Through this, we construct eight 2nd generation CARs with converted SEMA4A scFv’s and screen engineered T cells in vitro and in vivo, identifying lead SEMA4A CARs for future clinical developments. Further, we model BCMA-escaped relapse by using BCMA-calibrated MM cell lines and infusing limiting doses of BCMA CARs. We demonstrate efficacy of SEMA4A CAR T cells in killing primary bone marrow samples patient samples as well.

[0276] This work lays the rational foundation for preparing a phase I clinical trial with SEMA4A- directed CAR T cells for patients with relapsed / refractory MM.

[0277] Methods

[0278] Patient data

[0279] IRB protocol MCC 23222 is approved to obtain primary patient samples from the biobank.

[0280] Our cohort includes patients with plasma cell disorders who consent to Total Cancer Care® Moffitt Cancer Center (protocol#! 4690) and underwent standard of care BCMA-CAR T cell therapy with

[0281] 45791084.1 38 Attorney Docket No. MOF 24MB032 PCT cither idccabtagcnc viclcuccl or ciltacabtagcnc autolcuccl between June 2021 and April 2023 and whose bone marrow cells were available for analysis. IRB protocol Pro00014441 serves to consent patients, collect bone marrow aspirates for research protocols, positively select for CD 138+ cells with Miltenyi beads and submit ~1 million purified myeloma cells for bulk sequencing. We mine bulk RNASeq data from an institutional cohort of RRMM patients that was performed using the Illumina TruSeq RNA Exome with single library hybridization and an Illumina NovaSeq 6000 instrument to 100 million reads per sample. Raw FastQ files were aligned to the human genome reference GRCh38 / hg38 and the GENCODE transcriptome annotations (v32) using the STAR software (Dobin, A., et al. Bioinformatics 2013 29:15-21). Study participants provided written informed consent, in compliance with the Declaration of Helsinki.

[0282] MM Cells from patients with NDMM and RRMM were purified from bone marrow aspirates by CD 138 affinity chromatography (Miltenyi, Bergisch Gladbach, Germany), and nucleic acids were isolated from frozen tissue samples containing 1 million purified myeloma cells. RNA extraction was performed using Qiagen RNAeasy plus mini kits. RNASeq was performed using the Illumina TruSeq RNA Exome with single library hybridization, cDNA synthesis, library preparation, and sequencing performed on an Illumina NovaSeq 6000 instrument to 100 million reads per sample. The Moffitt Avatar cohort (N=829) raw FastQ files were aligned to the human genome reference GRCh38 / hg38 and the GENCODE transcriptome annotations (v32) using the Spliced Transcripts Alignment to a Reference (STAR) software54. RNA expression levels were calculated by RNASeq by Expectation Maximization (RSEM) from the aligned BAM files in terms of Transcript Per Million mapped reads (TPM) (Li, B., et al. BMC BioinformaKcs 2011 12:323). The TPM gene expression levels of MMRF-COMPASS cohort (NCT01454297, N=832) were obtained from GENOSPACE, which was annotated to GENCODE transcriptome annotation (vl9). The patient mutation information was obtained from GDC Data Portal. The log2-transformed TPM values (log2TPM) with a pseudo count of 1 were used for the statistical analysis. Student’s t-test was used to infer the significance with a cutoff as p<0.05. Pearson’s correlation was used to do the linear correlation analysis. Python ‘scipy’ and ‘seaborn’ package was used to perform the statistical analysis and visualize the data, respectively.

[0283] Survival analysis was performed using the R package Survival with log-rank test and hazard ratio statistical tests and the MMRF cohort. Patients with a SEMA4A expression above the median gene expression associate with a statistically significant worse survival.

[0284] Antigen density quantification by flow cytometry

[0285] The cell surface quantification of SEMA4A and BCMA in CD 138 positive cells from patient samples was enumerated by flow cytometry using BD QuantiBRITE beads (BD Biosciences

[0286] 45791084.1 39 Attorney Docket No. MOF 24MB032 PCT

[0287] #340495). FC Block (BD Human FC Block #564219) was added to prevent non-specific staining by FC receptor expressing cells and incubated with the antibody cocktail in Brilliant Stain buffer (BD Biosciences #340495) without washing and incubated on ice for 30 minutes. CD138 live cells cell populations were gated out based on CD138 antibody (Biolegend #352308) and DAPI (Biolegend #422801). In addition, SEMA4A (PE-conjugated, Biolegend #148404), BCMA (PE- conjugated, Miltenyi #130-118-970) were included in the panel and gated based on background staining on fluorescent minus one (FMO) and / or CHO negative control cells. In conjunction, BD QuantiBRITE-PE beads were run. These are pre-calibrated standard bead sets containing known numbers of fluorophore molecules bound per bead to calibrate and convert flow cytometry fluorescence signal into number of fluorophores. They allow for calculation of antigens per cell when using antibodies at saturating conditions and accounting for the corresponding Fluorophore to Protein Ratio (F:P) of each antibody. Molecules / cell were calculated post subtracting background signal emanating from a respective isotype control antibody (PE human IgGl antibody, Biolegend, Clone QA16A12).

[0288] Human normal tissue mic roar ray assembly and immunohistochemistry evaluation

[0289] The tissue microarray (TMA) was assembled using a receiver paraffin block created with a mold that leaves 36 empty spaces in a matrix array that was filled with archival tissue cores punched out from blocks from autopsy and surgical specimens from 6 individuals, 4 males and 2 females, ranging in age from 6-days to 59 years. The TMAs included the following tissues: bone marrow, bladder, lymph node, uterus, lung, pancreas, myocardium, spleen, colon, cerebellum, thymus, ovary, liver, skin, kidney, thyroid, aorta, adrenal, cervix, hippocampus, dermis, hypodermis, parietal cortex, prepuberal and adult testis, prostate, medulla oblongata, pons, cerebellum, and brain (hippocampus and parietal cortex). The TMA was tested with an anti- SEMA4A antibody (R&D Systems Inc, Catalog #: MAB46941, Minneapolis, MN, dilution 1:400). Immunohistochemistry was performed using a DAKO Link 48 automated Stainer (Agilent Technologies, Santa Clara CA) using EDTA buffer retrieval protocols and the EnVision FLEX visualization kit (Agilent Dako, Santa Clara, CA). Incubation times were: primary antibody 20 min; Linker 10 min; HRP (horseradish peroxidase)-secondary antibody 10 min; and DAB (3,3'- Diaminobenzidine) 5 min. Dilutions and retrieval methods were optimized using tonsil as controkpositive in B cells, negative in squamous epithelium. The antigenicity of the TMA was confirmed using the cytoplasmic epithelial marker keratin AE1 / AE3 (Figure 7). Specific patterns of expression evaluated were: cytoplasmic, membranous and nuclear.

[0290] 45791084.1 40 Attorney Docket No. MOF 24MB032 PCT

[0291] Cell lines

[0292] Normal cardiomyocyte (AGIO), Primary Renal Proximal Tubule Epithelial Cells (RPTEC) and liver epithelial cells (THLE-2) were obtained from ATCC and grown according to the manufacturer's directions. The following MM cell lines were used: NCI-H929, OPM-2, KMS-1 1, AMO-1, U266. Cell lines were obtained from Dr. Roodman’s laboratory, checked for Mycoplasma infection, and cultured at 37°C in a humidified atmosphere containing 5% CO2 and maintained in RPMI media supplemented with 10% fetal bovine serum (FBS).

[0293] CRISPR / Cas9 inducible system

[0294] Human KMS-11, U266, OPM2 and AM0.1 MM cells were infected with doxycycline- inducible Cas9-expressing lentivirus (pCW-Cas9, Addgene 50661). sgRNA sequences were designed using Synthego design tool. Each sgRNA was cloned into the lentiviral pLX-sgRNA (Addgene 50662) between the Xhol and Nhel sites according to Addgene individual sgRNA cloning protocol. SEMA4A targeting sgRNA (sgRNAl GCTCCAACAGGGGATGAACGT; sgRNA2 GTCCAACAGGGGATGAACGTA), MM cells expressing pCW-Cas9 were incubated with pLXsgRNA lentivirus expressing the desired sgRNA control sequence. Cells were treated with Ipg / ml doxycycline and 4 pg / ml blastycidine for 7 days. Knock-out cells were verified by flowcytometric analyses. For double KO we used pX330 plasmid (Addgene #58778) for BCMA targeting we used two sgRNA (sgRNAl GGTGTGACCAATTCAGTGAA; sgRNA2 GTATTAAGCTCAGTCCCAAAC).

[0295] Cell growth and migration assays

[0296] 3xl()5human knock-out and control MM cells were seeded in 12 well plates. After 24, 48, 72, 96, and 120 hours, cells were stained by Trypan Blue and viable cells counted. 2xl04human knock-out and control MM cells were seeded in 96 well plates. After 24, 48, 72, 96, and 120 hours 20 pl of CellTiter 96 AQueous One solution Reagent is added into each well. The plate is incubated for 2 hrs at 37°C. The absorbance at 490nm is read.

[0297] For migration assays, we used transwell inserts with 8 pm pores in a 24-wells format (Coring Costar, Cat: #3401). 1.5xl05knock-out and control MM cells were seeded in 300 pL of serum-free medium into the upper chamber. 500 pL of medium with 20% FBS was added in the lower chamber. After 48 h incubation at 37°C, the cells in the lower chamber were stained by Trypan Blue and viable cells counted or 80 pl of CellTiter 96 AQueous One solution Reagent is added into each well. The plate is incubated for 2 hrs at 37°C. The absorbance at 490nm is read.

[0298] Cell cycle analysis

[0299] 3xl05human knock-out and control MM cells were seeded in 6 well plates in FBS-free medium to arrest the cell cycle of all cells. Afterwards cells are grown in medium containing FBS

[0300] 45791084.1 41 Attorney Docket No. MOF 24MB032 PCT for 48h. After cells arc fixed with EtOH 70% treated with RNAsc, stained with PI, and analyzed by floweytometry.

[0301] Xenograft models

[0302] 6-8-week-old immunocompromised NOD scid gamma (NSG) mice were used for in vivo experiments. IxlO6human MM KO or control cells were resuspended in 200 pl PBS and injected in each mouse by tail vein injection. Mouse peripheral blood was collected, and serum was obtained by centrifugation at 2000g for 10 minutes at 4°C. Human light kappa chain levels were measured by Human Kappa ELISA Kit (Bethyl cat# E88-11) following manufactory protocol. Human light lambda chain levels were measured by Human Lambda ELISA Kit (Bethyl cat# E88-116) following manufactory protocol. For CAR T killing in vivo assay we used 8-12-week-old NSG mice. Mice were inoculated with 1x106 KMSl l-ffLuc KMS11 MM cells and injected with IxlO6CAR T cells, 7 days after MM cell injection. Tumor burden shown as bioluminescent signal quantified per animal every 2 weeks. Xenogen IVIS Imaging System (Xenogene) with Living Imgae software (Xenogene) was used for bioluminescence image acquisition.

[0303] Immunohistochemistry of mouse bone marrow samples.

[0304] Dissected femurs were fixed and decalcified before embedding paraffine. The tissue sections were deparaffinized and hydrated. Antigen retrieval was performed for 20 minutes using L.A.B solution (Polysciences, Inc., 24310). Subsequently, the sections were treated with 3% H2O2 for 10 minutes and then Background Buster (Innovex, NB306) for 20 minutes at room temperature, followed by primary antibody against rabbit anti-CD138 (2.5 pg / mL, Invitrogen, 36-2900) and rabbit control IgG (2.5 pg / mL, cell signaling, 2729P). The sections were incubated overnight and treated with biotinylated second antibody and Vectastain ABC reagent (Vector laboratories, PK- 4001) prior to color development by DAB solution (SK-4100) and hematoxylin. The images were captured under a light microscope (Olympus, 1X73). CD138 expression was quantified by H-score using QuPath-0.3.2 software.

[0305] Antibody generation

[0306] Generation of the novel high-quality monoclonal antibodies recognizing SEMA4A was performed at the Fred Hutchinson Antibody Technology Core Facility in Seattle, Washington. 12 female mice of 3 different strains (Balb / c CD1, Fl cross) were immunized by infusion of two million NIH / 3T3 cells expressing SEMA4A antigen in solution five times and a sixth boost with cells and SEM4A protein. Ten days following the sixth boost, blood was collected, and serum was tested by bead and cell-based ranking using a iQue high thruput flow cytometer (Intellicyte). The 6 best mice were given a final boost of IM cells and 5ug of protein. Three days later, mice were euthanatized, spleens were harvested, and splenocytes were fused 1 : 1 with a myeloma fusion

[0307] 45791084.1 42 Attorney Docket No. MOF 24MB032 PCT partner (FOXNY). Resultant hybridomas were resuspended in twelve 384-wcll plates and screened using simultaneous high-throughput against multiple cell lines (KMS11, H929, U266) and a bead- conjugated SEMA4A ECD (extracellular domain) antigen. Up to fifteen hybridoma clones were selected and subcloned by a ClonePix® 2 Colony Picker (Molecular Devices, CPII) in semi-solid methyl cellulose selection media containing fluorescently labeled secondary antibody to detected cells producing mouse IgG. These cells were screened using in a similar method using cells and protein on beads. 10-fold difference in MFI between SEMA4A+ HEK 293 and Parental HEK 293 was optimal. The positive cells from this cloning were rearrayed into 8 subclones and frozen. Epitope binning was performed using Carterra® LSA platform integrated surface plasmon resonance (SPR) and high-throughput kinetic analysis. Finally, variable domain sequencing was performed on each antibody.

[0308] 5xl05MM cells were stained with O.lpg, 0.2pg, 0.4pg and 0.8pg of purified monoclonal antibody for 30 min at 4°C. Then cells were washed with 500 pl of PBS twice. Cells were stained with the appropriate secondary antibody (APC Goat anti-mouse Biolegend #405308) for 30 min at 4°C according to the manufacturer's instructions. Washed twice and analyzed by flow cytometry. Isotype control IgGl : Miltenyi 130-113-196. Isotype control IgG2a: Miltenyi 130-113-269. Anti- SEMA4A antibody: APC Biolegend 7148406.

[0309] CAR cloning

[0310] The amino acid sequence for the single-chain variable fragment (scFv) specific for human SEMA4A was obtained from the heavy and light chain variable regions of the antibodies against human SEMA4A we generated. In the CARs construct, the anti-human SEMA4A scFv is preceded by a human CD8A leader peptide and followed by 4-1BB hinge-transmembrane-intracellular regions, and CD3z intracellular domains linked to a IRES sequence to induce co-expression of GFP.

[0311] CAR cytotoxicity

[0312] The cytotoxicity of T cells transduced with a CAR was determined by standard luciferasebased assay. In brief, U266 and KMS11 expressing firefly luciferase-TURBO-RFP served as target cells. The effector (E) and tumour target (T) cells were co-cultured in triplicates at the indicated E / T ratio using black-walled 96- well plates with 1.5 x 105target cells in a total volume of 100 pl per well in MM Medium. Target cells alone were plated at the same cell density to determine the maximal luciferase expression (relative light units; RLUmax). 16 h later, 100 pl luciferase substrate (Perkin Elmer #122799) was directly added to each well. Emitted light was detected in a luminescence plate reader or Xenogen IVIS Imaging System (Xenogen) and quantified using Living Image software (Xenogen). Lysis was determined as (1 - (RLUsample)Z(RLUmax)) x 100.

[0313] 45791084.1 43 Attorney Docket No. MOF 24MB032 PCT

[0314] In vivo models of BCMA-escaped disease

[0315] Male or female 8-12- week-old NOD.Cg-PrkdcscidI12rgtmWjl / SzJ (NSG) mice (Jackson Laboratory) were used, under a protocol approved by the Moffitt Institutional Animal Care and Use Committee, according to all relevant animal use guidelines and ethical regulations. A total of IxlO6FFLuc-GFP H929 cells were administered intravenously by tail vein injection (day 0). Seven days later, IxlO6or 0.2xl06BCMA CAR T cells were administered intravenously by tail vein injection. A second CAR T cell infusion was administered intravenously. All in vivo assays were performed with bulk transduced CAR T cells. Tumor burden was measured by Bioluminescence imaging used the Xenogen IVIS Imaging System (Xenogen). Living Image software (Xenogen) was used to analyze acquired bioluminescence data.

[0316] Cytokine release

[0317] CAR T cells are seeded 1 : 1 in co-culture with KMS 11 MM cells or alone for 16h, the supernatant is diluted 1 to 3 according to manufactory instruction. Diluted supernatant is loaded into the Simple Plex™ ELLA cartridge (32x8) and read with Ella Automated Immunoassay System #600-100.

[0318] T cell differentiation assay

[0319] 5xl03CAR T cells were seeded with 3x 10'’ MM KMS11 cells. For repeated stimulations during 48h CAR T cells were moved into well with other 3x10’ MM KMS11 cells, two or three times. After 48h cells were analyzed by flow cytometry with CD62L (Biolegend #304822) and CD45R (Biolegend #304136) antibodies.

[0320] Single cell analysis

[0321] Single-cell RNA sequencing was performed on bone marrow specimens from five healthy donors using lOx Chromium. Raw sequencing reads were processed with Cell Ranger analysis pipeline. Quality control and downstream analsyis were performed with the Seurat R package. Batch effects were corrected using Canonical Correlation Analysis (CCA) integration algorithm. After dimension reduction and cell clustering, cell types for each cluster were annotated with a combined approach taking account of 1) the expression of known cell markers, 2) cluster-specific gene markers, and 3) predicted cell identities by multiple bioinformatic tools. A total of 30,781 hematopoietic cells of high quality were subject to gene expression examination.

[0322] Statistical analysis

[0323] Data are expressed as the mean ± sem. For each experiment, overall significance of the differences among means was evaluated using Student t test. For mice experiment a sample size of n=5 mice per group allowed us to detect a difference at 92% and a magnitude of 10% standard deviation with power=0.80, at type I error level 0.05 using a two-sided Student’s t-test. ANOVA

[0324] 45791084.1 44 Attorney Docket No. MOF 24MB032 PCT ensure sample sizes arc conservative. Tukcy’s method was used to adjust for multiple comparisons within each experiment for a given outcome. Statistically significant difference comparing two groups are: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0325] Results

[0326] Target antigen density in MM relapses after BCMA CAR T cell therapy Through an institutional tissue protocol (IRB#Pro00014441), we collect 41 primary bone marrow (BM) samples purified for CD 138+ myeloma cells from 29 unique de-identified patients. Patient samples are selected based on the diagnosis of relapsed multiple myeloma after commercial BCMA CAR T cell therapy. In 6 of these patients, we can collect samples pre- and post-CAR T cell therapy from the same patients.

[0327] We quantitatively profile BCMA density (absolute number of surface molecules / cell) by flow cytometry. The antigen density on the surface of the tumor cell is a major factor in achieving durable antitumor efficacy with CAR T cells (Hamieh, M., et al. Nature 2019 568:112-116). Absolute surface antigen densities are determined by reading the median fluorescence intensity against a calibration curve based on external standard microsphere beads saturated with the relevant staining antibody at known antibody binding capacity (Haubner, S., et al. Cancer Cell. 2023 41(11): 1871 - 1891 ). QuantiBRITE™ PE (BD Biosciences #340495) beads are stained and acquired in parallel with the sample at identical flow cytometry instrument settings. We find that the density of BCMA averages 849 molecules / cell and that density is comparatively lower following BCMA- CAR T cell therapy. (Figs. 1A-1C). These data, consistent with emerging analyses from other groups (Zhou, X., et al. Haematologica 2023 108:958-968), suggest that downregulation of BCMA represents a mechanism of escape to BCMA CAR T cell therapy (Lee, L., et al. Blood 2018 131 :746-758).

[0328] We recently identified alternate immunotherapeutic targets through the mapping of the myeloma surface proteome (surfaceome) (Di Meo, F., et al. Cell Rep Med 2023 4:101110). To investigate whether these targets are upregulated in MM patients, we analyze a cohort of 829 MM patients treated at Moffitt Cancer Center. This cohort of patients includes 691 patients with RRMM. We find SEMA4A, a class 4 semaphorin involved in fine-tuning of the immune response and suggested to play a role in tumors including multiple myeloma (Di Meo, F., et al. Cell Rep Med 2023 4:101110; Ito, D., et al. Cell Adh Migr 2016 10:692-699; Nkyimbeng-Takwi, E., et al. Immunol Res 2011 50:10-21; Anderson, G.S.E, et al. Blood 2022 139:2471-2482), to be highly expressed in RRMM patients (Fig. ID). We consistently find SEMA4A to be frequently expressed in the Multiple Myeloma Research Foundation (MMRF) CoMMpass (NCT01454297) (Dong, C., et al. Oncogene. 2021 40(42):6130-6138) database of MM patients (n=832) (Fig. 6A).

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[0330] Given these findings, we quantitatively measure SEMA4A density in our cohort of 41 BM samples from patients who relapsed after BCMA-CAR T cell therapy (Figs. 1E-1F and 6B). Median SEMA4A number of molecules per myeloma cell averages 14,000 (range: 130 - 90,000), a level that would ensure optimal CAR T cell activation. Comparing SEMA4A expression in samples prior to and following the administration of the BMCA-CAR T cell treatment from the same patients, SEMA4A density is stable (Fig. 1G) and likely independent of BCMA expression; such independence is also suggested by the Pearson’s correlation from the Moffitt and MMRF datasets of MM patients (Fig. 1H-1I).

[0331] By using the latter repository, we perform survival analyses in 767 newly diagnosed multiple myeloma (NDMM) patients using the R package Survival with log-rank test and hazard ratio statistical tests. We find that high levels of SEMA4A are significantly associated with worse clinical outcomes (Fig. 1J).

[0332] By further stratifying the MM patient population by genetic abnormalities, SEMA4A expression is shown to be significantly higher in patients bearing p53 mutations compared to patients bearing wtTP53 (Fig. IK).

[0333] These data demonstrate that SEMA4A is highly and frequently expressed in p53-mutant MM and in RRMM especially post-BCMA-CAR T cell therapy, two disease states which generally do not respond to standard-of-care therapeutic approaches (Hu, J., et al. J Hematol Oncol. 2021 14(1): 157; Van Oekelen, O., et al. Blood 2023 141:756-765) and which are in urgent need of novel therapies.

[0334] SEMA4A on the surfaceome of normal tissues and organs

[0335] Characterizing the expression profile of novel antigens in normal tissues is important to better predict on-target off-tumor effects. The safety profile of CD19-CAR T cell therapy benefits greatly from the exclusivity of CD19 expression to B cells, the depletion of which is tolerable (Santomasso, B.D., et al. Blood. 2023 141(20):2443-2451). Examples of emerging toxicities from targeting antigens other than CD19 include parkinsonism after BCMA-CAR T cell therapy, due to BCMA expression in the patient’s basal ganglia (Van Oekelen, O., et al. Nat Med 2021 27:2099- 2103) or cerebellar toxicity after the infusion of high-dose GPRC5D CAR T cells due to GPRC5D expression in the medulla oblongata (Mailankody, S„ et al. N Engl J Med 2022 387:1196-1206).

[0336] Protein annotation of SEMA4A from multiple proteomic datasets shows absent expression in non-hematopoietic tissues (Di Meo, F., et al. Cell Rep Med 2023 4: 101110; Walker, I.G., et al. Leukemia. 2024 38(8): 1848-1852). To validate SEMA4A expression in normal tissues and organs, we build a normal tissue microarray (TMA) assembled in a matrix array filled with archival tissue cores punched out from blocks from autopsy and surgical specimens from 6 individuals, 4 males

[0337] 45791084.1 46 Attorney Docket No. MOF 24MB032 PCT and 2 females, ranging in age from 6-days to 59 years (Fig. 2A). Immunohistochemical expression of SEMA4A in the TMA was tested using a commercially available anti-SEMA4A antibody (R&D, cat#MAB46941) in a DAKO Link48 automated Stainer using the EnVision FLEX visualization kit (Agilent). Dilutions and retrieval methods were optimized using tonsil as a control. Tissue antigenicity of the TMA was confirmed using the cytoplasmic markers keratin AE1 / AE3 (epithelia) and CD45 (leukocytes). SEMA4A expression was negative in aorta, testis, colon, prostate, skin, medulla oblongata, ovary, dermis, and subcutaneous fat, endomyometrium, bladder, parietal cortex, hippocampus, cerebellum, adrenal, and small intestine (Figs. 2B and 7A). Positivity in macrophages was observed in the thyroid, lung and thymus (Fig. 2C). Liver, kidney, and myocardium show equivocal expression and / or subcellular localization and thus, we precisely measure antigen density on these tissue by flowcytometry (Fig. 2D). All performed analyses indicate low expression outside the hematopoietic system.

[0338] To map SEMA4A expression in the hematopoietic lineage, we perform single-cell RNAseq analyses from >30,000 bone marrow cells and 5 healthy donors suggesting that SEMA4A is expressed at low levels in a small percentage (<25%) of dendritic and myeloid cells (Figs. 2E-2F).

[0339] SEMA4A impacts myeloma cell biology

[0340] A previous study showed that MM cells cannot downregulate SEMA4A to avoid detection (Anderson, G.S.E, et al. Blood 2022 139:2471-2482). We first genetically inactivate SEMA4A with an inducible CRISPR / Cas9 system in KMS-11, U266, OPM2 and AMO. 1 MM cell lines and two single-guide RNAs (sg-RNA) (Fig. 8A). SEMA4A-KO cells show decreased MM cell proliferation in vitro (Figs. 3A-3B and 8B) by culturing SEMA4A-KO and control cells for 24, 48, 72, 96, and 120 hours. At each time point, we observe decreased number of live cells in the KO groups compared to controls, consistently with delayed G1 phase of cell cycle and decreased cell viability measured with the CellTiter96® AQueous One solution Cell Proliferation assay (MTS) (Figs. 8C-8D). Annexin V and PI staining revealed no increase in apoptosis in the KO cell lines compared to controls.

[0341] To determine potential effects on MM cell migration (Sun, T., et al. J Cell Biol 2017 216:199-215), we culture KO and control cells in hollow plastic chambers that are sealed at one end with a porous membrane and filled with serum free media, as reported (Chen, R., et al. Oncotarget 2016 7:73101-731 13; Neri, P., et al. Blood 201 1 117:6202-6213). This chamber is suspended over a larger well containing media supplemented with 10% fetal bovine serum. While control cells are attracted to the lower chambers and migrate across the membrane, SEMA4A deletion leads to impaired migration (Figs. 3C and 8E). Similar results are obtained in BCMA-KO MM cells (Fig. 3F).

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[0343] In vivo, the lack of SEMA4A significantly prolongs overall survival of immunocompromised NSG mice injected with IxlO6human MM KO or control cells by tail vein injection (Fig. 3D). By measuring the serum concentration of human kappa (if KMS11 cells are injected) or lambda (if U266 cells are injected) free light chain at multiple time points post injection as a read-out of the tumor burden, we find significantly reduced human kappa or lambda free light chain in recipient animals (Figs. 3E-3F). To further assess tumor bone marrow infiltration and define myeloma bone disease associated with increased osteoclastic bone resorption, mice bone sections are stained with CD138 and tartrate-resistant acid phosphatase (TRAP) highlighting osteoclasts. Myeloma bone disease is associated with an increase in osteoclastic bone resorption and a reduction in osteoblastic bone formation, as we previously showed (Roodman, G.D. Discov Med 2004 4:144-148). SEMA4A-KO masses have decreased malignant infiltration and osteoclasts compared to controls (Fig. 3G). As shown in Figs. 3H-3I, the bones of mice injected with SEMA4AKO MM cells exhibit reduced number of MM cells and osteoclasts.

[0344] These data demonstrate that SEMA4A plays critical roles in MM cell proliferation, migration, and osteoclast activation.

[0345] SEMA4A-directed CAR T cells

[0346] We generate novel purified monoclonal antibodies recognizing SEMA4A through the immunization of ATX-GXTM mice by infusion of NIH / 3T3 cells overexpressing human SEMA4A antigen and of recombinant peptides covering the extracellular domain (ECD) of SEMA4A. Obtained hybridomas are screened using simultaneous high-throughput against three MM cell lines (KMS11, H929, U266) and a bead-conjugated SEMA4A ECD antigen. Up to fifteen hybridoma clones are selected and subcloned by a ClonePix® 2 Colony Picker in semi-solid methylcellulose selection media containing fluorescently labeled secondary antibody to detect cells producing mouse IgG. These cells are screened with cells and protein on beads. 10-fold difference in MFI between SEMA4A+ HEK293 cells and parental HEK293 are considered optimal. The positive cells from this cloning are re-arrayed into 8 subclones and frozen (Fig. 4A). As part of this antibody discovery campaign, we test 13 binders by using different concentrations (0.1 pg. 0.2pg, 0.4pg and 0.8pg) in flow cytometry on two MM cell lines (U266 and KMS11) expressing the target at different levels (Figs. 4B and 9A-9B). We also use SEMA4A-KO MM cells, three normal cell types and the commercial antibody as controls.

[0347] 8 high-quality scFv sequences recognizing ECDs of SEMA4A are cloned into 2nd generation lentiviral CARs, followed by CD8a hinge and transmembrane regions and 4- IBB and CD3^ signaling domain, a configuration present in idecabtagene vicleucel and ciltacabtagene autoleucel (Fig. 4C). We determine the cytotoxicity of engineered T cells transduced with single

[0348] 45791084.1 48 Attorney Docket No. MOF 24MB032 PCT

[0349] SEMA4A-CARs by standard luciferase-based assay, as previously described (Zhao, Z., ct al. Cancer Cell 2015 28:415-428; Themeli, M., et al. Nat Biotechnol 2013 31:928-933). KMS11 and U266 MM cell lines expressing SEMA4A at different levels or depleted of SEMA4A (negative control) serve as target cells. The effector (E) and tumor target (T) cells are co-cultured in triplicate at different E / T ratios using IxlO5target cells in 100 pl per well. Target cells alone are plated at the same cell density. After 18hrs, luciferase substrate is added, and emitted light quantified. This screening identifies 5 CAR constructs specifically killing SEMA4A-positive cells (Fig. 4D). The construct integrating the scFv from clone #2 shows the highest killing percentage (at T:E equal to 1 :10, clone#2 killing activity is 70%, clone#7,65%, clone#3,60%, clone#5,50% and clone#6,30%). The CAR construct integrating the scFv derived from clone #1 is excluded from further analyses because it killed cells bearing very low levels of SEMA4A expression, raising potential toxicity concerns. The CAR constructs integrating the scFv’s derived from clones #4 and #8 are excluded because of suboptimal killing efficacy. These five selected CAR constructs bearing the scFv’s derived from clones #2, 7, 3, 5 and 6 are equally stimulated in response to weekly exposure to KMS11 MM cells (Fig. 4E). By analyzing cytokine release with the ELLA cytokine release assay (Ella™ Assays; R&D Systems), we show that CAR T cells engineered with the scFv’s derived from clone #2 and #6 produce higher concentrations of IL-2 and TNFa. Similar production of Granzyme B, IFNg, Granzyme A, and Perforin is observed across all samples (Fig. 4F).

[0350] We also evaluate potential differences in the T-cell phenotype driven by distinct scFv’s and / or CAR expression levels. The CAR construct containing the scFv derived from clone #6 shows greater differentiation toward a naive state (Figs. 9C-9D).

[0351] Given that SEMA4A is expressed in -20% of normal T cells 14, we also test SEMA4A- directed CAR T cells for fratricide killing and show no fratricide activity (Fig. 9E).

[0352] In vivo, we use 8- 12- week-old NSG mice, inoculated with IxlO6FFLuc-i- MM KMS11 cells by i.v. injection, followed by IxlO6or 3xl06CAR T cells injected 7 days later. Two CAR constructs (bearing scFv#2 and scFv#7) lead to longer mice survival at the lowest (I xlO6) CAR T cells dose, suggesting also significant superior efficacy than BCMA CAR T cells (Figs. 4G-4H and 9F). Thus, from an initial set of 13 purified monoclonal antibodies, we select 8 SEMA4A-specific antibodies that we use for CAR construction. Five CARs effectively work in in vitro killing assays and two lead SEMA4A-directed CARs lead to superior efficacy in eliminating myeloma at the lowest (IxlO6CAR T cells) tested dose in vivo (Fig. 41). Add in vitro killing on patient samples here

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[0354] Efficacy of SEMA4A CAR T cells in models of BCMA-escape driven disease

[0355] To further assess the efficacy of SEMA4A CAR T cells, we model BCMA CAR therapy relapse by infusing sub-curative doses of BCMA CAR T cells in NSG immunocompromised mice injected with FFLuc-i- H929 MM cells (Fig. 5A). BCMA CARs effectively control H929 cells at the dose of IxlO6CAR T cells but allow for myeloma relapse at the dose of 0.2xl06cells (Fig. 5B). BCMA expression in progressing H929 MM cells is reduced in the group treated with 0.2xl06CAR T cells compared to untreated mice (Fig. 5C). In this setting of relapse-prone mice, a second treatment of CAR therapy with SEMA4A CAR T cells eradicates the disease (Figs. 5D-5E).

[0356] Further, we treat additional models of BCMA-escape driven myeloma with SEMA4A CAR T cells. We generate H929BCMA-null and MMlSBCMA-null tumors through biallelic disruption of the BCMA gene with the CRISPR / Cas9 technology and, we create a panel of H929 MM cells bearing different levels of BCMA expression through the overexpression of BCMA under a weak PGK promoter in BCMA KO cells, resembling the levels that we observe in patients relapsed after BCMA CAR therapy (Fig. 6A-6B). SEMA4A-directed CAR T cells are effective at killing MM cells expressing low levels of BCMA, with superior efficacy than BCMA CAR T cells especially when BCMA is at the lowest levels (Fig. 6D-6E). In vivo, we show that SEMA4A CAR T cells can maintain long remission of disease compared to BCMA CAR T cells when NSG immunocompromised mice are injected with BCMAKO H929 cells (Fig. 6F-6G).

[0357] These studies confirm that under low BCMA-antigen density conditions, the SEMA4Adirected CAR T cells afford durable responses.

[0358] Discussion

[0359] CAR therapy is effective against B-cell malignancies, but tumor escape associated with low / negative target antigen expression is emerging as one limitation.

[0360] Prior studies with CD19-targeted CAR T cells have shown that CARs require a few thousand (Mansilla-Soto, J., et al. Nat Med 2022 28:345-352; Spiegel, J.Y., et al. Nat Med 2021 27:1419-14 1) target molecules per cell for effective tumor recognition and control (Salem, D.A., et al. Leuk Res 2018 71: 106-111). In multiple myeloma (MM), BCMA CAR T cell therapy leads to high overall response rate, but response durability for many patients is limited (Raje, N., et al. N Engl J Med 2019 380: 1726-1737). While complete loss of BCMA after CAR T cell therapy is uncommon, relapsed disease frequently expresses low levels of antigen3. In a unique cohort of myeloma patients relapsed after BCMA CAR T cell therapy, we demonstrate that BCMA number of molecules per cell is dramatically decreased, generally at levels below the threshold required for optimal CAR T cell function.

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[0362] One approach to address this unmet clinical scenario with dismal prognosis and limited therapeutic options, relies on the identification of alternate targets. SEMA4A is frequently expressed in most patients, especially in those with relapsed disease and p53 mutation, two major roadblocks in myeloma treatment. While the expression seems similar to BCMA (Figs. ID and IF), these two molecules are independently expressed of each other (Figs. 1 H- II), and the number of SEMA4A molecules per cell is much higher than BCMA after BCMA CAR T cell therapy (Figs. IE and 1G). As importantly, SEMA4A is minimally expressed in normal non-hematopoietic tissues (Fig. 2B), minimizing the risk of on-target off-tumor effects and confirming recent findings from another group (Anderson, G.S.F., et al. Blood 2022 139:2471-2482; Walker, I.G., et al. Leukemia. 2024 38(8): 1848- 1852). Our data from scRNA-seq data on bone marrow samples from 5 healthy donors demonstrates that SEMA4A is expressed at low levels on myeloid and dendritic cells (Fig. 7B). Further, we show that SEMA4A expression on normal T cells (20%) (Di Meo, F., et al. Cell Rep Med 2023 4: 101110) does not cause fratricide killing by SEMA4A-directed CAR T cells (Fig. 9E). Finally, SEMA4A plays a role in the disease biology and thus, a therapy targeting this antigen may tackle critical mechanisms of cancer survival. It was previously shown that MM cells cannot downregulate SEMA4A to avoid detection (Anderson, G.S.F., et al. Blood 2022 139:2471-2482) and we further show that a) SEMA4A expression levels impact clinical prognosis (Fig. 1 J) and genetic deletion of SEMA4A decreases MM cell proliferation (Figs. 3A-3B), migration (Fig. 3C), tumor tissue infiltration (Figs. 3G-3H), osteoclast formation (Figs. 3G and 31) and significantly prolongs animal survival (Fig. 3D).

[0363] Of note, a previous report suggested a role for SEMA4A in cancer migration (Sun, T., et al. J Cell Biol 2017 216:199-215) and we show that this function also applies to multiple myeloma (Fig. 3C). It was reported that SEMA4Adeficient T cells display defective Th differentiation (Kumanogoh, A., et al. Immunity 2005 22:305-316), however, other family members may compensate for the absence of SEMA4A. Semaphorin receptors, like neuropilins and plexins, are known to be shared by several semaphorin family members (Kumanogoh, A., et al. Immunity 2005 22:305-316; Kolodkin, A.L., et al. Cell 1997 90:753-762; Pasterkamp, R.J., et al. Nature 2003 424:398-405; Toyofuku, T., et al. Genes Dev 2004 18:435-447; Kumanogoh, A., et al. Adv Immunol 2003 81: 173-1980; Kumanogoh, A., et al. Immunity 2000 13:621-631: Shi, W., et al. Immunity 2000 13:633-642), suggesting the existence of functional redundancy. It was shown that SEMA4A binding to Nrpl potentiates regulatory T cell (Treg) function and survival52,53, further supporting the biological and therapeutic relevance of this molecule.

[0364] We generate novel purified monoclonal antibodies recognizing SEMA4A with an antibody discovery campaign based on the immunization of ATX-GXTM mice. These best-in-class

[0365] 45791084.1 51 Attorney Docket No. MOF 24MB032 PCT humanized transgenic mice arc optimized for human antibody sequence dcvclopability and diversity, with a robust immune response comparable to wild-type mice. We select and convert these new antibodies into single chain variable fragments (scFv) that are fusion proteins of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, representing the antigen-binding domains on CARs. SEMA4A-directed CAR T cells effectively eradicate MM cells from a variety of xenograft models (Figs. 4G-4H). Such studies show that under lower BCMA- antigen density conditions, the SEMA4A CARs afford the most durable responses.

[0366] While it is not yet firmly established whether or how the degree of BCMA expression may affect the efficacy of BCMA-targeted CAR T cell therapy (Brudno, J.N., et al. J Clin Oncol 2018 36:2267-2280; Maude, S.L., et al. N Engl J Med 2018 378:439-448; Orlando, E.J., et al. Nat Med 2018 24: 1504-1506; SoKllo, E., et al. Cancer Discov 2015 5: 1282-1295), it is possible that targeting an alternate antigen with an independent expression pattern, such as SEMA4A, may increase the depth and / or duration of responses in patients harboring BCMA-low, BCMA-negative and BCMA-positive MM plasma cell reservoirs. Thus, our study sets the rational basis for development of a CAR T cell therapy for patients with a BCMA-positive or BCMA-negative disease.

[0367] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0368] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0369] 45791084.1 52

Claims

1. Attorney Docket No. MOF 24MB032 PCTCLAIMS1. A chimeric antigen receptor (CAR) polypeptide, comprising a TRP1 antigen binding domain, a transmembrane domain, an intracellular signaling domain, and a co- stimulatory signaling region, wherein the TRP1 antigen binding domain is a single-chain variable fragment (scFv) of an antibody comprising a variable heavy (VH) domain having CDR1, CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1, CDR2 and CDR3 sequences, and wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GFTFNSFT (SEQ ID NO:22) the CDR2 sequence of the VH domain comprises the amino acid sequence ISSSGYNT (SEQ ID NO:23), the CDR3 sequence of the VH domain comprises the amino acid sequence ARHGKIYDGYPFAMDF (SEQ ID NO:24), the CDR1 sequence of the VLcomprises the amino acid sequence ENIYSN (SEQ ID NO:25), the CDR2 sequence of the VL domain comprises the amino acid sequence AAT, and the CDR3 sequence of the VL domain comprises the amino acid sequence QHFWGTPWT (SEQ ID NO:26); wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GYTFTSYW (SEQ ID NO:27) the CDR2 sequence of the VH domain comprises the amino acid sequence IDPSDSET (SEQ ID NO:28), the CDR3 sequence of the VH domain comprises the amino acid sequence ARHYDGYAVDY (SEQ ID NO:29), the CDR1 sequence of the VL comprises the amino acid sequence QSVTTSRYSY (SEQ ID NO:30), the CDR2 sequence of the VL domain comprises the amino acid sequence YAS, and the CDR3 sequence of the VL domain comprises the amino acid sequence QHSWEIPWT (SEQ ID NO:31); wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GFHIKDTY (SEQ ID NO:32) the CDR2 sequence of the VH domain comprises the amino acid sequence IDPANGNS (SEQ ID NO:33), the CDR3 sequence of the VH domain comprises the amino acid sequence AESNYFGSSPYALDY (SEQ ID NO:34), the CDR1 sequence of the VL comprises the amino acid sequence QSISNN (SEQ ID NO:35), the CDR2 sequence of the VL domain comprises the amino acid sequence YAS, and the CDR3 sequence of the VL domain comprises the amino acid sequence QQSNSWPLT (SEQ ID NO:36); or wherein the CDR1 sequence of the VH domain comprises the amino acid sequence GFTFSGYT (SEQ ID NO:37) the CDR2 sequence of the VH domain comprises the amino acid sequence ISGSGGNI (SEQ ID NO:38), the CDR3 sequence of the VH domain comprises the amino acid sequence TRQEGGLRGFAY (SEQ ID NO:39), the CDR1 sequence of the VL comprises the amino acid sequence ENIYNN (SEQ ID NO:40), the CDR2 sequence of the VL domain comprises the amino acid sequence AAT, and the CDR3 sequence of the VL domain comprises the amino acid sequence QHFWGIPYT (SEQ ID NO:41).45791084.1 53Attorney Docket No. MOF 24MB032 PCT2. The polypeptide of claim 1, wherein the VH domain comprises the amino acid sequence: EVKLVESGGGLVKPGGSLNLSCAASGFTFNSFTMSWVRQTPAKRLEWVATISSSGYNTYY PDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCARHGKIYDGYPFAMDFWGQGTSVT VSS (SEQ ID NO:1), and the VL domain comprises the amino acid sequence:DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYHQKQGKSPQLLVYAATNLADGVPSR FSGNGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIK (SEQ ID NO:2);3. The polypeptide of claim 1, wherein the VH domain comprises the amino acid sequence: QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNIDPSDSETH YNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARHYDGYAVDYWGQGTSVTVSS (SEQ ID NO: 3), and the VL domain comprises the amino acid sequence:DIVLTQSPASLPVSLGQRATISCRASQSVTTSRYSYMHWYQQKPGQPPKLLIMYASNLESG VPARFSGSGSGTDFTLNIHPVEEEDIAIYYCQHSWEIPWTFGGGTKLEIK (SEQ ID NO:4);4. The polypeptide of claim 1 , wherein the VH domain comprises the amino acid sequence: EVQLHQSGADLVKPGASVKLSCTASGFHIKDTYIHWMKQRPEQGLEWIGRIDPANGNSEY DPKFQGKATITADTSSNTAYLHLSSLTSEDTAVYYCAESNYFGSSPYALDYWGQGTSVTVSS (SEQ ID NO: 5), and the VL domain comprises the amino acid sequence:DIILTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGS GSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLEL (SEQ ID NO:6); or5. The polypeptide of claim 1, wherein the VH domain comprises the amino acid sequence: EVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPAKRLEWVATISGSGGNIYY PDTVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCTRQEGGLRGFAYWGQGTLVTVSA AKTTPPSVYPL (SEQ ID NOY), and the VL domain comprises the amino acid sequence:DIQMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQLLVYAATNLADGVPSR FSGSGSGTQFSLKINSLQSEDFGSYYCQHFWGIPYTFGGGTKLEIT (SEQ ID NO:8).

6. The polypeptide of claim 1, wherein the scFv comprises the amino acid sequence SEQ ID NO:10 or 11.

7. The polypeptide of claim 1, wherein the scFv comprises the amino acid sequence SEQ ID NO:12 or 13.

8. The polypeptide of claim 1, wherein the scFv comprises the amino acid sequence SEQ ID NO:14 or 15.

9. The polypeptide of claim 1, wherein the scFv comprises the amino acid sequence SEQ ID NO: 17 or 17.

10. The polypeptide of claim 1, wherein the costimulatory signaling region comprises the cytoplasmic domain of a costimulatory molecule selected from the group consisting of CD27,45791084.1 54Attorney Docket No. MOF 24MB032 PCTCD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof11. The polypeptide of any one of claims 1 to 10, wherein the CAR polypeptide is defined by the formula:SP-TRP1-HG-TM-CSR-ISD; orSP- TRP1-HG-TM-ISD-CSR wherein “SP” represents a signal peptide, wherein “TRP1” represents a TRP1 -binding region, wherein “HG” represents and optional hinge domain, wherein “TM” represents a transmembrane domain, wherein “CSR” represents a co- stimulatory signaling region, wherein “ISD” represents an intracellular signaling domain, and whereinrepresents a bivalent linker.

12. The polypeptide of claim 1 , wherein the intracellular signaling domain comprises a CD3 zeta (CD3Q signaling domain.

13. The polypeptide of claim 1, wherein the polypeptide contains only an intracellular signaling domain or a co-stimulatory domain, but not both.

14. An isolated nucleic acid sequence encoding the recombinant polypeptide of claim 1.

15. A vector comprising the isolated nucleic acid sequence of claim 14.

16. A cell comprising the vector of claim 15.

17. The cell of claim 16, wherein the cell is selected from the group consisting of an aPT cell, y5T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.

18. A method of providing an anti-cancer immunity in a subject with a melanoma, the method comprising administering to the subject an effective amount of an immune effector cell genetically modified to express the CAR polypeptide of claim 1, thereby providing an anti -tumor immunity in the mammal.

19. The method of claim 18, wherein the immune effector cell is selected from the group consisting of an a T cell, y3T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T (Treg) cell, or any combination thereof.45791084.1 55Attorney Docket No. MOF 24MB032 PCT20. The method of claim 19, further comprising administering to the subject a checkpoint inhibitor.

21. The method of claim 20, wherein the checkpoint inhibitor comprises an anti-PD- 1 antibody, anti-PD-Ll antibody, anti-CTLA-4 antibody, or a combination thereof.45791084.1 56

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