Therapeutic combinations comprising anti-STEAP2 chimeric antigen receptor t cells

Abstract

The present disclosure provides therapeutic combinations of chimeric antigen receptor T cells (e.g., AZD0754) that specifically bind to human STEAP2 with an androgen receptor antagonist (e.g., enzalutamide). Methods of administering the combination to treat cancer (e.g., prostate cancer) are also provided.

Description

[0001] References to sequence listings submitted electronically via the EFS network

[0002] The contents of the sequence list (CARTSTEAP2-101-US-PSP_ST26; size: 30,921 bytes; and creation date: September 21, 2023) submitted electronically in this application are incorporated herein by reference in their entirety. Background Technology

[0003] Prostate cancer (PC) is the second most common cancer worldwide and a leading cause of cancer-related deaths in men. Approximately one in eight men will be diagnosed with prostate cancer, and about one in 41 men will die from it (American Cancer Society, 2023). Fortunately, when appropriate treatment is initiated early and the tumor is confined to prostate tissue, PC progression is usually slow and the prognosis is generally favorable. On the other hand, for patients who do not respond to effective primary interventions such as radical prostatectomy, androgen deprivation therapy (ADT) can control the tumor burden for years, but castration-resistant and eventually metastatic prostate cancer often develops. In the United States, prostate cancer claims tens of thousands of lives each year.

[0004] For patients with castration-resistant prostate cancer (CRPC) and detectable metastatic disease, standard care (chemotherapy using a combination of docetaxel and prednisone) prolongs survival but is not curative (Adamo et al., Front. Endocrinol., 3:73, 2012; Saad and Hotte, Can. Urol. Assoc. J., 4(6):380-384, 2010). A potential reason may be related to the mechanism of action of docetaxel, a microtubule stabilizer that preferentially kills rapidly proliferating cells but may spare slowly circulating cancer cells. In the latter case, the cells could be cancer stem cells, which are thought to produce most of the malignant cells within the tumor and appear to be particularly resistant to therapy (Kong et al., Cancers (Basel), 3(l):716-729, 2011; Lang et al., J Pathol, 217(2):299-306, 2009).

[0005] Therefore, there is a need in the art for improved treatment and prevention strategies for prostate cancer. This invention addresses this need, as well as other needs. This document describes a novel approach to treating cancers (e.g., prostate cancer) using a combination of T cells expressing a chimeric antigen receptor (CAR) targeting human STEAP2 and an androgen receptor antagonist. Summary of the Invention

[0006] This disclosure relates to a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of the following that effectively inhibits tumor cell growth: T cells comprising (i) a chimeric antigen receptor (CAR) encoding a polynucleotide encoding an epitope binding to human prostatic six-transmembrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist; wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in NO: 6.

[0007] Some aspects of this disclosure relate to a method for treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the following: T cells comprising (i) a chimeric antigen receptor (CAR) encoding a polynucleotide encoding an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist; wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in ID NO: 6.

[0008] In some respects, VH comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and VL comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0009] In some respects, VH contains the amino acid sequence shown in SEQ ID NO:7, and VL contains the amino acid sequence shown in SEQ ID NO:8.

[0010] In some respects, the polynucleotide also encodes armor molecules, and said armor molecules include dominant-negative TGF-β receptor type 2 (TGFβRIIDN).

[0011] In some respects, the armor molecule contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0012] In some respects, the armor molecule contains the amino acid sequence shown in SEQ ID NO: 10.

[0013] In some respects, the polynucleotide encoding CAR comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 11.

[0014] In some respects, the polynucleotide encoding CAR contains the nucleotide sequence shown in SEQ ID NO: 11.

[0015] In some respects, the polynucleotide encoding CAR contains the nucleotide sequence shown in SEQ ID NO: 11, and the polynucleotide encoding the armor molecule contains the nucleotide sequence shown in SEQ ID NO: 9.

[0016] In some respects, the polynucleotide encoding CAR and the polynucleotide encoding the armor molecule are operatively linked under the control of a single promoter.

[0017] In some respects, polynucleotides encoding CAR and polynucleotides encoding armor molecules are operatively linked via IRES.

[0018] In some aspects, the polynucleotide encoding the CAR and the polynucleotide encoding the armor molecule are linked by a nucleotide sequence encoding a cleavable peptide linker. In some aspects, the cleavable peptide linker is a self-cleaving peptide linker. In some aspects, the cleavable peptide linker includes the T2A peptide. In some aspects, the cleavable peptide linker comprises SEQ ID NO: 13.

[0019] In some respects, the polynucleotide comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14.

[0020] In some respects, the polynucleotide contains the nucleotide sequence shown in SEQ ID NO: 14.

[0021] In some respects, T cells contain an amino acid sequence, including the amino acid sequence shown in SEQ ID NO: 12.

[0022] In some respects, T cells contain an amino acid sequence, including the amino acid sequence shown in SEQ ID NO: 15.

[0023] In some respects, androgen receptor antagonists are enzalutamide, apalutamide, darolutamide, abiraterone, bicalutamide, nilutamide, flutamide, proxalutamide, or combinations thereof.

[0024] In some respects, the androgen receptor antagonist is enzalutamide.

[0025] In some respects, the androgen receptor antagonist is abiraterone.

[0026] In some respects, androgen receptor antagonists are administered orally.

[0027] In some respects, T cells are administered intravenously.

[0028] In some respects, T cells and androgen receptor antagonists are administered sequentially or simultaneously, and in any order.

[0029] In some respects, the administration of androgen receptor antagonists does not inhibit T cell activity or T cell killing ability.

[0030] In some respects, the administration of androgen receptor antagonists does not inhibit the release of interferon-γ (IFNγ) in tumor cells.

[0031] In some respects, the administration of androgen receptor antagonists does not reduce CAR expression on T cells.

[0032] In some respects, T cells are CD8+ T cells.

[0033] In some respects, the tumor cells are prostate tumor cells, optionally wherein the prostate tumor cells are metastatic, recurrent, or recurrent.

[0034] In some respects, this administration increased STEAP2 expression in tumor cells compared to T cell administration alone. In some respects, this administration increased STEAP2 expression in tumor cells by approximately 50% to approximately 200% compared to T cell administration alone. In some respects, STEAP2 expression increased by approximately 100%.

[0035] Some aspects of this disclosure relate to the use of T cells and androgen receptor antagonists in a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of T cells and at least one androgen receptor antagonist that effectively inhibits tumor cell growth, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule, wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 ... The amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 6. In some respects, the tumor cells are prostate tumor cells. In some respects, the prostate tumor cells are metastatic, recurrent, or relapsed.

[0036] Some aspects of this disclosure relate to the use of T cells and androgen receptor antagonists in a method of treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of T cells and at least one androgen receptor antagonist, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule, wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in IDNO: 6. In some respects, the tumor cells are prostate tumor cells. In some respects, the prostate tumor cells are metastatic, recurrent, or relapsed. Attached Figure Description

[0037] Figures 1A to 1C The response of three LuCaP prostate cancer patient-derived xenograft (PDX) models to enzalutamide treatment over time is shown. In LuCaP73 ( Figure 1A ), LuCaP70 ( Figure 1B ) and LuCaP86.2 ( Figure 1C In the PDX model, tumor volume was measured after treatment with a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg) for up to 42 days.

[0038] Figures 2A to 2B . Figure 2A The response of the LuCaP73 PDX model to different doses of enzalutamide over time is shown. Tumor volume was measured after treatment with a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg) for up to 42 days. This indicates that p < 0.001 and This indicates that p < 0.0001 (two-sided unadjusted p value). Figure 2B The body weights of LuCaP73 PDX models after treatment with mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg doses) are shown.

[0039] Figures 3A to 3B The androgen receptor (AR) profile was shown on day 42 after treatment with the mediator or 10 mg / kg enzalutamide (Enza). Figure 3A ) and STEAP2 ( Figure 3B LuCaP73 tumor expression was detected. AR and STEAP2 expression levels were normalized to GAPDH levels.

[0040] Figures 4A to 4B . Figure 4A The response of the LuCaP70 PDX model to different doses of enzalutamide over time is shown. Tumor volume was measured after treatment with a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg) for 21 days. This indicates that p < 0.001 and This indicates p < 0.05 (two-sided, unadjusted p value). Figure 4B The body weights of LuCaP 70 PDX models after treatment with mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg dose) are shown.

[0041] Figures 5A to 5E . Figures 5A to 5C The androgen receptor (AR) profile is shown on day 21 after treatment with the mediator or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg). Figure 5A STEAP2 Figure 5B ) and KLK3 ( Figure 5C LuCaP70 tumor expression. This indicates that p < 0.0001; p < 0.01; and ns indicates no significance (determined by one-way ANOVA followed by Dunnett's multiple comparison test). Figures 5D to 5E Immunohistochemistry (IHC) (H score) based on the use of anti-STEAP2 antibody and calculated tumor membrane staining intensity and percentage of stained cells is shown. Figure 5D And by the percentage of membrane staining in the tumor () Figure 5E The expression level of STEAP2 in LuCaP70 tumors was obtained. This indicates p < 0.05 (confirmed by one-way ANOVA followed by Dunnett's multiple comparison test).

[0042] Figures 6A to 6E . Figure 6A The response of the LuCaP86.2 PDX model to different doses of enzalutamide over time is shown. Tumor volume was measured for 25 days after treatment with a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg). This indicates that P < 0.01, and This indicates p < 0.05 (two-sided, unadjusted p value). Figure 6B The body weights of LuCaP86.2 PDX models after treatment with mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg dose) are shown. Figures 6C to 6E The androgen receptor (AR) profile is shown on day 25 after treatment with the mediator or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg). Figure 6C STEAP2 Figure 6D ) and KLK3 ( Figure 6E LuCaP86.2 tumor expression.

[0043] Figures 7A to 7D The response of enzalutamide to serum prostate-specific antigen (PSA) levels in a castration-sensitive LuCaP PDX model is shown. LuCaP73 was treated with the mediator or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg dose). Figure 7A ), LuCaP70 ( Figure 7B ) and LuCaP86.2 ( Figure 7C The PDX model was used, and serum PSA levels were determined by ELISA at the end of the study (LuCaP73 = day 42, LuCaP70 = day 21, and LuCaP86.2 = day 25). This indicates that p < 0.0001 and This indicates p < 0.05 (confirmed by one-way ANOVA followed by Dunnett's multiple comparison test). Figure 7D The effects of continuous enzalutamide treatment on STEAP2 antigen binding capacity (ABC) up to day 21 and up to day 28 in LNCAP cell lines are shown.

[0044] Figures 8A to 8B The efficacy of AZD0754 CAR T cells in the LuCaP73 PDX model is shown. Measurements over time were performed using untransduced T cells (UT; 5 × 10⁻⁶). 6 CAR T cells (5 × 10⁵ cells / mouse), 40A3 dnTFGβRII (i.e., AZD0754) CAR T cells (CAR T; ... 5 (5 × 10⁵ cells / mouse) or 40A3 dnTFGβRII CAR T cells (CAR T; 5 × 10⁵ cells / mouse) 6 Tumor volume in mice treated with (cells / mouse) Figure 8A ) and weight ( Figure 8B UT / CAR T treatment was administered at the designated time point (day 34). For each treatment group, N=12. This indicates that p < 0.0001 and This indicates that p < 0.05.

[0045] Figures 9A to 9B The response to AZD0754 CAR T cell and enzalutamide combination therapy is shown in the LuCaP73 PDX model. Treatment-naïve mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁻⁶) were measured over time. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 Tumor volume in mice treated with enzalutamide (10 mg / kg) and cells / mouse Figure 9A ) and weight ( Figure 9B For each treatment group, N=9. This indicates that p < 0.05.

[0046] Figures 10A to 10BThe figures show that on day 42, untreated mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁶) were compared. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 The percentage of AZD0754 CAR-T cells in the blood of mice treated with enzalutamide (10 mg / kg) and mice (cells / mouse). Figure 10A ) and cell number ( Figure 10B ).

[0047] Figures 11A to 11B The figures show that on day 42, untreated mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁶) were compared. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 The percentage of hCD45+ complemented TGFβRII+ CAR-T cells in the blood of mice treated with enzalutamide (10 mg / kg) and mice (cells / mouse) Figure 11A ) and cell number ( Figure 11B ).

[0048] Figure 12 The figures show that on day 42, untreated mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁶) were compared. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 Serum PSA levels in mice treated with (cells / mouse) and enzalutamide (10 mg / kg).

[0049] Figures 13A to 13B It shows abiraterone ( Figure 13A ) and enzalutamide ( Figure 13B The effects of treatment on the proliferation of LNCAP or 22RV1 prostate cell lines.

[0050] Figures 14A to 14B The results showed that abiraterone ( ) 24 hours, 48 ​​hours, or 72 hours after treatment Figure 14A ) and enzalutamide ( Figure 14B The effect of treatment on the proliferation of 40A3 dnTFGβRII CAR T cells.

[0051] Figures 15A to 15B The levels of abiraterone (Abiraterone) as measured by flow cytometry at 24, 48, and 72 hours post-treatment are shown. Figure 15A ) and enzalutamide ( Figure 15B The effect of treatment on STEAP2 expression in LNCAP cells.

[0052] Figures 16A to 16C This study demonstrates the effect of abiraterone treatment on the cytotoxic ability of 40A3 dnTFGβRII CART cells co-cultured with LNCAP cells. Figure 16A ) or IFN of the CAR T cells The effects of release ( Figure 16B Treatment with 5 μM abiraterone was compared with DMSO, and CAR T cells were compared with untransduced T cells (UT), where LNCAP and / or CAR T or UT cells were pretreated with abiraterone (PreTx). Androgen receptor inhibition in LNCAP cells treated with culture medium, DMSO, or 5 μM abiraterone was validated by qPCR of downstream target expression. Figure 16C ).

[0053] Figures 17A to 17CThis study demonstrates the effect of enzalutamide treatment on the cytotoxic ability of 40A3 dnTFGβRII CART cells co-cultured with LNCAP cells. Figure 17A ) or IFN of the CAR T cells The effects of release ( Figure 17B Treatment with 5 μM enzalutamide was compared with DMSO, and CAR T cells were compared with untransduced T cells (UT), where LNCAP and / or CAR T or UT cells were pretreated with enzalutamide (PreTx). Androgen receptor inhibition in LNCAP cells treated with culture medium, DMSO, or 5 μM enzalutamide was validated by qPCR of downstream target expression. Figure 17C ). Detailed Implementation

[0054] This disclosure relates to an antigen-binding portion that specifically binds to an epitope on human prostatic six-transmembrane epithelial antigen-2 (STEAP2). Some aspects of this disclosure relate to a polynucleotide comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain that binds to an epitope on human STEAP2. Some aspects of this disclosure relate to a host cell comprising the polynucleotide. Other aspects of this disclosure relate to an antibody or antigen-binding portion thereof that specifically binds to an epitope on human STEAP2. In some aspects, the antigen-binding domain binds to an epitope on the extracellular loop of human STEAP2. Further aspects of this disclosure relate to a method of treating a subject in need, the method comprising administering to the subject the polynucleotide, the cell, and / or the antibody or antigen-binding portion thereof. In some aspects, the subject has prostate cancer or a tumor derived from prostate cancer.

[0055] I. Terminology

[0056] To facilitate understanding of this description, certain terms are first defined. Additional definitions are set forth throughout the specific implementation.

[0057] It should be noted that the terms “an” or “a type” refer to one or more of that entity; for example, “nucleotide sequence” should be understood to mean one or more nucleotide sequences. Therefore, the terms “an” (or “a type”), “one or more” and “at least one” are used interchangeably in this document.

[0058] Furthermore, as used herein, “and / or” is considered to refer to each of two specified features or components, whether or not they are specifically disclosed with the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0059] It should be understood that wherever the term "comprise" is used to describe aspects herein, other similar aspects described by "consisting of" and / or "substantially consisting of" are also provided. As used herein, the terms "comprise" and "include" and their variations (e.g., "comprises", "comprising", "includes", and "including") will be understood to indicate that a component, feature, element, or step or group of components, features, elements, or steps stated therein is included, but does not exclude any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms "comprise", "substantially consisting of", and "consisting of" may be replaced by any of the other two terms while retaining their ordinary meaning.

[0060] Unless otherwise defined, 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 pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0061] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the values ​​that define that range. Unless otherwise indicated, nucleotide sequences are written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino-to-carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the entire specification. Therefore, the terms that are immediately defined below are more fully defined by reference to the entire specification.

[0062] The term “about” is used herein to mean approximately, roughly, about, or in the range of… When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value. Generally, the term “about” can modify values ​​above and below the stated value by varying upwards or downwards (higher or lower), for example, by 10%.

[0063] As used herein, the term “approximately” when applied to one or more values ​​of interest means a value similar to the stated reference value. In some respects, unless otherwise stated or otherwise apparent from the context, the term “approximately” means a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the stated reference value (except where such a number would exceed 100% of the possible value).

[0064] In some respects, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains: CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise stated herein, amino acids in the variable regions are numbered using the Kabat numbering system, and those in the constant regions are numbered using the EU system.

[0065] Immunoglobulins can originate from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are subclassed in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some respects, the antibodies described herein are IgG1 subtypes. Immunoglobulins (e.g., IgG1) exist in several allotypes, differing from each other by at most a few amino acids. "Antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0066] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human STEAP2). The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody (e.g., the anti-STEAP2 antibody described herein) include (i) Fab fragments (fragments derived from papain cleavage) or fragments derived from V... L V H(ii) A similar monovalent fragment consisting of the LC and CH1 domains; (iii) an F(ab')2 fragment (a fragment derived from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; H (iv) The Fd fragment composed of the CH1 domain; L and V H The Fv segment, composed of structural domains, (v) is composed of V H The dAb fragment consists of domains (Ward et al., (1989) Nature 341:544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs optionally connected by a synthetic linker. Furthermore, although the two domains V of the Fv fragment... L and V H Encoded by individual genes, they can be linked together using recombination methods via synthetic linkers, which allow them to form a single protein chain, where V... L District and V H Regions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding moiety" of antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and screening for these fragments in the same manner as intact antibodies is ineffective. Antigen-binding moieties can be generated via recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0067] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered antigen-binding polypeptide comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Expression of a CAR on the surface of a cell (e.g., an immune cell) allows the cell to target and bind to a specific antigen. In some respects, CARs are expressed by immune cells, such as T cells. In some respects, the antigen-binding domain comprises Fab, Fab', F(ab')2, Fd, Fv, a single-chain variable fragment (scFv), a single-chain antibody, VHH, vNAR, a nanobody (a single-domain antibody), or any combination thereof. In some respects, the transmembrane domain comprises a transmembrane domain selected from CD4, CD8α, or CD28. In some respects, the intracellular domain comprises a co-stimulatory domain or a portion thereof. In some respects, the intracellular domain includes a co-stimulatory domain selected from the group consisting of: the intracellular domain of CD3z, the CD28 co-stimulatory domain, the CD27 co-stimulatory domain, the 4-1BB co-stimulatory domain, the ICOS co-stimulatory domain, the OX-40 co-stimulatory domain, the GITR co-stimulatory domain, the CD2 co-stimulatory domain, the IL-2Rβ co-stimulatory domain, the MyD88 / CD40a CD28 co-stimulatory domain, and any combination thereof. The CAR may also include a “hinge” or “spacer” domain. Non-limiting examples of hinge / spacer domains include immunoglobulin hinge / spacer domains, such as the IgG1 hinge domain and the IgG2 hinge domain, the IgG3 hinge domain, or the IgG4 hinge domain.

[0068] As used herein, the term "armor" refers to the molecular manipulation of CAR-expressing cells (e.g., CAR-T cells) to further express one or more "armor molecules" that can counteract immunosuppression. For example, researchers recently reported modifying CAR-T cells to secrete single-chain variable fragments (scFv) that block PD-1, which improved the antitumor activity of CAR-T cells in mouse models of PD-L1+ hematologic malignancies and solid tumors (Rafiq, S., Yeku, O., Jackson, H. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36, 847–856 (2018)). Other studies have demonstrated the effectiveness of armoring T cells with dominant-negative TGF-β receptor type 2 (TGFβRIIDN) armor molecules to neutralize the inhibitory effect of TGF-β on T cells (Bollard et al., Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma, J Clin Oncol 36(11):1128-1139 (2018)). Currently, at least one clinical study is investigating the effectiveness of armoring PSMA-CAR-T cells with TGFβRIIDN armor molecules for the treatment of castration-resistant prostate cancer (NCT03089203).

[0069] As used herein, the term “STEAP2” refers to prostatic six-span membrane epithelial antigen 2. STEAP2 is a complete six-span membrane protein that is highly expressed in prostatic epithelial cells and is a cell surface marker for prostate cancer; for example, STEAP2 has been found to be expressed at significant levels in the LNCaP prostate cell line (Porkka et al., Lab Invest 2002, 82:1573-1582). STEAP2 (UniProtKB / Swiss-Prot: Q8NFT2.3) is a 490-amino acid protein encoded by the STEAP2 gene located in the human chromosome 7q21 region.

[0070] "Drug-resistant androgen receptors" are modified (relative to wild-type) androgen receptors that are less inhibited by drugs compared to wild-type androgen receptors. "Drug-resistant human androgen receptors" are modified (relative to wild-type) human androgen receptors that are less inhibited by drugs compared to wild-type human androgen receptors. Examples of "drug-resistant human androgen receptors" include human androgen receptors with less activity levels inhibited by competitive inhibitors (e.g., cassoxat, flutamide, MDV3100, or ARN-509) compared to wild-type human androgen inhibitors, human androgen receptors active without ligand binding, and human androgen receptors active without some or all of their ligand-binding domains.

[0071] The term "androgen receptor," or "AR," or "NR3C4" refers to a nuclear receptor activated by the binding of the androgens testosterone or dihydrotestosterone. The term "androgen receptor" can refer to the nucleotide or protein sequence of a human androgen receptor (e.g., Entrez 367, Uniprot P10275, RefSeq NM_000044, or RefSeq NP_000035 (SEQ ID NO:2)). The term "androgen receptor" includes the wild-type form of the nucleotide sequence or protein, as well as any mutants thereof. In some aspects, "androgen receptor" is a wild-type androgen receptor. In some aspects, "androgen receptor" is one or more mutant forms. The term "androgen receptor XYZ" refers to the nucleotide sequence or protein of a mutant androgen receptor, wherein the Y-numbered amino acid of an androgen receptor that normally has the X amino acid in the wild type is alternatively replaced by the Z amino acid in the mutant. In all aspects, the androgen receptor is a human androgen receptor. In all aspects, the androgen receptor has a nucleotide sequence corresponding to reference number GI:349501065. In all respects, the androgen receptor has the nucleotide sequence corresponding to RefSeq NM_000044.3. In all respects, the androgen receptor has the protein sequence corresponding to reference number GI:21322252. In all respects, the androgen receptor has the protein sequence corresponding to RefSeq NP_000035.2.

[0072] As used herein, the term "affinity" refers to a measure of the strength of binding between an antigen or target (such as an epitope) and its homologous binding domain (such as a complementary site). As used herein, the term "affinity" refers to the overall stability of the complex between a population of epitopes and complementary sites (i.e., antigens and antigen-binding domains).

[0073] The term "epitope" refers to a site on an antigen (e.g., STEAP2) where a chimeric antigen receptor, immunoglobulin, or antibody specifically binds, as defined by a particular method used to identify the site. Epitopes can be formed from consecutive amino acids (typically linear epitopes) or from discontinuous amino acids juxtaposed through the ternary folding of a protein (typically conformational epitopes). Epitopes formed from consecutive amino acids are generally, but not always, retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0074] The term "binds to the same epitope" for two or more antigen-binding moieties means that the antigen-binding moieties bind to the same amino acid residue segment. An antigen-binding moiety that "competes with another antibody to bind to the target" refers to an antigen-binding moiety that inhibits (partially or completely) the binding of other antibodies to the target.

[0075] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specifically binding” refer to the binding of an antigen-binding moiety (e.g., a CAR or antibody) to an epitope on a predetermined antigen. Generally, an antigen-binding moiety (e.g., a CAR or antibody): (i) when, for example, through BIACORE ® When using surface plasmon resonance (SPR) technology, or Scatchard analysis of antibody-antigen-positive cell binding, with a predetermined antigen (e.g., human STEAP2) as the analyte and antibody as the ligand, in the 2000 instrument, the result is determined to be approximately less than 10. -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K) D (i) binding, and (ii) binding to the predetermined antigen with an affinity at least twice that of binding to nonspecific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). Therefore, the antigen-binding portion (e.g., CAR or antibody) of “specifically binding to human STEAP2” refers to binding to the predetermined antigen with an affinity at least twice that of binding to nonspecific antigens (e.g., BSA, casein). -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Binds to the antigen-binding portion of human STEAP2 (e.g., CAR or antibody).

[0076] As used herein, the term "peptide" is intended to cover both the singular and plural "peptide" and any one or more chains containing two or more amino acids. Therefore, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," or any other term used to refer to one or more chains containing two or more amino acids is included in the definition of "peptide," even though each of these terms may have a more specific meaning. The term "peptide" may be used in place of any of these terms or used interchangeably with any of these terms. The term also includes peptides that have undergone post-translational or post-synthetic modifications, such as palmitoyl group conjugation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, disulfide bond formation, proteolytic cleavage, or modifications by non-naturally occurring amino acids. As used herein, the term "peptide" covers full-length peptides and their fragments, variants, or derivatives. As used herein, a "peptide" can be a portion of a fusion peptide that includes additional components (such as, for example, albumin or PEG moieties) to increase its half-life. Peptides as used herein can also be derivatized in a variety of different ways. Peptides may contain modifications, including, for example, conjugations of palmitoyl groups. As used herein, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0077] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, predicted non-essential amino acid residues in the STEAP2 binding moiety (e.g., anti-STEAP2 CARs or antibodies) are replaced by another amino acid residue from the same side chain family.

[0078] The percentage identity between two sequences is a function of the number of common positions shared by the sequences (i.e., % homology = number of common positions / total number of positions × 100), taking into account the number of gaps required for optimal alignment of the two sequences and the length of each gap. As described in the following non-restrictive example, mathematical algorithms can be used to compare sequences and determine the percentage identity between two sequences.

[0079] The percentage of identity between two nucleotide sequences can be determined using the GAP procedure in the GCG software package (available at worldwideweb.gcg.com) with the NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN procedure (version 2.0), using the PAM120 weighted residue table, vacancy length penalty of 12, and vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm in the GAP program, which has been incorporated into the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0080] The nucleic acid and protein sequences described herein can be further used as “query sequences” to search public databases for, for example, to identify relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, vacancy BLAST, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using BLAST and vacancy BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0081] As used herein, the term "promoter" refers to a DNA sequence recognized by cellular mechanisms or introduced synthetic mechanisms required for the specific transcription of a gene. The term "promoter" is also intended to encompass those nucleic acid elements sufficient for use in controlling cell-type-specific, tissue-specific, or inducible expression of promoter-dependent genes through external signals or agents; such elements may be located in the 5' or 3' region of a natural gene. In some respects, a promoter can be a constitutively active promoter, a cell-type-specific promoter, or an inducible promoter.

[0082] As used herein, the term “IRES” refers to an element that facilitates direct entry of the internal ribosome into the cistron (protein-coding region) of the start codon (such as ATG), thereby leading to cap-independent translation of the gene. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83 (199); Jackson RJ and Kaminski, A. RNA 1(10):985-1000 (1995). Under the translational control of IRES, translation proceeds in a cap-independent manner.

[0083] As used herein, the term "termination signal sequence" can refer to any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence. The polyadenylation signal sequence is a recognition region essential for endonuclease cleavage of the RNA transcript, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation signal sequence provides a "polyA site," a site on the RNA transcript where adenine residues are added post-transcriptionally via polyadenylation.

[0084] As used herein, the terms “operably linked,” “operably inserted,” “operably positioned,” “under control,” or “under transcriptional control” mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term “operably linked” means that the DNA sequence and the regulatory sequence are linked in such a way that gene expression is permitted when an appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence. The term “operably inserted” means that the DNA of interest introduced into the cell is located near the DNA sequence that guides the transcription and translation of the introduced DNA (i.e., promotes, for example, the production of a polypeptide encoded by the DNA of interest).

[0085] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is the viral vector, in which the additional DNA segment can be linked to the viral genome. Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attached mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Moreover, some vectors can direct the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors that are practically useful in recombinant DNA technology are often in plasmid form. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors that perform equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0086] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acids that are not naturally present in cells, and may be a cell in which a recombinant expression vector has been introduced. It should be understood that these terms refer not only to the specific test cell, but also to the progeny of such cells. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may not be identical to the parent cells, but are still included within the scope of the term "host cell" as used herein.

[0087] "Immune response," as understood in the art, and generally refers to a biological response within a vertebrate against exogenous factors or abnormal cells (e.g., cancerous cells), which protects the organism against these factors and the diseases they cause. Immune responses are mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation within the vertebrate body. Immune responses include, for example, T cells (e.g., effector T cells, Th cells, CD4+). + Cells, CD8 + The activation or suppression of T cells (or Treg cells), or the activation or suppression of any other cell of the immune system (e.g., NK cells).

[0088] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that represents a major component of the innate immune system. NK cells repel tumors and virus-infected cells by inducing apoptosis, or programmed cell death, in target cells. They are called "natural killers" because NK cells do not require activation to kill target cells. T cells play a major role in cell-mediated immunity. T cell receptors (TCRs) expressed on the surface of T cells distinguish them from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for T cell maturation. There are six types of T cells: helper T cells (e.g., CD4+ cells); cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ cells, etc.). T cells or killer T cells); memory T cells ((i) stem memory TCM cells (such as naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2R.p, CXCR3 and LFA-1 and exhibit many functional properties specific to memory cells); (ii) central memory TCM cells express L-selectin and CCR7, they secrete IL-2 but not IFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines (such as IFNy and IL-4)); regulatory T cells (Treg, suppressor T cells or CD4+CD25+ regulatory T cells); natural killer T cells (NKT); and γδ T cells.

[0089] "Immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting a disease or experiencing a relapse of a disease by means of methods including inducing, enhancing, inhibiting or otherwise altering the immune system or immune response.

[0090] As used herein, the term "link" refers to the association of two or more molecules. Links can be covalent or non-covalent. Links can also be genetic (i.e., recombinant fusion). Such links can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0091] As used herein, the term "treatment," when used in the context of treating cancer, refers to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of symptoms and / or preventing or delaying the onset of the disease or illness.

[0092] As used herein, “cancer” refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0093] Terms such as “effective amount,” “therapeutic effective amount,” and “adequate amount” for CAR T cells and at least one androgen receptor antagonist or composition as described herein refer to an amount sufficient, when administered to a subject, including a human, to achieve a beneficial or desired outcome including: symptom relief; reduction of the severity of a symptom, condition, or disease; attainment of a stable (i.e., non-worsening) state of a symptom, condition, or disease; delay of the onset of or slowing of the progression of a symptom, condition, or disease; improvement or relief of a symptom, condition, or disease state (whether partial or complete), whether detectable or undetectable; improvement of at least one measurable bodily parameter, which is not necessarily identifiable by the patient; or enhancement or improvement of a symptom, condition, or disease. In some respects, treatment includes causing a clinically significant response without excessive levels of side effects. Therefore, “therapeutic effective amount” or its synonyms depend on the context in which they are applied. In some respects, a therapeutically effective amount of a drug (e.g., a T-cell adaptor molecule or composition described herein) is an amount that produces a beneficial or desired outcome in a subject compared to a control not receiving the drug. The amount of a given agent (e.g., CAR T cells and at least one androgen receptor antagonist or combination described herein) will vary depending on various factors, such as the given agent, the drug formulation, the route of administration, the type of disease or condition, the identity of the subject (e.g., age, sex, and / or weight) or the host being treated.

[0094] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly mammalian subjects. Mammal subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc.

[0095] "Control," "controlled experiment," or "standard control" is used in its ordinary, common sense and refers to an experiment in which the subjects or reagents are treated as in a parallel experiment, except that the procedures, reagents, or variables of the experiment are omitted. In some cases, a control is used as a comparative standard in the evaluation of experimental effects. In all respects, a control is the same experiment or the same conditions without the application of a compound (e.g., the compound described herein). In all respects, inhibition of activity compared to a control is the inhibition of activity by the compound (e.g., as described herein) compared to the activity in the absence of the compound (e.g., as described herein).

[0096] The term "contact" is used in its ordinary sense and refers to a process that allows at least two different substances (e.g., chemical compounds including biomolecules, or cells) to come close enough to react, interact, or physically contact. However, it should be recognized that the resulting reaction products can be produced directly from the reaction between the added reagents, or from intermediates from one or more added reagents that can be generated in the reaction mixture. The term "contact" can include allowing two substances to react, interact, or physically contact, where the two substances can be a compound and a protein or enzyme as described herein. In some aspects, contact includes causing the compound described herein to interact with a protein or enzyme.

[0097] As used herein, the term "administration" means oral administration to a subject, administration in suppository form, local contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration, or administration via implanted sustained-release device, such as a micro-osmotic pump. Administration can be performed via any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. "Co-administration" means that the compositions described herein are administered simultaneously, immediately before, or immediately after the administration of one or more other therapies (e.g., anticancer agents). The compounds of the present invention can be administered to a patient alone or co-administered. Co-administration is intended to include the simultaneous or sequential administration of compounds, alone or in combination (more than one compound or agent). Therefore, when desired, the formulations can also be combined with other active substances (e.g., to reduce metabolic degradation, to increase prodrug degradation, and to release the drug, detectable agent). The compositions of the present invention can be delivered via transdermal, local routes and formulated as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jelly preparations, paints, powders, and aerosols. Oral formulations include tablets, pills, powders, sugar-coated pills, capsules, liquids, lozenges, capsules, gels, syrups, slurries, suspensions, etc., suitable for patient ingestion. Solid form formulations include powders, tablets, pills, capsules, capsules, suppositories, and dispersible particles. Liquid form formulations include solutions, suspensions, and emulsions, such as water or water / propylene glycol solutions. The compositions of the present invention may additionally contain components that provide sustained release and / or comfort. Such components include high molecular weight, anionic viscous polymers, gelling polysaccharides, and finely fragmented drug carrier substrates. These components are discussed in more detail in U.S. Patent Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference for all purposes. The compositions of the present invention may also be delivered in the form of microspheres for slow release in vivo. For example, the microspheres may be administered by intradermal injection of drug-containing microspheres that release the drug slowly under the skin (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, (1995); in the form of biodegradable and injectable gel formulations (see, for example, Gao Pharm. Res. 12:857-863, 1995); or in the form of microspheres for oral administration (see, for example, Eyles, J. Pharm. Pharmacol. 49:669-674, 1997).In another aspect, formulations of the compositions of the present invention can be delivered using liposomes fused to or endocytosed by cell membranes, i.e., by using receptor ligands attached to the liposomes, which bind to cell surface membrane protein receptors thereby causing endocytosis. By using liposomes, particularly when the liposomes carry receptor ligands specific to target cells or otherwise preferentially targeting specific organs, the compositions of the present invention can be delivered in vivo to target cells in a concentrated manner. (See, for example, Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0098] As used in this article, “survival” means that a patient remains alive and includes both overall survival and progression-free survival. 1-year survival and 2-year survival are KM estimates of the proportion of subjects alive at 12 or 24 months.

[0099] "Extended survival" means an increase in overall survival and / or progression-free survival relative to control treatment regimens (such as treatment using antibody-drug conjugates described herein). Survival is monitored for at least approximately one month, two months, four months, six months, nine months, or at least approximately one year, or at least approximately two years, or at least approximately three years, or at least approximately four years, or at least approximately five years, or at least approximately ten years after the start of treatment or after initial diagnosis.

[0100] "Reduction or inhibition" refers to the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduction or inhibition can refer to the symptoms of the treated condition, the presence or size of metastases, or the size of the primary tumor.

[0101] As used herein, the term “inhibition” in relation to cell proliferation (e.g., cancer cell proliferation) refers to a negative impact (e.g., reduced proliferation) or killing of cells. In some respects, inhibition refers to the reduction of a disease or disease symptom (e.g., cancer, cancer cell proliferation). Thus, inhibition includes at least in part partially or completely blocking stimulation, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating the amount of signal transduction or enzymatic activity or protein. Similarly, an “inhibitor” is a compound or protein that inhibits a receptor or another protein, for example, by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating its activity (e.g., receptor activity or protein activity).

[0102] The term "pharmaceutically acceptable salt" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds, either purely or in a suitable inert solvent, with a sufficient amount of the desired base. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds, either purely or in a suitable inert solvent, with a sufficient amount of the desired acid. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrophosphoric acid, dihydrophosphoric acid, sulfuric acid, monohydrosulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Also included are salts of amino acids such as arginine and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Certain compounds of the present invention contain both basic and acidic functional groups, allowing the compounds to be converted into basic addition salts or acid addition salts. Other pharmaceutically acceptable carriers known to those skilled in the art are suitable for use in the present invention. Salts tend to be more soluble in aqueous or other protic solvents than their corresponding free base forms. In other cases, the formulation may be a lyophilized powder in 1 mM–50 mM histidine, 0.1%–2% sucrose, and 2%–7% mannitol, at a pH of 4.5–5.5, mixed with buffer solution prior to use.

[0103] As used in this article, the terms “ug” and “uM” are used interchangeably with “μg” and “μM”, respectively.

[0104] As used herein, the term "salt" refers to an acid or base salt of a compound used in the methods of the present invention. Exemplary examples of acceptable salts are inorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid, etc.) salts, and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts.

[0105] The various aspects described in this article are described in more detail in the following sections.

[0106] I. The Method of This Disclosure

[0107] The chimeric antigen receptor (CAR) T-cell and androgen receptor antagonists described in this article can be used for therapeutic applications, such as the treatment of cancer, for example, prostate cancer.

[0108] In some respects, CAR T cells and at least one androgen receptor antagonist, as described herein, can be used in methods of treating human or animal bodies. Relevant aspects of this disclosure provide:

[0109] (i) CAR T cells and at least one androgen receptor antagonist as described herein, used as a drug;

[0110] (ii) CAR T cells and at least one androgen receptor antagonist as described herein, in a method of treating a disease or condition;

[0111] (iii) A method for inhibiting the growth of tumor cells, wherein the method comprises contacting tumor cells with CAR T cells as described herein and at least one androgen receptor antagonist;

[0112] (iv) CAR T-cell and androgen receptor antagonists as described herein, used in the manufacture of drugs for the treatment of diseases or conditions; and,

[0113] (v) A method of treating a subject’s disease or condition, wherein the method comprises administering to the subject in need a therapeutically effective amount of a CAR T-cell and androgen receptor antagonist as described herein.

[0114] Subjects can be patients, preferably human patients, such as patients with prostate cancer.

[0115] Treatment can be any therapy or treatment that achieves some desired therapeutic effect, such as inhibiting or delaying the progression of a symptom, and includes reducing the rate of progression, stopping the rate of progression, improving the symptom, curing or alleviating (whether partially or completely) the symptom, preventing, improving, delaying, reducing or stopping one or more symptoms and / or signs of the symptom, or prolonging the survival of an individual or patient beyond the survival expected without treatment. In some respects, this method is a method of treating cancer.

[0116] Treatments as a preventative measure (i.e., prevention) are also included. For example, individuals who are susceptible to diseases such as cancer or who are at risk of developing or relapsing into disease can be treated, as described herein. Such treatment can prevent or delay the onset or recurrence of disease in an individual.

[0117] Some aspects of this disclosure relate to a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of the following that effectively inhibits the growth of tumor cells: T cells comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist. In some aspects, the CAR includes an antigen-binding domain containing VH and VL, wherein VH includes VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL includes VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 includes the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 includes the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 includes the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0118] Some aspects of this disclosure relate to a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of the following that effectively inhibits tumor cell growth: T cells comprising (i) a chimeric antigen receptor (CAR) encoding an epitope binding to human prostatic six-transmembrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist; wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 6.

[0119] Some aspects of this disclosure relate to a method for treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the following: T cells comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist. In some aspects, the CAR includes an antigen-binding domain containing VH and VL, wherein VH includes VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL includes VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 includes the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 includes the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 includes the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0120] Some aspects of this disclosure relate to a method for treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the following: T cells comprising (i) a chimeric antigen receptor (CAR) encoding a polynucleotide encoding an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist; wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in ID NO: 6.

[0121] In some respects, VH comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and VL comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0122] In some respects, VH contains the amino acid sequence shown in SEQ ID NO:7, and VL contains the amino acid sequence shown in SEQ ID NO:8.

[0123] In some respects, the polynucleotide also encodes armor molecules, and said armor molecules include dominant-negative TGF-β receptor type 2 (TGFβRIIDN).

[0124] In some respects, the armor molecule contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0125] In some respects, the armor molecule contains the amino acid sequence shown in SEQ ID NO: 10.

[0126] In some respects, the polynucleotide encoding CAR comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 11.

[0127] In some respects, the polynucleotide encoding CAR contains the nucleotide sequence shown in SEQ ID NO: 11.

[0128] In some respects, the polynucleotide encoding CAR contains the nucleotide sequence shown in SEQ ID NO: 11, and the polynucleotide encoding the armor molecule contains the nucleotide sequence shown in SEQ ID NO: 9.

[0129] In some respects, the polynucleotide encoding CAR and the polynucleotide encoding the armor molecule are operatively linked under the control of a single promoter.

[0130] In some respects, polynucleotides encoding CAR and polynucleotides encoding armor molecules are operatively linked via IRES.

[0131] In some aspects, the polynucleotide encoding the CAR and the polynucleotide encoding the armor molecule are linked by a nucleotide sequence encoding a cleavable peptide linker. In some aspects, the cleavable peptide linker is a self-cleaving peptide linker. In some aspects, the cleavable peptide linker includes the T2A peptide. In some aspects, the cleavable peptide linker comprises SEQ ID NO: 13.

[0132] In some respects, the polynucleotide comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14.

[0133] In some respects, the polynucleotide contains the nucleotide sequence shown in SEQ ID NO: 14.

[0134] In some respects, T cells contain an amino acid sequence, including the amino acid sequence shown in SEQ ID NO: 12.

[0135] In some respects, T cells contain an amino acid sequence, including the amino acid sequence shown in SEQ ID NO: 15.

[0136] In some respects, androgen receptor antagonists are enzalutamide, apalutamide, dalostamide, abiraterone, bicalutamide, nilumet, flutamide, proxalutamide, or combinations thereof.

[0137] In some respects, the androgen receptor antagonist is enzalutamide.

[0138] In some respects, the androgen receptor antagonist is abiraterone.

[0139] In some aspects, the method includes administering two or more androgen receptor antagonists. In some aspects, the method includes administering two androgen receptor antagonists. In some aspects, the method includes administering enzalutamide and different androgen receptor antagonists. In some aspects, the method includes administering abiraterone and different androgen receptor antagonists. In some aspects, the method includes administering abiraterone and enzalutamide.

[0140] In some aspects, the method also includes administering to the subject at least one additional treatment besides CAR T cells and at least one androgen receptor antagonist. Therefore, the CAR T cells and at least one androgen receptor antagonist described herein can be administered to the subject alone or in combination with one or more other treatments. When CAR T cells and at least one androgen receptor antagonist are administered to the subject in combination with another treatment, the additional treatment can be administered to the individual simultaneously, sequentially, or separately from the administration of CAR T cells and at least one androgen receptor antagonist. When the additional treatment is administered simultaneously with CAR T cells and at least one androgen receptor antagonist, the CAR T cells, at least one androgen receptor antagonist, and the additional treatment can be administered to the subject as a combination formulation. For example, the additional therapy can be a known therapy or treatment agent for the disease to be treated (e.g., prostate cancer).

[0141] In some aspects, CAR T cells and / or at least one androgen receptor antagonist are administered in the form of a pharmaceutical composition, which may contain at least one component other than CAR T cells and / or at least one androgen receptor antagonist. Therefore, another aspect of the invention provides a pharmaceutical composition comprising CAR T cells and at least one androgen receptor antagonist as described herein. A method is also provided comprising formulating CAR T cells and / or at least one androgen receptor antagonist into a pharmaceutical composition.

[0142] In addition to CAR T-cells and / or at least one androgen receptor antagonist, the pharmaceutical composition may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. As used herein, the term “pharmaceuticalally acceptable” refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic reactions, or other problems or complications, to the extent reasonably medically permissible, and that are commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc., must also be “acceptable” in the sense of compatibility with the other components of the formulation. The exact nature of the carrier or other material will depend on the route of administration, which, as discussed below, may be infusion, injection, or any other suitable route.

[0143] In some respects, CAR T cells and / or at least one androgen receptor antagonist may be provided in lyophilized form for reconstitution prior to administration. For example, lyophilized CAR T cells and / or at least one androgen receptor antagonist may be reconstituted in sterile water and mixed with saline prior to administration to a subject.

[0144] Administration may be at a “therapeuticly effective amount”, which is sufficient to demonstrate benefit in the subjects. The actual dosage, as well as the rate and timing of administration, will depend on the nature and severity of the condition being treated, the specific individual being treated, the individual’s clinical condition, the cause of the condition, the site of delivery of the composition, the type of antibody molecule, the method of administration, the schedule of administration, and other factors known to the medical practitioner.

[0145] In some respects, androgen receptor antagonists are administered orally.

[0146] In some respects, T cells are administered intravenously.

[0147] In some respects, T cells and androgen receptor antagonists are administered sequentially or simultaneously, and in any order.

[0148] In some respects, the administration of androgen receptor antagonists does not inhibit T cell activity or T cell killing ability.

[0149] In some respects, the administration of androgen receptor antagonists does not inhibit the release of interferon-γ (IFNγ) in tumor cells.

[0150] In some respects, the administration of androgen receptor antagonists does not reduce CAR expression on T cells.

[0151] In some respects, T cells are CD8+ T cells.

[0152] In some respects, the tumor cells are prostate tumor cells, optionally wherein the prostate tumor cells are metastatic, recurrent, or recurrent.

[0153] In some respects, this administration increased STEAP2 expression in tumor cells compared to T cell administration alone. In some respects, this administration increased STEAP2 expression in tumor cells by approximately 50% to approximately 200% compared to T cell administration alone. In some respects, STEAP2 expression increased by approximately 100%.

[0154] Dosing regimens can be adjusted to provide the best desired response (e.g., therapeutic or preventative response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in a dosage unit form that is easy to administer and achieves uniform dosing. As used herein, a dosage unit form refers to a physically discrete unit suitable as a single dose for use in a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and exposure, along with the required drug delivery system. The specifications of the dosage unit form are determined by and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic or preventative effect to be achieved, and (b) the inherent limitations in the art regarding the formulation of such active compound for the treatment of individual allergies.

[0155] Some aspects of this disclosure relate to the use of T cells and androgen receptor antagonists in a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of T cells and at least one androgen receptor antagonist that effectively inhibits the growth of tumor cells, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule. In some aspects, the CAR includes antigen-binding domains containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the tumor cells are prostate tumor cells. In some aspects, the prostate tumor cells are metastatic, recurrent, or relapsed.

[0156] Some aspects of this disclosure relate to the use of T cells and androgen receptor antagonists in a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of T cells and at least one androgen receptor antagonist that effectively inhibits tumor cell growth, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule, wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 ... The amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 6. In some respects, the tumor cells are prostate tumor cells. In some respects, the prostate tumor cells are metastatic, recurrent, or relapsed.

[0157] Some aspects of this disclosure relate to the use of T cells and androgen receptor antagonists in a method of treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of T cells and at least one androgen receptor antagonist, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule. In some aspects, the CAR includes antigen-binding domains containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the tumor cells are prostate tumor cells. In some aspects, the prostate tumor cells are metastatic, recurrent, or relapsed.

[0158] Some aspects of this disclosure relate to the use of T cells and androgen receptor antagonists in a method of treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of T cells and at least one androgen receptor antagonist, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule, wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in IDNO: 6. In some respects, the tumor cells are prostate tumor cells. In some respects, the prostate tumor cells are metastatic, recurrent, or relapsed.

[0159] Certain aspects of this disclosure relate to methods of treating a disease or condition in a subject in need, the method comprising administering to the subject the compositions disclosed herein. In some aspects, the disease or condition includes cancer. In some aspects, the cancer is prostate cancer. In some aspects, the cancer includes tumors originating from prostate cancer (e.g., tumors resulting from metastases of prostate cancer). In some aspects, the cancer (e.g., prostate cancer) is locally advanced. In some aspects, the cancer (e.g., prostate cancer) is metastatic. In some aspects, the cancer (e.g., prostate cancer) is recurrent. In some aspects, the cancer (e.g., prostate cancer) is recurrent.

[0160] The compositions disclosed herein, such as the T cells comprising polynucleotides encoding CARs disclosed herein, can be used in combination with other anticancer therapies, including one or more additional immunotherapies. In some aspects, the compositions disclosed herein are administered concurrently with other anticancer agents. In some aspects, the compositions disclosed herein and other anticancer agents are administered sequentially (e.g., on the same day or on different days).

[0161] In various aspects, the disclosed anti-STEAP2 CAR T cells and at least one androgen receptor antagonist can be applied to any cell or tissue expressing STEAP2, such as STEAP2-expressing neoplastic cells or tissues. Exemplary aspects include methods for inhibiting STEAP2-mediated cell signaling or methods for killing cells. These methods can be used with any cell or tissue expressing STEAP2, such as cancerous cells or metastatic lesions. Non-limiting examples of cancers expressing STEAP2 include prostate cancer, bladder cancer, colon cancer, pancreatic cancer, ovarian cancer, testicular cancer, Ewing's sarcoma, or cervical cancer.

[0162] An exemplary method includes the step of contacting cells with an effective amount (i.e., an amount sufficient to kill cells) of CAR T cells as described herein and at least one androgen receptor antagonist. This method can be used, for example, with cells cultured in vitro, in vivo, ex vivo, or in situ. For example, cells expressing STEAP2 (e.g., cells collected via biopsy of tumors or metastatic lesions; cells from established cancer cell lines; or recombinant cells) can be cultured in vitro in a culture medium, and the contact step can be achieved by adding CAR T cells and at least one androgen receptor antagonist to the culture medium. This method will result in the killing of STEAP2-expressing cells, particularly tumor cells expressing STEAP2. Alternatively, CAR T cells and at least one androgen receptor antagonist can be administered to a subject via any suitable route of administration (e.g., intravenous, subcutaneous, or direct contact with tumor tissue) to exert their effect in vivo.

[0163] The in vivo effects of the disclosed CAR T-cell and androgen receptor antagonist therapeutic compositions can be evaluated in suitable animal models. For example, xenogeneic cancer models can be used, in which cancer explants or passaged xenograft tissues are introduced into immunocompromised animals, such as nude mice or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Patient-derived xenografts (PDXs), in which tumor tissue from a patient is implanted into immunocompromised or humanized mice (see, e.g., Liu Y et al. Signal Transduct. Target Ther. 2023; 8(1):160), can also be used to evaluate the in vivo effects of the disclosed CAR T-cell and androgen receptor antagonist therapeutic compositions. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression, or metastasis.

[0164] To determine cytotoxicity, cell necrosis or apoptosis (programmed cell death) can be measured. Cell necrosis is typically accompanied by increased plasma membrane permeability; cell swelling; and plasma membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercially available photometric assays for the in vitro quantitative determination of DNA fragmentation are available. Examples of such assays include TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, described in Biochemica (1999), Vol. 2, pp. 34–37 (Roche Molecular Biochemicals).

[0165] Apoptosis can also be determined by measuring morphological changes in cells. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described in Duke and Cohen, Current Protocols in Immunology (Coligan et al., ed. (1992), pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and chromatin condensation and marginalization along the nuclear membrane can be observed. In some respects, apoptosis can also be determined by screening for caspase activity. In some respects, caspase-globulin... ® This assay can be used to measure the activities of caspase-3 and caspase-7. In some respects, the assay provides luminescent caspase-3 / 7 substrates in reagents optimized for caspase activity, luciferase activity, and cell lysis. In some respects, Caspase-Glo is added in an "add-mix-measure" manner. ® The 3 / 7 reagent induces cell lysis, followed by caspase cleavage of the substrate and the generation of a "glow-like" luminescence signal by luciferase. In some respects, the luminescence can be proportional to the amount of caspase activity present and can be used as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane bubbling, and cell contraction. The determination of any of these effects on cancer cells suggests that ADCs may be used for cancer treatment.

[0166] Cell viability can be determined, for example, by using indicators such as neutral red, trypan blue, crystal violet, or ALAMAR. ™The uptake of dyes such as blue in cells is measured (see, for example, Page et al. (1993) Intl J Oncology 3:473-6). In such assays, cells are incubated in a dye-containing medium, washed, and the remaining dye, reflecting cellular uptake, is measured spectrophotometrically. Cell viability can also be measured, for example, by quantifying ATP (an indicator of metabolically active cells). In some respects, as described in the examples provided herein, CellTiter-Glo ® A luminescent cell viability assay is used to evaluate the in vitro potency and / or cell viability of the prepared ADC. In this assay, in some respects, a single reagent (CellTiter-Globe) is used. ® The reagent was added directly to cells cultured in a serum-supplemented medium. The addition of the reagent resulted in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82:1107-12).

[0167] In vivo assays can also be used to assess mechanisms such as apoptosis that promote tumor cell death and tumor shrinkage. In one aspect, the presence of apoptotic foci in xenografts from tumor-bearing mice treated with the therapeutic composition can be examined and compared with untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of treated mice provides an indication of the therapeutic efficacy of the composition.

[0168] One exemplary aspect is a method for reducing or inhibiting the growth of a tumor (e.g., a tumor expressing STEAP2), the method comprising administering a therapeutically effective amount of CAR T cells and at least one androgen receptor antagonist. In some aspects, the treatment is sufficient to reduce or inhibit the growth of a patient's tumor, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival, and / or maintain or improve quality of life. In some aspects, when administered alone, the tumor is resistant to or refractory to treatment using CAR T cells (e.g., anti-STEAP2 CAR T cells).

[0169] In each respect, treatment involves a single or repeated administration of CAR T cells and at least one androgen receptor antagonist formulation via an acceptable route of administration.

[0170] The level of the target antigen (e.g., the level of cells expressing the target antigen) in a given sample from a patient can be assessed to help determine the most effective dosing regimen, etc. One exemplary aspect is a method for determining whether a patient will respond to treatment using CAR T cells and at least one androgen receptor antagonist disclosed herein, the method comprising providing a biological sample from the patient and contacting the biological sample with CAR T cells and at least one androgen receptor antagonist. Exemplary biological samples include tissue, stool samples, or tumor biopsies (e.g., tumor biopsies derived from patients with cancer expressing the target antigen (e.g., cancer expressing STEAP2) or at risk of such cancer). In some aspects, samples (e.g., tissue) can be obtained from the subject, and the protein expression of the target antigen (e.g., STEAP2) can be detected and / or measured using appropriate immunological methods. Such assessments are also used for monitoring purposes throughout the therapy and, in combination with assessments of other parameters, can be used to measure treatment success.

[0171] In some respects, the efficacy of CAR T cells and at least one androgen receptor antagonist can be assessed by exposing a tumor sample from a subject to CAR T cells and at least one androgen receptor antagonist and evaluating the tumor growth rate or volume. In some respects, CAR T cells and at least one androgen receptor antagonist can be administered to a subject when they have been determined to be effective.

[0172] In some aspects, co-administration includes administering an active agent (e.g., CAR T cells) in any order within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours of at least one androgen receptor antagonist. Co-administration also includes administering two active agents simultaneously, substantially simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes of each other), or sequentially in any order. In some aspects, co-administration can be achieved through co-formulation, i.e., preparing a single pharmaceutical composition comprising two active agents. In other aspects, the active agents can be formulated individually. In another aspect, the active agents and / or adjuvants can be linked or conjugated to each other. In some aspects, the compounds described herein can be combined with cancer treatments such as radiation or surgery.

[0173] Specific in vivo clinical protocols regarding the route of administration, excipients, diluents, dosage, and timing can be determined by those skilled in the art based on clinical circumstances. The dosage and frequency (single or multiple doses) administered to mammals can vary depending on various factors, such as whether the mammal suffers from another disease and the route of administration; the recipient's body type, age, sex, health, weight, body mass index, and diet; the nature and severity of the symptoms of the disease being treated (e.g., cancer symptoms), the types of concurrent treatments, complications arising from the treated disease, or other health-related problems. Other treatment regimens or agents can be used in conjunction with the methods and compounds of the applicant's invention. Adjustments and manipulations of established dosages (e.g., frequency and duration) are entirely within the capabilities of those skilled in the art.

[0174] II. The polynucleotides disclosed herein

[0175] Some aspects of this disclosure relate to polynucleotides comprising a nucleotide sequence encoding a CAR that specifically binds to human STEAP2. In some aspects, the CAR comprises (i) an antigen-binding domain that binds to an epitope on STEAP2, (ii) a transmembrane domain, and (iii) an intracellular domain. In some aspects, the CAR also comprises a hinge / spacer region domain. In some aspects, the hinge / spacer region domain is located between the antigen-binding domain and the transmembrane domain.

[0176] In some respects, the polynucleotide also contains a nucleotide sequence encoding an armor molecule. In some respects, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the armor moiety are expressed under the control of the same promoter. In some respects, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the armor moiety are expressed under the control of two promoters. In some respects, the two promoters are different promoters. In some respects, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the armor moiety are expressed as a single continuous polypeptide. In some respects, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the armor moiety are expressed as two separate polypeptides. In some respects, the CAR and the nucleotide sequence encoding the armor moiety are linked by a nucleotide sequence encoding a linker. In some respects, the linker is a peptide linker. In some respects, the linker is a cleavable linker. In some respects, the linker is a self-cleaving peptide linker, such as containing the T2A peptide.

[0177] IA antigen-binding domain

[0178] This document discloses polynucleotides comprising nucleotide sequences encoding CARs, wherein the CAR comprises (i) an antigen-binding domain that binds to an epitope on human STEAP2, (ii) an intracellular signaling domain, and (iii) a transmembrane domain. Any antigen-binding domain may be used in the compositions disclosed herein. In some aspects, the antigen-binding domain comprises Fab, Fab', F(ab')2, Fd, Fv, a single-chain variable fragment (scFv), a single-chain antibody, VHH, vNAR, a nanobody (a single-domain antibody), or any combination thereof. In some aspects, the antigen-binding domain comprises scFv.

[0179] In some aspects, the antigen-binding domain of the CAR includes a variable heavy chain region (VH) and a variable light chain region (VL), wherein the VH includes a VH complementarity-determining region (CDR) 1, VH-CDR2, and VH-CDR3; and wherein the VL includes VL-CDR1, VL-CDR2, and VL-CDR3. In some aspects, the antigen-binding domain includes VH-CDR3 containing an amino acid sequence selected from SEQ ID NO: 6. In some aspects, the antigen-binding domain includes VH-CDR2 containing an amino acid sequence selected from SEQ ID NO: 5. In some aspects, the antigen-binding domain includes VH-CDR1 containing an amino acid sequence selected from SEQ ID NO: 4.

[0180] In some aspects, the antigen-binding domain comprises VL-CDR3 containing an amino acid sequence selected from SEQ ID NO: 3. In some aspects, the antigen-binding domain comprises VL-CDR2 containing an amino acid sequence selected from SEQ ID NO: 2. In some aspects, the antigen-binding domain comprises VL-CDR1 containing an amino acid sequence selected from SEQ ID NO: 1.

[0181] In some aspects, the antigen-binding domain includes VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0182] In some aspects, the CAR includes an antigen-binding domain containing a VH, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7. In some aspects, the antigen-binding domain includes a VH containing an amino acid sequence selected from SEQ ID NO: 7.

[0183] In some aspects, the CAR includes an antigen-binding domain containing a VL, the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8. In some aspects, the antigen-binding domain includes a VL containing an amino acid sequence selected from SEQ ID NO: 8.

[0184] In some aspects, the CAR comprises an antigen-binding domain containing VH and VL, wherein the VH comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the CAR comprises an antigen-binding domain containing VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8.

[0185] In some aspects, the CAR comprises an antigen-binding domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the CAR comprises an antigen-binding domain having an amino acid sequence shown in SEQ ID NO: 16.

[0186] IB Intracellular Domain

[0187] This document discloses polynucleotides comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises (i) an antigen-binding domain that binds to an epitope on human STEAP2, (ii) an intracellular signaling domain, and (iii) a transmembrane domain. Any intracellular signaling domain may be used in the compositions disclosed herein. In some aspects, the intracellular signaling domain comprises a co-stimulatory domain or a portion thereof.

[0188] In some respects, the intracellular domain includes costimulatory domains selected from the group consisting of: the intracellular domain of CD3z, the CD28 costimulatory domain, the CD27 costimulatory domain, the 4-1BB costimulatory domain, the ICOS costimulatory domain, the OX-40 costimulatory domain, the GITR costimulatory domain, the CD2 costimulatory domain, the IL-2Rβ costimulatory domain, the MyD88 / CD40a CD28 costimulatory domain, and any combination thereof.

[0189] In some aspects, the intracellular domain includes a 4-1BB co-stimulatory domain. In some aspects, the CAR includes (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (ii) a 4-1BB co-stimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 7 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; and (ii) a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17.

[0190] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; and (ii) a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17.

[0191] In some aspects, the intracellular domain comprises the intracellular domain of CD3z and the CD28 co-stimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (ii) the intracellular domain of CD3z and the CD28 co-stimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; and a CD28 co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 19.

[0192] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; and (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and (iii) an intracellular domain of CD28 having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and (iv) an intracellular domain of CD28 having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 9 The amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In some aspects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; and (ii) an intracellular domain of CD3z comprising the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 19.

[0193] In some aspects, the intracellular domain comprises the intracellular domain of CD3z and the 4-1BB costimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (ii) the intracellular domain of CD3z and the 4-1BB costimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 17.

[0194] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; and (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and (iii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and (iv) an intracellular domain of CD3z having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or The amino acid sequence shown in SEQ ID NO: 17 has an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; and (ii) an intracellular domain of CD3z comprising the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17.

[0195] In some aspects, the intracellular domain comprises the intracellular domain of CD3z, the CD28 co-stimulatory domain, and the 4-1BB co-stimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (ii) the intracellular domain of CD3z, the CD28 co-stimulatory domain, and the 4-1BB co-stimulatory domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; and a 4-1BB co-stimulatory domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17.In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO:7 and a VL containing the amino acid sequence shown in SEQ ID NO:8; and (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO:18; a CD28 co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO:19; and a 4-1BB co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO:17.

[0196] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; and (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; The amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. In some respects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; and (ii) an intracellular domain of CD3z comprising the amino acid sequence shown in SEQ ID NO: 18; a CD28 co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 19; and a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17.

[0197] In some aspects, the CAR comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12. In some aspects, the CAR comprises the amino acid sequence shown in SEQ ID NO: 12.

[0198] IC transmembrane domain

[0199] This document discloses polynucleotides comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises (i) an antigen-binding domain that binds to an epitope on human STEAP2, (ii) an intracellular signaling domain, and (iii) a transmembrane domain. Any transmembrane domain may be used in the compositions disclosed herein. In some aspects, the transmembrane domain comprises a transmembrane domain selected from CD4, CD8α, or CD28. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain.

[0200] In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) a 4-1BB co-stimulatory domain; and (iii) a transmembrane domain comprising a transmembrane domain of CD28. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20.

[0201] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; The amino acid sequence shown in SEQ ID NO: 16 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; (ii) a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20.

[0202] In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) an intracellular domain of CD3z and a co-stimulatory domain of CD28; and (iii) a transmembrane domain comprising a transmembrane domain of CD28. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and a CD28 co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20.In some respects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 19; and (iii) a transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 20.

[0203] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain ... The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) a transmembrane domain comprising an amino acid sequence having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. In some respects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 19; and (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20.

[0204] In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) an intracellular domain of CD3z and a 4-1BB co-stimulatory domain; and (iii) a transmembrane domain comprising a transmembrane domain of CD28. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 17 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20.In some respects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 20.

[0205] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain ... The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. In some respects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20.

[0206] In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) an intracellular domain of CD3z, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain; and (iii) a transmembrane domain comprising a transmembrane domain of CD28.In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; and a 4-1BB co-stimulatory domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane ... an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO: 18; a CD28 co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 19; and a 4-1BB co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 20.

[0207] In some respects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; The amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20. In some respects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising the amino acid sequence shown in SEQ ID NO: 18; a CD28 co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 19; and a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20.

[0208] In some respects, CAR contains the amino acid sequence shown in SEQ ID NO: 12.

[0209] ID Spacing Area / Hinge Structural Domain

[0210] This document discloses polynucleotides comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises (i) an antigen-binding domain that binds to an epitope on human STEAP2, (ii) an intracellular signaling domain, (iii) a transmembrane domain, and (iv) a hinge / spacer region domain. Any hinge / spacer region domain may be used in the compositions disclosed herein. In some aspects, the hinge / spacer region domain comprises a human immunoglobulin hinge / spacer region domain. In some aspects, the hinge / spacer region domain comprises an IgG hinge domain. In some aspects, the hinge / spacer region domain comprises an IgG1 hinge domain and an IgG2 hinge domain, an IgG3 hinge domain, or an IgG4 hinge domain. In some aspects, the hinge / spacer region domain comprises an IgG4 hinge domain. In some aspects, the IgG hinge domain is a variant hinge domain. In some aspects, the IgG4 hinge domain is a variant IgG4 hinge domain. In some aspects, the variant IgG4 hinge domain comprises an S228P mutation. In some respects, the IgG4 hinge domain contains the amino acid sequence shown in SEQ ID NO: 21.

[0211] In some aspects, the CAR includes (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) a 4-1BB co-stimulatory domain; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain comprising the amino acid sequence shown in SEQ ID NO: 21.

[0212] In some respects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) a 4-1BB co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. The amino acid sequence shown in SEQ ID NO: 21 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iv) an IgG hinge domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21. In some respects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO: 16; (ii) a 4-1BB co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 17; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain comprising the amino acid sequence shown in SEQ ID NO: 21.

[0213] In some aspects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) an intracellular domain of CD3z and a co-stimulatory domain of CD28; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain.In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; (iii) a transmembrane domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain having an amino acid sequence having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; The amino acid sequence shown in SEQ ID NO: 7 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) an intracellular domain of CD3z containing the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain containing the amino acid sequence shown in SEQ ID NO: 19; (iii) a transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain containing the amino acid sequence shown in SEQ ID NO: 21.

[0214] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain ... The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21. In some respects, the CAR comprises (i) an antigen-binding domain containing the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z and a CD28 co-stimulatory domain; (iii) a transmembrane domain containing a transmembrane domain of CD28; and (iv) an IgG hinge domain.

[0215] In some respects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) an intracellular domain of CD3z and a 4-1BB co-stimulatory domain; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain.In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; (iii) a transmembrane domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain having an amino acid sequence having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; The amino acid sequence shown in 21 has an amino acid sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) an intracellular domain of CD3z and a 4-1BB co-stimulatory domain; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain.

[0216] In some aspects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a 4-1BB co-stimulatory domain ... The amino acid sequence shown in SEQ ID NO: 20 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; (iii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21. In some respects, the CAR comprises (i) an antigen-binding domain containing the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z and a 4-1BB co-stimulatory domain; (iii) a transmembrane domain containing a transmembrane domain of CD28; and (iv) an IgG hinge domain.

[0217] In some respects, the CAR comprises (i) an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; (ii) an intracellular domain of CD3z, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain.In some aspects, the CAR comprises (i) an antigen-binding domain comprising a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (ii) an intracellular domain of CD3z containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; and a 4-1BB co-stimulatory domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; (iii) a transmembrane domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; The amino acid sequence shown in SEQ ID NO: 21 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (iv) an IgG hinge domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.In some respects, the CAR comprises (i) an antigen-binding domain comprising a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8; (ii) an intracellular domain of CD3z, a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain.

[0218] In some respects, the CAR comprises (i) an antigen-binding domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; (ii) an intracellular domain of CD3z comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; and a CD28 co-stimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; The amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; (iii) a transmembrane domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and (iv) an IgG hinge domain having an amino acid sequence ... an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid sequence having an amino acid The amino acid sequence shown in NO:21 has an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the CAR comprises (i) an antigen-binding domain comprising the amino acid sequence shown in SEQ ID NO:16; (ii) an intracellular domain of CD3z, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain; (iii) a transmembrane domain comprising a transmembrane domain of CD28; and (iv) an IgG hinge domain.

[0219] In some aspects, the nucleotide sequence encoding the CAR has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 22. In some aspects, the nucleotide sequence encoding the CAR comprises the nucleotide sequence shown in SEQ ID NO: 22.

[0220] IE Armored Motives

[0221] This article discloses a polynucleotide comprising (a) a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain that binds to an epitope on human STEAP2, and (b) a nucleotide sequence encoding an armor molecule. One approach to preparing CAR-T cells that are more resistant to tumor-associated immunosuppression is called “armoring.” Armoring is a molecular manipulation of CAR-T cells to express one or more “armor molecules” that can combat immunosuppression. For example, researchers have reported modifying CAR-T cells to secrete a single-stranded variable fragment (scFv) that blocks PD-1, which improves the antitumor activity of CAR-T cells in mouse models of PD-L1+ hematologic malignancies and solid tumors (Rafiq, S., Yeku, O., Jackson, H. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36, 847–856 (2018)). Other studies have demonstrated the effectiveness of armoring T cells with dominant-negative TGF-β receptor type 2 (TGFβRIIDN) armor molecules to neutralize the inhibitory effect of TGF-β on T cells (Bollard et al., Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma, J Clin Oncol 36(11):121-1139 (2018)). Currently, at least one clinical study is investigating the effectiveness of armoring PSMA-CAR-T cells with TGFβRIIDN armor molecules for the treatment of castration-resistant prostate cancer (NCT03089203).

[0222] In some aspects, the armor molecule comprises a dominant-negative TGF-β receptor type 2 (TGFβRIIDN). In some aspects, the armor molecule comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the armor molecule comprises the amino acid sequence shown in SEQ ID NO: 10.

[0223] In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 105. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0224] In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in 10 has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0225] In some aspects, the armor molecule comprises a dominant-negative TGF-β receptor type 2 (TGFβRIIDN). In some aspects, the armor molecule comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the armor molecule comprises the amino acid sequence shown in SEQ ID NO: 10.

[0226] In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0227] In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in 10 has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0228] In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain having an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0229] In some respects, the polynucleotide comprises (a) a nucleotide sequence encoding an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12, and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. In some respects, the polynucleotide comprises (a) a nucleotide sequence encoding an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0230] In some respects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR comprising the nucleotide sequence shown in SEQ ID NO: 22; and (b) a nucleotide sequence encoding an armor molecule comprising the nucleotide sequence shown in SEQ ID NO: 9.

[0231] In some respects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR comprising the nucleotide sequence shown in SEQ ID NO: 11; and (b) a nucleotide sequence encoding an armor molecule comprising the nucleotide sequence shown in SEQ ID NO: 9.

[0232] In some aspects, the nucleotide encoding the CAR and the nucleotide encoding the armor molecule are linked by a third nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable peptide linker. In some aspects, the cleavable peptide linker comprises a T2A peptide. In some aspects, the cleavable peptide linker comprises SEQ ID NO: 13.

[0233] In some aspects, the polynucleotide comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14. In some aspects, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0234] III. The anti-STEAP2 antibody disclosed herein

[0235] Some aspects of this disclosure relate to antibodies or antigen-binding moieties thereof that specifically bind to human STEAP2. In some aspects, the antibody or antigen-binding moieties thereof comprise a variable heavy chain region (VH) and a variable light chain region (VL), wherein the VH comprises a VH complementarity-determining region (CDR) 1, VH-CDR2, and VH-CDR3; and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3. In some aspects, the antibody or antigen-binding moieties thereof comprise VH-CDR3 containing an amino acid sequence selected from SEQ ID NO: 6. In some aspects, the antibody or antigen-binding moieties thereof comprise VH-CDR2 containing an amino acid sequence selected from SEQ ID NO: 5. In some aspects, the antibody or antigen-binding moieties thereof comprise VH-CDR1 containing an amino acid sequence selected from SEQ ID NO: 4.

[0236] In some aspects, the antibody or its antigen-binding portion comprises VL-CDR3 containing an amino acid sequence selected from SEQ ID NO: 3. In some aspects, the antibody or its antigen-binding portion comprises VL-CDR2 containing an amino acid sequence selected from SEQ ID NO: 2. In some aspects, the antibody or its antigen-binding portion comprises VL-CDR1 containing an amino acid sequence selected from SEQ ID NO: 1.

[0237] In some aspects, the antibody or its antigen-binding portion comprises VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0238] In some aspects, the antibody or its antigen-binding portion comprises a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7. In some aspects, the antibody or its antigen-binding portion comprises a VH containing an amino acid sequence selected from SEQ ID NO: 7.

[0239] In some aspects, the antibody or its antigen-binding portion comprises a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8. In some aspects, the antibody or its antigen-binding portion comprises a VL containing an amino acid sequence selected from SEQ ID NO: 8.

[0240] In some aspects, the antibody or its antigen-binding portion comprises a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the antibody or its antigen-binding portion comprises a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8.

[0241] In some aspects, the antibody or its antigen-binding portion comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the antibody or its antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 16.

[0242] In some respects, the antibody or its antigen-binding moiety cross-competes with the antibody or its antigen-binding moiety disclosed herein for binding to human STEAP2. In some respects, the antibody or its antigen-binding moiety binds to the same epitopes on human STEAP2 as the antibody or its antigen-binding moiety disclosed herein. In some respects, the antibody or its antigen-binding moiety binds to overlapping epitopes on human STEAP2 as the antibody or its antigen-binding moiety disclosed herein.

[0243] IV. Androgen receptor antagonists disclosed herein

[0244] Androgen receptors are members of the nuclear hormone receptor family, activated by androgens such as dihydrotestosterone (DHT). Androgen receptors are a primary therapeutic target for prostate cancer. Several compounds have been developed as chemotherapy therapies for prostate cancer.

[0245] Androgen receptor antagonists (antiandrogens) are drugs used to treat hormone-based syndromes and prostate cancer. Currently used drugs for prostate cancer include flutamide, bicalutamide, nilumet, enzalutamide, and ARN-509. Each of these inhibitors binds to the hormone-binding pocket (HBP) of the androgen receptor. This is the same site where the natural physiological steroids testosterone (TES) and dihydrotestosterone (DHT) bind. The drugs work by competing with the natural hormone for the binding pocket and thus reducing receptor activation.

[0246] Castration-resistant prostate cancer has shown sensitivity to androgen / AR axis manipulation without resistance. The androgen axis can be manipulated using antiandrogens (nilumid, enzalutamide), androgen synthesis inhibitors (ketoconazole, abiraterone acetate), corticosteroids (dexamethasone, prednisone), or estrogen therapy. In cases of castration-resistant disease, taxane-based chemotherapy has shown efficacy and prolonged survival. Patients who have progressed on docetaxel have been shown to benefit from abiraterone acetate, a selective cytochrome P450 17A1 inhibitor that requires co-administration with glucocorticoids to reduce side effects. Enzalutamide (MDV-3100) is a novel AR antagonist that blocks AR signaling more effectively than currently available AR antagonists (Tran et al., Science 2009; 324(5928):787-790.) and has shown impressive antitumor activity and similar effects on overall survival as abiraterone.

[0247] Some aspects of this disclosure relate to a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of the following that effectively inhibits the growth of tumor cells: T cells comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist. In some aspects, the CAR includes an antigen-binding domain containing VH and VL, wherein VH includes VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL includes VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 includes the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 includes the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 includes the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0248] Some aspects of this disclosure relate to a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of the following that effectively inhibits tumor cell growth: T cells comprising (i) a chimeric antigen receptor (CAR) encoding an epitope binding to human prostatic six-transmembrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist; wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4, and VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 6.

[0249] Some aspects of this disclosure relate to a method for treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the following: T cells comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist. In some aspects, the CAR includes an antigen-binding domain containing VH and VL, wherein VH includes VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL includes VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 includes the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 includes the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 includes the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0250] Some aspects of this disclosure relate to a method for treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the following: T cells comprising (i) a chimeric antigen receptor (CAR) encoding a polynucleotide encoding an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and at least one androgen receptor antagonist; wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 5. The amino acid sequence shown in ID NO: 6.

[0251] In all respects, the androgen receptor is a mutant androgen receptor. In all respects, the mutant androgen receptor is associated with a disease unrelated to the wild-type androgen receptor. In all respects, the androgen receptor includes at least one amino acid mutation compared to the above sequence (e.g., mutations in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In all respects, the mutant androgen receptor is a splice variant. In all respects, the mutant androgen receptor lacks a portion of the ligand-binding domain. In all respects, the mutant androgen receptor is active in the absence of a binding ligand. In all respects, the mutant androgen receptor lacks the ligand-binding domain. In all respects, the splice variant androgen receptor is AR variant 1 (e.g., GI: 21322252). In all aspects, the splice variant androgen receptor is AR variant 2 (AR45) (e.g., GI: 21713434). In all aspects, the splice variant androgen receptor is AR variant 3 (AR-V7) (e.g., GI: 224181614). In all aspects, the splice variant androgen receptor is AR variant 4 (AR-V1) (e.g., GI: 224181616). In all aspects, the splice variant androgen receptor is AR variant 5 (AR-V4) (e.g., GI: 224181620). In all aspects, the splice variant androgen receptor is AR variant 6 (AR-V3) (e.g., GI: 224181622). In all aspects, the splice variant androgen receptor is AR v567es (e.g., GI: 270358642).

[0252] In all respects, androgen receptor antagonists are antagonists of nuclear receptors. In all respects, androgen receptor antagonists are antagonists of human androgen receptors. In all respects, androgen receptor antagonists are antagonists of wild-type human androgen receptors. In all respects, androgen receptor antagonists are antagonists of mutant human androgen receptors. In all respects, androgen receptor antagonists are antagonists of drug-resistant human androgen receptors. In all respects, androgen receptor antagonists are antagonists of cassoxat-resistant human androgen receptors. In all respects, androgen receptor antagonists are antagonists of flutamide-resistant human androgen receptors. In all respects, androgen receptor antagonists are antagonists of MDV3100-resistant human androgen receptors. In all respects, androgen receptor antagonists are antagonists of ARN-509-resistant human androgen receptors. In all respects, androgen receptor antagonists are antagonists of non-ligand-activated androgen receptors. In all respects, androgen receptor antagonists are antagonists of the N-terminal activated, non-ligand-activated androgen receptor. In all respects, androgen receptor antagonists are antagonists of non-ligand-activated androgen receptor splice variants. In all respects, androgen receptor antagonists are antagonists of the HER2-activated, non-ligand-activated androgen receptor. In all respects, androgen receptor antagonists are antagonists of the IL-6-activated, non-ligand-activated androgen receptor.

[0253] In all respects, androgen receptor antagonists do not inhibit the binding of ligands (e.g., DHT) to the androgen receptor. In all respects, androgen receptor antagonists do not bind to the ligand-binding domain of the androgen receptor. In all respects, androgen receptor antagonists bind to the DNA-binding domain of the androgen receptor. In all respects, androgen receptor antagonists do not increase the degradation of the androgen receptor compared to the absence of the androgen receptor. In all respects, androgen receptor antagonists do not reduce the nuclear localization of the androgen receptor compared to the absence of the androgen receptor. In all respects, androgen receptor antagonists do not prevent the androgen receptor from binding to DNA. In all respects, androgen receptor antagonists do not reduce the binding of the androgen receptor to DNA. In all respects, androgen receptor antagonists prevent RNA polymerase II from being recruited to DNA. In all respects, androgen receptor antagonists prevent the binding of RNA polymerase II to the transcriptional complex, which includes the androgen receptor bound to the androgen receptor. In all respects, androgen receptor antagonists prevent the initiation of transcription mediated by the androgen receptor. In all aspects, androgen receptor antagonists inhibit (e.g., compared to control) the recruitment of RNA pol II to DNA. In all aspects, androgen receptor antagonists inhibit (e.g., compared to control) the binding of RNA pol II to a transcriptional complex comprising an androgen receptor that binds to the androgen receptor antagonist. In all aspects, androgen receptor antagonists inhibit (e.g., compared to control) the initiation of transcription induced by the androgen receptor. In all aspects, androgen receptor antagonists reduce androgen receptor-induced transcription relative to a control (e.g., in the absence of an androgen receptor antagonist). In all aspects, androgen receptor antagonists reduce the binding of co-activators to the androgen receptor. In all aspects, androgen receptor antagonists bind to both the androgen receptor and DNA. In all aspects, androgen receptor antagonists bind to the androgen receptor while binding to DNA. In all aspects, androgen receptor antagonists bind to the minor groove of DNA. In all aspects, androgen receptor antagonists contact one or more of Lys609, Asn610, Pro612, Phe582, Ala586, Tyr593, and / or Arg615. In all aspects, androgen receptor antagonists contact one or more of Lys609, Asn610, and Pro612. In all aspects, androgen receptor antagonists contact the minor groove of DNA. In all aspects, androgen receptor antagonists contact one or more amino acids corresponding to Lys609, Asn610, Pro612, Phe582, Ala586, Tyr593, and / or Arg615 of the human androgen receptor. In all aspects, androgen receptor antagonists contact one or more amino acids corresponding to Lys609, Asn610, and / or Pro612 of the human androgen receptor. In all aspects, androgen receptor antagonists contact amino acids corresponding to Lys609 and Pro612 of the human androgen receptor.In all aspects, androgen receptor antagonists contact amino acids corresponding to Lys609 and Pro612 of the human androgen receptor, but not amino acids corresponding to Asn610. In all aspects, androgen receptor antagonists contact amino acids corresponding to either Lys609 or Pro612 of the human androgen receptor, but not amino acids corresponding to Asn610. In all aspects, androgen receptor antagonists contact amino acids corresponding to either Lys609 or Pro612 of the human androgen receptor. In all aspects, androgen receptor antagonists contact amino acids Lys609 or Pro612 of the human androgen receptor. In all aspects, androgen receptor antagonists contact amino acids Lys609 and Pro612 of the human androgen receptor. In all aspects, androgen receptor antagonists contact amino acids Lys609 and Pro612 of the human androgen receptor, but not Asn610. In all aspects, androgen receptor antagonists contact amino acids Lys609 or Pro612 of the human androgen receptor without contacting Asn610.

[0254] In some respects, androgen receptor antagonists inhibit androgen receptor activity in the prostate and / or bone to a greater extent than in other tissues (e.g., compared to controls).

[0255] In some respects, androgen receptor antagonists are soluble in aqueous solutions. In other respects, androgen receptor antagonists are soluble in dextrins (e.g., hydroxypropyl β and γ).

[0256] In some respects, androgen receptor antagonists are flutamide, nilumet, enzalutamide, bicalutamide, ketoconazole, abiraterone, abiraterone acetate, orteronel, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, episteride, dexamethasone, prednisone, leuprorelin, goserelin, triptorelin, histidinerelin, estrogens, or combinations thereof.

[0257] In some respects, androgen receptor antagonists are enzalutamide, apalutamide, dalostamide, abiraterone, bicalutamide, nilumet, flutamide, proxalutamide, or combinations thereof.

[0258] In some respects, androgen receptor antagonists are enzalutamide (Tran et al., Science 2009, 324(5928): 787-790). Enzalutamide can be marketed, for example, under the name Xtandi ® Obtained from Medivation or Astellas. The structure of enzalutamide is shown below (Xtandi ® Product label; 08 / 2012 revised version):

[0259]

[0260] In some respects, androgen receptor antagonists are abiraterone, for example, in the form of abiraterone acetate (Agarwal et al., Future Oncology 2010, 6(5): 665-679). Abiraterone can be obtained, for example, from Janssen Biotech, Inc. The structure of abiraterone acetate is shown below (ZYTIGA). ® Product label; 03 / 2015 revised version):

[0261]

[0262] In some respects, androgen receptor antagonists are administered orally.

[0263] In some respects, the administration of androgen receptor antagonists does not inhibit T cell activity or T cell killing ability (e.g., the activity or killing ability of anti-STEAP2 CAR T cells).

[0264] In some respects, the administration of androgen receptor antagonists does not inhibit the release of interferon-γ (IFNγ) from CAR T cells in tumor cells (e.g., anti-STEAP2 CAR T cells).

[0265] In some respects, the administration of androgen receptor antagonists does not reduce CAR expression on T cells (e.g., anti-STEAP2 CAR on T cells).

[0266] V. Cells disclosed herein

[0267] Some aspects of this disclosure relate to cells comprising the polynucleotides or polypeptides disclosed herein. Some aspects of this disclosure relate to a cell comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to human STEAP2. In some aspects, the cell also comprises (ii) a polynucleotide encoding an armor molecule. In some aspects, the cell is an immune cell. In some aspects, the cell is selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, tumor-infiltrating lymphocytes, and any combination thereof. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a human cell.

[0268] The cells disclosed herein can be obtained from any source. For example, T cells can be differentiated from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. Any number of techniques known to those skilled in the art (such as FICOLL) can also be used. ™ T cells are obtained from blood units collected from the subject via apheresis (isolation and / or apheresis). In some respects, cells collected via apheresis are washed to remove plasma fractions and placed in an appropriate buffer or medium for subsequent processing. In other respects, cells are washed with PBS. As will be recognized, washing steps such as those using a semi-automated flow-through centrifuge, such as COBE, can be used. ™ 2991 Cell Processing Machine, Baxter Cytomate ™ In some aspects, the washed cells are resuspended in one or more biocompatible buffers or other saline solutions with or without buffers. In other aspects, unwanted components of the apheresis sample are removed. Further methods for isolating T cells for T-cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0269] In some aspects, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example by centrifugation achieved via a PERCOLL™ gradient. In some aspects, specific subsets of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, are further isolated using positive or negative selection techniques known in the art. For example, enriching T cell populations by negative selection can be accomplished using a combination of antibodies targeting surface markers specific to the negatively selected cells. In some aspects, cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry can be used, employing a mixture of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some aspects, flow cytometry and cell sorting are used to isolate cell populations of interest for use in this disclosure.

[0270] In some respects, PBMCs are used directly with immune cells (such as CARs) for genetic modification, using methods as described herein. In other respects, after isolating PBMCs, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subsets before or after genetic modification and / or expansion.

[0271] In some respects, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying each associated cell surface antigen in these types of CD8+ cells. In some respects, central memory T cells express phenotypic markers including CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and are negative for granzyme B. In some respects, central memory T cells are CD45RO+, CD62L+, and CD8+ T cells. In some respects, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In some respects, CD4+ T cells are further sorted into subsets. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with cell surface antigens.

[0272] In some aspects, immune cells (e.g., T cells) are genetically modified after isolation using known methods, or are activated and expanded (or differentiated, in the case of progenitor cells) in vitro prior to genetic modification. In other aspects, immune cells (e.g., T cells) are genetically modified with the CAR described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding the CAR), followed by activation and / or expansion in vitro. Methods for activating and expanding T cells are known in the art and described, for example, in U.S. Patent Nos. 6,905,874; 6,867,041; and 6,797,514; and PCT Publication No. WO 2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. Typically, such methods involve contacting PBMCs or isolated T cells with stimulants and co-stimulants (such as anti-CD3 and anti-CD28 antibodies, which typically attach to beads or other surfaces) in a culture medium containing appropriate cytokines (such as IL-2). Anti-CD3 and anti-CD28 antibodies attached to the same beads are used as "alternative" antigen-presenting cells (APCs). One example is The Dynabeads. ®The system is a CD3 / CD28 activator / stimulator system for the physiological activation of human T cells. In other aspects, T cells are activated and stimulated to proliferate using methods such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012 / 129514 (the contents of which are hereby incorporated in their entirety by reference).

[0273] In some cases, T cells are obtained from donor subjects. In some cases, the donor subject is a patient with cancer or a tumor. In other cases, the donor subject is a patient without cancer or a tumor.

[0274] In some respects, the cell contains a polynucleotide containing a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0275] In some respects, the cell contains a polynucleotide containing a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain containing VH and VL, the VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0276] In some respects, the cell contains a polynucleotide containing a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0277] In some respects, the cell contains a polynucleotide containing a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain containing VH and VL, the VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the polynucleotide comprises a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0278] In some respects, the cell contains a polynucleotide comprising (a) a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0279] In some aspects, the cell contains a polynucleotide comprising (a) a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain comprising VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in 10 has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising the amino acid sequence shown in SEQ ID NO: 10.

[0280] In some respects, the cell contains a polynucleotide comprising (a) a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 9. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) an armor molecule comprising the nucleic acid sequence shown in SEQ ID NO: 9.

[0281] In some aspects, the cell contains a polynucleotide comprising (a) a nucleotide sequence encoding a CAR, wherein the CAR contains an antigen-binding domain comprising VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) a nucleotide sequence encoding TGFβRIIDN. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; The amino acid sequence shown in 9 has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some aspects, the polynucleotide comprises (a) a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain containing VH and VL, the VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and (b) an armor molecule comprising the nucleic acid sequence shown in SEQ ID NO: 9.

[0282] In some respects, the cell contains a polynucleotide comprising (a) a nucleotide sequence encoding a CAR containing the nucleotide sequence shown in SEQ ID NO: 22; and (b) a nucleotide sequence encoding an armor molecule containing the nucleotide sequence shown in SEQ ID NO: 9.

[0283] In some aspects, the cell contains a polynucleotide comprising a nucleotide sequence encoding a polypeptide containing an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15. In some aspects, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 15.

[0284] In some respects, the cell contains a CAR containing an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.

[0285] In some aspects, the cell contains a polypeptide comprising (i) a CAR containing an antigen-binding domain comprising VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 6; and (b) the amino acid sequence shown in SEQ ID NO: 10.

[0286] In some respects, the cell contains a CAR containing the amino acids shown in SEQ ID NO: 12 and an armor molecule containing the amino acids shown in SEQ ID NO: 10.

[0287] VI. The vector, host cell, and pharmaceutical composition disclosed herein

[0288] In some aspects, the polynucleotides of this disclosure are present in a vector. Therefore, vectors comprising the polynucleotides of this disclosure are provided herein. In some aspects, this disclosure relates to vectors or vector families comprising polynucleotides encoding CARs, as described herein. In other aspects, this disclosure relates to vectors or vector families comprising polynucleotides encoding antibodies or antigen-binding molecule thereof that specifically bind to STEAP2, as disclosed herein.

[0289] In some respects, the vector set comprises a first vector and a second vector, wherein the first vector contains a nucleic acid sequence encoding the CAR disclosed herein, and the second vector contains a nucleic acid sequence encoding the armor molecule disclosed herein.

[0290] Any vector known in the art is suitable for this disclosure. In some aspects, the vector is a viral vector. In other aspects, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0291] In other aspects, this document provides host cells comprising the polynucleotides or vectors of this disclosure. In some aspects, this disclosure relates to host cells, such as in vitro cells, that contain polynucleotides encoding CARs or TCRs, as described herein. In some aspects, this disclosure relates to host cells, such as in vitro cells, that contain polynucleotides encoding antibodies or antigen-binding molecule thereof that specifically bind to STEAP2, as disclosed herein. In other aspects, this disclosure relates to in vitro cells that contain polypeptides encoded by polynucleotides encoding CARs that specifically bind to STEAP2. In other aspects, this disclosure relates to cells, in vitro cells, that contain polypeptides encoded by polynucleotides encoding antibodies or antigen-binding molecule thereof that specifically bind to STEAP2, as disclosed herein.

[0292] Any cell can be used as the host cell for the polynucleotides, carriers, or polypeptides disclosed herein. In some respects, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., E. coli); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella (e.g., Salmonella typhimurium); Serratia (e.g., Serratia marcescans) and Shigella; Bacilli (e.g., B. subtilis and B. licheniformis); Pseudomonas (e.g., P. aeruginosa); and Streptomyces. In some respects, the cell is a human cell.

[0293] Other aspects of this disclosure relate to compositions comprising the polynucleotides, carriers, peptides, or cells described herein. In some aspects, the composition comprises a pharmaceutically acceptable load, diluent, solubilizer, emulsifier, preservative, and / or adjuvant. In some aspects, the composition comprises an excipient. In one aspect, the composition comprises a polynucleotide encoding a CAR, wherein the CAR comprises an antigen-binding molecule that specifically binds to STEAP2. In another aspect, the composition comprises a CAR encoded by the polynucleotide of this disclosure, wherein the CAR comprises an antigen-binding molecule that specifically binds to STEAP2. In another aspect, the composition comprises a T cell containing a polynucleotide encoding a CAR, wherein the CAR comprises an antigen-binding molecule that specifically binds to STEAP2. In another aspect, the composition comprises an antibody or antigen-binding molecule thereof that specifically binds to STEAP2, as described herein. In another aspect, the composition comprises a cell (e.g., a T cell, such as a CAR-T cell) containing a polynucleotide encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to STEAP2, as disclosed herein. In another aspect, the composition further comprises at least one androgen receptor antagonist.

[0294] This disclosure also provides pharmaceutical compositions comprising anti-STEAP2 CAR T cells and a pharmaceutically acceptable loading agent. In some aspects, the pharmaceutical compositions further comprise at least one androgen receptor antagonist. Pharmaceutical compositions comprising anti-STEAP2 CAR T cells are intended for, but not limited to, diagnosing, detecting, or monitoring a condition, preventing, treating, managing, or improving a condition or one or more of its symptoms, and / or for research purposes. In one aspect, this document provides a pharmaceutical composition comprising anti-STEAP2 CAR T cells as described herein and a pharmaceutically acceptable loading agent. In some aspects, this document provides a pharmaceutical composition comprising at least one androgen receptor antagonist as described herein and a pharmaceutically acceptable loading agent.

[0295] This disclosure also provides pharmaceutical compositions comprising anti-STEAP2 CAR T cells, at least one androgen receptor antagonist, and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising anti-STEAP2 CAR T cells and at least one androgen receptor antagonist are intended for, but not limited to, diagnosing, detecting, or monitoring a condition, preventing, treating, managing, or improving a condition or one or more of its symptoms, and / or for research purposes. In one aspect, this document provides a pharmaceutical composition comprising anti-STEAP2 CAR T cells as described herein, at least one androgen receptor antagonist, and a pharmaceutically acceptable carrier.

[0296] In other aspects, the composition is formulated for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art. In some aspects, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8. In some aspects, when contemplated for parenteral administration, the composition is in the form of a pyrogen-free, parenteral-acceptable aqueous solution containing the desired antigen-binding molecule of BCMA (with or without an additional therapeutic agent) in a pharmaceutically acceptable medium. In some aspects, the medium for parenteral injection is sterile distilled water in which the antigen-binding molecule of BCMA (with or without at least one additional therapeutic agent) is formulated as a suitably preserved sterile isotonic solution. In some aspects, preparation involves formulating the desired molecule with a polymeric compound (such as polylactic acid or polyglycolic acid), beads, or liposomes that provide controlled or sustained release of the product, followed by delivery via a reservoir injection. In some aspects, an implantable drug delivery device is used to introduce the desired molecule.

[0297] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, intranasal (e.g., inhalation), transdermal (e.g., topical), mucosal, and rectal administration. In one specific aspect, the composition is formulated according to standard procedures to be suitable for intravenous, subcutaneous, intramuscular, intranasal, or topical administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer solutions. Where necessary, the composition may also contain a solubilizer and a local anesthetic, such as lidocaine, to reduce pain at the injection site.

[0298] In all respects, the kits described herein for laboratory and therapeutic applications are within the scope of this disclosure. Such kits may include carriers, packaging, or containers divided to receive one or more containers (such as vials, tubes, etc.), each containing one of the individual elements to be used in the methods disclosed herein; and labels or inserts containing instructions for use (such as those described herein). Kits may include containers containing a pharmaceutical portion. This disclosure also provides one or more of anti-STEAP2 CAR T cells and at least one androgen receptor antagonist or pharmaceutical compositions thereof packaged in an airtight container (such as an ampoule or pouch) indicating the amount of the pharmaceutical agent.

[0299] The kit may include the containers described above, as well as one or more other containers associated therewith, including materials that are commercially and user-appropriate, including buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials and / or tube labels listing the contents and / or instructions for use; and packaging inserts with instructions for use.

[0300] Labels may be present on or with the container to indicate that the composition is intended for specific therapeutic or non-therapeutic applications, such as prognostic, preventative, diagnostic, or laboratory applications. Labels may also indicate instructions for in vivo or in vitro use, as described herein. Instructions and other information may also be included in inserts or labels that are included within or on the kit. Labels may be on or associated with the container. A label may be on the container when the letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with the container when it is present within a receiver or carrier that also contains the container, such as as a packaging insert. Labels may indicate that the composition is intended for the diagnosis or treatment of conditions, such as cancer as described herein.

[0301] Table 1. Sequences

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] Unless otherwise stated, the practice of this disclosure will employ conventional techniques within the scope of the art, including those of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are well explained in the literature. See, for example, Sambrook et al., eds., (1989) Molecular Cloning: A Laboratory Manual (2nd edition; Cold Spring Harbor Laboratory Press); Sambrook et al., eds., (1992) Molecular Cloning: A Laboratory Manual (Cold Springs Harbor Laboratory, NY); DN Glover, ed., (1985) DNA Cloning, Volumes I and II; Gait, ed., (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Patent No. 4,683,195; Hames and Higgins, eds., (1984) Nucleic Acid Hybridization; Hames and Higgins, ed., (1984) Transcription and Translation; Freshney (1987) Culture of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells and Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide to Molecular Cloning. Cloning; the treatise, Methods In Enzymology (AcademicPress, Inc., NYMiller and Calos (eds., 1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al. (eds., Methods in Enzymology, Vol. 154 & 155); Mayer and Walker (eds., 1987) Immunochemical Methods in Cell And Molecular Biology (Academic Press, London); Weir and Blackwell (eds., 1986) Handbook of Experimental Immunology, Vol. I–V; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooks, Antisense Drug Technology: Principles, Strategies and Applications, 2nd ed., CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0309] The following examples are provided in an illustrative rather than restrictive manner.

[0310] Example

[0311] Example 1: Effects of androgen receptor antagonists on STEAP2-positive prostate cancer

[0312] Compared with normal prostate, prostatic six-transmembrane epithelial antigen-2 (STEAP2) expression is increased in prostate cancer, suggesting that STEAP2 may drive prostate cancer progression. STEAP2 drives invasive prostate cancer traits by utilizing downstream targets to promote proliferation, migration, and invasion, including metalloproteinases MMP3, MMP10, MMP13, and MMP7, which are known to be associated with driving invasive and metastatic potential (Burnell S et al., Sci. Rep. 2018; 8:6252). STEAP2 overexpression increases the migration and invasion capabilities of prostate cancer cells and is associated with advanced cancer stage and histological grade (Whiteland H et al., Clin. Exp. Metastasis. 2014; 31(8):909-20).

[0313] Androgen receptor antagonists (antiandrogens) are drugs used to treat hormone-based syndromes and prostate cancer. Currently used drugs for prostate cancer include flutamide, bicalutamide, nilumet, enzalutamide, and ARN-509. Each of these inhibitors binds to the ligand-binding domain (LBD) of the androgen receptor. This is the same site where the natural physiological steroids testosterone (TES) and dihydrotestosterone (DHT) bind. The drugs work by competing with the natural hormones for binding to the LBD and thus reducing receptor activation.

[0314] To determine the effects of androgen receptor antagonists on prostate cancer cell lines in the STEAP2 background, patient-derived xenografts (PDX) from LuCaP prostate cancer patients were utilized (see Nguyen HM et al., Prostate. 2017; 77(6): 654-671, which is incorporated herein by reference). Specifically, the responses of LuCaP73, LuCaP70, and LuCaP86.2 PDX models to enzalutamide treatment were evaluated. As described in Nguyen, the LuCaP73 PDX model represents a more castration-sensitive model (+++) than the LuCaP70 PDX model (++), while the LuCaP86.2 PDX model showed minimal responsiveness to castration therapy.

[0315] like Figures 1A to 1C As shown, three LuCaP prostate cancer patient-derived xenograft (PDX) models were evaluated across 42 days after enzalutamide treatment. Enzalutamide was administered orally once daily. The drug carrier consisted of 5% DMSO, 1% carboxymethyl cellulose, and 0.1% Tween 80. In LuCaP73 ( Figure 1A ), LuCaP70 ( Figure 1B ) and LuCaP86.2 ( Figure 1CIn PDX models (n=8 per group), tumor volume was measured after treatment with either a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg) for up to 42 days. Enzalutamide treatment resulted in a dose-dependent reduction in tumor volume, with the most significant reduction observed in the most castration-sensitive LuCaP73 PDX model. Figure 2A () The indicator is p < 0.001, and Indicating p < 0.0001). Following enzalutamide treatment, the body weight of mice implanted with LuCaP73 cells also showed a slight improvement (…). Figure 2B ).

[0316] Figures 3A to 3B The androgen receptor (AR) profile was shown on day 42 after treatment with the mediator or 10 mg / kg enzalutamide (Enza). Figure 3A ) and STEAP2 ( Figure 3B LuCaP73 tumor expression was analyzed. AR and STEAP2 expression levels were normalized to GAPDH levels. Both AR and STEAP2 expression increased in LuCaP73 tumors after enzalutamide treatment.

[0317] like Figure 4A As shown, the castration-sensitive LuCaP70 PDX model also exhibited a significant reduction in tumor volume over time due to treatment with different doses of enzalutamide. Tumor volume was measured after treatment with a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg) for 21 days. This indicates that p < 0.001 and This indicates that p < 0.05. Figure 4B The body weight of the LuCaP-70 PDX model after treatment with the mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg) is shown, indicating that enzalutamide treatment improves body weight.

[0318] Figures 5A to 5C The androgen receptor (AR) profile is shown on day 21 after treatment with the mediator or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg or 80 mg / kg). Figure 5A STEAP2 Figure 5B ) and KLK3 ( Figure 5C LuCaP70 tumor expression. This indicates that p < 0.0001; p < 0.01; and ns indicates no significance. Significant dose-dependent increases in AR and STEAP2 levels were observed, while KLK3 expression levels showed no significant change.

[0319] Figures 5D to 5E Immunohistochemistry (IHC) (H score) based on the use of anti-STEAP2 antibody and calculated tumor membrane staining intensity and percentage of stained cells is shown. Figure 5D And by the percentage of membrane staining in the tumor () Figure 5E The STEAP2 expression level in LuCaP70 tumors obtained ( (p<0.05). The highest dose of enzalutamide (80 mg / kg) had a significant effect on the STEAP2 H score (p<0.05), indicating an increase in STEAP2 membrane staining. Enzalutamide treatment had a dose-dependent effect on the STEAP2 membrane tumor distribution score (e.g., tumors treated with the medium resulted in approximately 8.75% STEAP2 1+, approximately 72.5% STEAP2 2+, and approximately 18.75% STEAP2 3+, while enzalutamide 80 mg / kg treatment resulted in approximately 2% STEAP2 1+, approximately 52% STEAP2 2+, and approximately 46% STEAP2 3+ distribution). These results indicate the effect of enzalutamide treatment on STEAP2 membrane expression.

[0320] like Figure 6A As shown, the castration-resistant LuCaP86.2 PDX model showed a significantly lower response to different doses of enzalutamide over time compared to the responses observed in the LuCaP73 and LuCaP70 PDX models. Tumor volume was measured 25 days after treatment with either the mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg). This indicates that P < 0.01, and This indicates that P < 0.05. Figure 6B The body weights of LuCaP 86.2 PDX models after treatment with a mediator control or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg) are shown, with no significant change in body weight when treated with enzalutamide compared to treatment with a mediator.

[0321] Figures 6C to 6E The androgen receptor (AR) profile is shown on day 25 after treatment with the mediator or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg). Figure 6C STEAP2 Figure 6D ) and KLK3 ( Figure 6E Enzalutamide was used to detect the expression of AR, STEAP2, or KLK3 in LuCaP86.2 tumors. No significant differences were observed in AR, STEAP2, or KLK3 expression levels at most doses of enzalutamide, with only a slight increase in STEAP2 levels observed at 80 mg / kg dose. The impaired response to enzalutamide may be related to the expression of AR in LuCaP 86.2 tumors. V567 The variant is a constitutive active splicing variant of AR.

[0322] Serum prostate-specific antigen (PSA) levels were measured in a castration-sensitive LuCaP PDX model after enzalutamide treatment. LuCaP73 was treated with a mediator or enzalutamide (10 mg / kg, 30 mg / kg, 50 mg / kg, or 80 mg / kg dose). Figure 7A ), LuCaP70 ( Figure 7B ) and LuCaP86.2 ( Figure 7C The PDX model was used, and serum PSA levels were determined by ELISA at the end of the study (LuCaP73 = day 42, LuCaP70 = day 21, and LuCaP86.2 = day 25). This indicates that p < 0.0001 and This indicates p < 0.05. In mice implanted with castration-sensitive LuCaP73 and LuCaP70 tumors, serum PSA was significantly reduced at all doses of enzalutamide, while only 80 mg / kg of enzalutamide resulted in a significant reduction in serum PSA in mice implanted with castration-resistant LuCaP86.2 tumors.

[0323] STEAP2 receptor density and antigen-binding capacity (ABC) were measured in LNCAP cells after continuous enzalutamide exposure over a 28-day period. In full-length AR castration-sensitive LNCAP cell lines, continuous enzalutamide treatment increased STEAP2 receptor density levels by mean fluorescence intensity (MFI), lasting up to 21 days. After day 21, STEAP2 levels returned to baseline upon removal of enzalutamide.

[0324] These results indicate that androgen receptor antagonists such as enzalutamide are more likely to target castration-sensitive prostate cancer PDX models. Furthermore, enzalutamide treatment was found to significantly increase STEAP2 expression in castration-sensitive prostate cancer PDX models.

[0325] Example 2: In vivo efficacy of anti-STEAP2 CAR T and androgen receptor antagonist combination therapy in a mouse model effect

[0326] As described in Example 1, treatment of castration-sensitive prostate cancer cells with androgen receptor antagonists (e.g., enzalutamide) resulted in a significant increase in STEAP2 expression. AZD0754 is an anti-STEAP2 chimeric antigen receptor (CAR) T cell (clone 40A3) that expresses the dominant-negative form of TFGβRII (dnTFGβRII) to enhance tumor microenvironment activity. In NSG-MHC I / II double knockout mice (also known as NSG-(K... b D b ) null (IA) null NSG-(K b D b ) null (IA null The therapeutic efficacy of AZD0754 treatment was evaluated using the LuCaP73 PDX model in The Jackson Laboratory (strain number: 025216).

[0327] Measurements over time were performed using untransduced T cells (UT; 5 × 10⁶). 6 CAR T cells (5 × 10⁵ cells / mouse), 40A3 dnTFGβRII (i.e., AZD0754) CAR T cells (CAR T; ... 5 1 cell / mouse) or 40A3 dnTFGβRII CAR T cells (CAR; 5×10 6 Tumor volume in mice treated with (cells / mouse) Figure 8A ) and weight ( Figure 8B UT / CAR T treatment was administered at the designated time point (day 34). For each treatment group, N=12. This indicates that p < 0.0001 and This indicates p < 0.05. (In 5 × 10⁻⁶) 5 1 cell / mouse or 5×10 6 Treatment with AZD0754 per cell / mouse resulted in a significant reduction in tumor volume, compared to only 5×10⁻⁶ cells / mouse. 6 Significant weight gain was observed at a dose of AZD0754 per cell / mouse.

[0328] Figures 9A to 9B The response to AZD0754 CAR T cell and enzalutamide combination therapy is shown in the LuCaP73 PDX model. Treatment-naïve mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁻⁶) were measured over time. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 Tumor volume in mice treated with enzalutamide (10 mg / kg) and cells / mouse Figure 9A ) and weight ( Figure 9B For each treatment group, N=9. This indicates p < 0.05. A significant reduction in tumor volume was observed in the AZD0754 + enzalutamide combination therapy group compared to the enzalutamide-only group.

[0329] Figures 10A to 10B The figures show that on day 42, untreated mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁶) were compared. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 The percentage of AZD0754 CAR-T cells in the blood of mice treated with enzalutamide (10 mg / kg) and mice (cells / mouse). Figure 10A ) and cell number ( Figure 10BThese results indicate that at the end of the study, the percentage and number of AZD0754 CAR T cells were comparable, suggesting that enzalutamide had no negative impact on CAR T cell viability.

[0330] Figures 11A to 11B The figures show that on day 42, untreated mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁶) were compared. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5 The percentage of hCD45+ complemented TGFβRII+ CAR-T cells in the blood of mice treated with enzalutamide (10 mg / kg) and mice (cells / mouse) Figure 11A ) and cell number ( Figure 11B These results indicate that hCD45 was present at the end of the study. + complementary position + TGFβRII + The percentage and number of CAR T cells were comparable, indicating that enzalutamide had no negative impact on CAR T cell viability and phenotype.

[0331] Figure 12 The figures show that on day 42, untreated mice, mice treated with the vector, and mice treated with untransduced T cells (UT; 5 × 10⁶) were compared. 5 Mice treated with 5 × 10⁵ cells / mouse and mice treated with untransduced T cells (UT; 5 × 10⁵ cells / mouse) 5 Mice treated with a control group (cells / mouse) and a vector, mice treated with enzalutamide (10 mg / kg), and mice treated with untransduced T cells (UT; 5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and enzalutamide (10 mg / kg), and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) were also included. 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) 5 Mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, as well as mice treated with AZD0754 (5 × 10⁻⁶ cells / mouse) and the vector control, were also included. 5Serum PSA levels in mice treated with AZD0754 (cells / mouse) and enzalutamide (10 mg / kg) were measured. Results showed that serum PSA levels decreased after combination treatment with AZD0754 and enzalutamide, reaching levels similar to or lower than those achieved with enzalutamide alone.

[0332] To evaluate the effects of the androgen receptor antagonists abiraterone and enzalutamide on the proliferation of prostate cell lines and AZD0754 CAR T cells. Figures 13A to 13B It shows abiraterone ( Figure 13A ) and enzalutamide ( Figure 13B Treatments affected the proliferation of LNCAP or 22RV1 prostate cell lines in a dose-dependent manner. Figures 14A to 14B The results showed that abiraterone ( ) 24 hours, 48 ​​hours, or 72 hours after treatment Figure 14A ) and enzalutamide ( Figure 14B The effects of treatment on the proliferation of 40A3 dnTFGβRII CAR T cells were investigated. LNCAP cells were found to be more sensitive to androgen receptor antagonist treatment compared to 22RV1 cells. Approximately 5 μM and higher doses of abiraterone and enzalutamide were found to inhibit AZD0754 proliferation.

[0333] Figures 15A to 15B The levels of abiraterone (Abiraterone) as measured by flow cytometry at 24, 48, and 72 hours post-treatment are shown. Figure 15A ) and enzalutamide ( Figure 15B The effect of treatment on STEAP2 expression in LNCAP cells. No effect on STEAP2 expression was observed after treatment with abiraterone or enzalutamide.

[0334] The effects of abiraterone and enzalutamide treatments on AZD0754 activity during co-culture with LNCAP cells were evaluated. LNCAP cells were pretreated in complete culture medium with 0 or 5 μM abiraterone, enzalutamide, or DMSO for 24 h. Then, AZD0754 or untransduced T cells were added at a concentration of 5 μM with or without abiraterone or enzalutamide. IFN was also measured by harvesting the cell culture supernatant 24 h after CAR T cell treatment. Release. Furthermore, LNCAP cells treated with 0 or 5 μM abiraterone, enzalutamide, or DMSO in complete culture medium for 72 hours were used to analyze gene expression of downstream androgen receptor targets by qPCR. Cells were subsequently harvested and RNA was extracted for qPCR analysis.

[0335] Figures 16A to 16C This study demonstrates the effect of abiraterone treatment on the cytotoxic ability of 40A3 dnTFGβRII CART cells co-cultured with LNCAP cells. Figure 16A) or IFN of the CAR T cells The effects of release ( Figure 16B Treatment with 5 μM abiraterone was compared with DMSO, and CAR T cells were compared with untransduced T cells (UT), where LNCAP and / or CAR T or UT cells were pretreated with abiraterone (PreTx). Androgen receptor inhibition in LNCAP cells treated with culture medium, DMSO, or 5 μM abiraterone was validated by qPCR of downstream target expression. Figure 16C When co-cultured with LNCAP cells, abiraterone appears to have reduced CAR T-killing ability or IFN-γ cytotoxicity. The release has no effect.

[0336] Figures 17A to 17C This study demonstrates the effect of enzalutamide treatment on the cytotoxic ability of 40A3 dnTFGβRII CART cells co-cultured with LNCAP cells. Figure 17A ) or IFN of the CAR T cells The effects of release ( Figure 17B Treatment with 5 μM enzalutamide was compared with DMSO, and CAR T cells were compared with untransduced T cells (UT), where LNCAP and / or CAR T or UT cells were pretreated with enzalutamide (PreTx). Androgen receptor inhibition in LNCAP cells treated with culture medium, DMSO, or 5 μM enzalutamide was validated by qPCR of downstream target expression. Figure 17C Enzalutamide appeared to inhibit CAR T-cell killing ability or IFN-γ when co-cultured with LNCAP cells. The release has no effect.

[0337] The foregoing description of specific aspects so fully reveals the general nature of this disclosure that others, by applying knowledge of the art, can readily modify and / or adapt such specific aspects for various applications without excessive experimentation, without departing from the general conception of this disclosure. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology used herein is for descriptive rather than limiting purposes, and that the terminology or terminology of this specification should be interpreted by those skilled in the art based on the teachings and guidance.

[0338] Other aspects of this disclosure will be apparent to those skilled in the art from the description and practice disclosed herein. This specification and embodiments are intended to be considered merely exemplary, and the true scope and spirit of this disclosure are indicated by the following claims.

[0339] All publications, patents and patent applications disclosed herein are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited.

Claims

1. A method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of the following that effectively inhibits tumor cell growth: a) T cells, said T cells comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and b) At least one androgen receptor antagonist; The CAR comprises an antigen-binding domain containing VH and VL, wherein the VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

2. A method for treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the following: a) T cells, said T cells comprising (i) a polynucleotide encoding a chimeric antigen receptor (CAR) that binds to an epitope on human prostatic six-span membrane epithelial antigen-2 (STEAP2); and b) At least one androgen receptor antagonist; The CAR comprises an antigen-binding domain containing VH and VL, wherein the VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; wherein VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

3. The method according to claim 1 or 2, wherein the VH comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

8.

4. The method according to any one of claims 1 to 3, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO:

8.

5. The method according to any one of claims 1 to 4, wherein the polynucleotide further encodes an armor molecule and the armor molecule comprises a dominant-negative TGF-β receptor type 2 (TGFβRIIDN).

6. The method according to any one of claims 1 to 5, wherein the armor molecule comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

10.

7. The method according to any one of claims 1 to 6, wherein the armor molecule comprises the amino acid sequence shown in SEQ ID NO:

10.

8. The method according to any one of claims 1 to 7, wherein the polynucleotide encoding the CAR comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO:

11.

9. The method according to any one of claims 1 to 8, wherein the polynucleotide encoding the CAR comprises the nucleotide sequence shown in SEQ ID NO:

11.

10. The method according to any one of claims 1 to 9, wherein the polynucleotide encoding the CAR comprises the nucleotide sequence shown in SEQ ID NO: 11, and the polynucleotide encoding the armor molecule comprises the nucleotide sequence shown in SEQ ID NO:

9.

11. The method according to any one of claims 1 to 10, wherein the polynucleotide encoding the CAR and the polynucleotide encoding the armor molecule are operably linked under the control of a single promoter.

12. The method according to any one of claims 1 to 11, wherein the polynucleotide encoding the CAR and the polynucleotide encoding the armor molecule are operatively linked via IRES.

13. The method according to any one of claims 1 to 12, wherein the polynucleotide encoding the CAR and the polynucleotide encoding the armor molecule are linked by a nucleotide sequence encoding a cleavable peptide linker.

14. The method of claim 13, wherein the cleavable peptide adapter is a self-cleaving peptide adapter.

15. The method of claim 13 or 14, wherein the cleavable peptide linker comprises a T2A peptide.

16. The method according to any one of claims 13 to 15, wherein the cleavable peptide adapter comprises SEQ ID NO:

13.

17. The method according to any one of claims 1 to 16, wherein the polynucleotide comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the nucleotide sequence shown in SEQ ID NO:

14.

18. The method according to any one of claims 1 to 17, wherein the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:

14.

19. The method according to any one of claims 1 to 18, wherein the T cell comprises an amino acid sequence including the amino acid sequence shown in SEQ ID NO:

12.

20. The method according to any one of claims 1 to 19, wherein the T cell comprises an amino acid sequence including the amino acid sequence shown in SEQ ID NO:

15.

21. The method according to any one of claims 1 to 20, wherein the androgen receptor antagonist is enzalutamide, apalutamide, dalostamide, abiraterone, bicalutamide, nilutetamide, flutamide, proxalutamide, or a combination thereof.

22. The method according to any one of claims 1 to 21, wherein the androgen receptor antagonist is enzalutamide.

23. The method according to any one of claims 1 to 21, wherein the androgen receptor antagonist is abiraterone.

24. The method according to any one of claims 1 to 23, wherein the androgen receptor antagonist is administered orally.

25. The method according to any one of claims 1 to 24, wherein the T cells are administered intravenously.

26. The method according to any one of claims 1 to 25, wherein the T cells and the androgen receptor antagonist are administered sequentially or simultaneously and in any order.

27. The method according to any one of claims 1 to 26, wherein the administration of the androgen receptor antagonist does not inhibit T cell activity or T cell killing ability.

28. The method according to any one of claims 1 to 27, wherein the administration of the androgen receptor antagonist does not inhibit the release of interferon-γ (IFNγ) in tumor cells.

29. The method according to any one of claims 1 to 28, wherein the administration of the androgen receptor antagonist does not reduce CAR expression on the T cells.

30. The method according to any one of claims 1 to 29, wherein the T cell is a CD8+ T cell.

31. The method according to any one of claims 1 to 30, wherein the tumor cells are prostate tumor cells, optionally wherein the prostate tumor cells are metastatic, recurrent, or recurrent.

32. The method according to any one of claims 1 to 31, wherein the administration increases the expression of STEAP2 in the tumor cells compared to the expression of STEAP2 in the tumor cells when the T cells are administered alone.

33. The method of claim 32, wherein the administration increases the expression of STEAP2 in the tumor cells by about 50% to about 200% compared to the expression of STEAP2 in the tumor cells when the T cells are administered alone.

34. The method of claim 33, wherein the STEAP2 expression is increased by about 100%.

35. Use of T cells and androgen receptor antagonists in a method for inhibiting the growth of tumor cells, the method comprising contacting the tumor cells with an amount of T cells and at least one androgen receptor antagonist that effectively inhibits the growth of tumor cells, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule, wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein the VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein the VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, the VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, and the VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO:

4. The amino acid sequence shown in SEQ ID NO: 5, and the VH-CDR3 contains the amino acid sequence shown in SEQ ID NO:

6.

36. Use of T cells and androgen receptor antagonists in a method of treating a subject with cancer containing tumor cells, the method comprising administering to the subject in need a therapeutically effective amount of the T cells and at least one androgen receptor antagonist, wherein the T cells comprise (i) a polynucleotide encoding a CAR that binds to an epitope on human STEAP2 and (ii) a polynucleotide encoding an armor molecule, wherein the CAR comprises an antigen-binding domain containing VH and VL, wherein the VH comprises VH-CDR1, VH-CDR2, and VH-CDR3, and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3; and wherein the VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, the VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, the VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, and the VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO:

4. The amino acid sequence shown in SEQ ID NO: 5, and the VH-CDR3 contains the amino acid sequence shown in SEQ ID NO:

6.

37. The use according to claim 35 or 36, wherein the tumor cells are prostate tumor cells.

38. The use according to claim 37, wherein the prostate tumor cells are metastatic, recurrent, or recurrent.

Images