Chimeric antigen receptor binding to STEAP1

Abstract

Disclosed herein is a chimeric antigen receptor (CAR) having a binding domain that binds to STEAP1. The CAR disclosed herein can be used to treat prostate cancer, Ewing's sarcoma family of tumors (EFT), bladder cancer, ovarian cancer and rhabdomyosarcoma. The CAR disclosed herein can bind to STEAP1 and induce cytotoxicity even when the density of STEAP1 antigen is low.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 309,389, filed February 11, 2022, the contents of which are incorporated by reference in their entirety as if set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. W81XWH-21-1-0581 awarded by the U.S. Army Medical Research and Development Command. The U.S. Government has certain rights in this invention.

[0003] Sequence Listing Reference The sequence listing accompanying this application is provided in XML format rather than hard copy, and is incorporated herein by reference. The name of the XML file containing this sequence listing is F053-0149PCT Sequence Listing.xml. The size of this XML file is 196KB, was created on February 10, 2023, and was submitted electronically via the Patent Center.

[0004] The present disclosure provides a chimeric antigen receptor (CAR) having a binding domain that binds to STEAP1. The CAR disclosed herein can bind to STEAP1 and induce cytotoxicity even when the density of STEAP1 antigen is low. The CAR disclosed herein can be used to treat cancers that express STEAP1, such as prostate cancer, Ewing's sarcoma family tumors (EFT), bladder cancer, ovarian cancer, and rhabdomyosarcoma. [Background technology]

[0005] According to the World Health Organization, cancer is the second leading cause of death worldwide, with an estimated 9.6 million cancer deaths in 2018.

[0006] STEAP1 is a protein whose expression is upregulated in many cancers, including prostate cancer, Ewing Sarcoma Family Tumors (EFT), bladder cancer, ovarian cancer, and rhabdomyosarcoma. Prostate cancer is the most commonly diagnosed cancer in men, after skin cancer. Prostate cancer is the second leading cause of cancer death in men. Ewing Sarcoma Family Tumors (EFT) are a family of small round blue cell tumors that arise from bone or soft tissue. This family is the second most common malignant bone tumor in children and adolescents, with an incidence rate of 200 cases per year in the United States.

[0007] For many years, the treatment of choice for cancer has been surgery, chemotherapy and / or radiation therapy. Recently, more targeted therapies have been developed to specifically target cancer cells by identifying and exploiting specific molecular and / or immunophenotypic changes primarily found in cancer cells. For example, many cancer cells selectively express certain markers on their cell surface, and these markers are used as targets for antibody-based therapies.

[0008] Great strides have been made in engineering immune system cells to target and kill unwanted cells, such as cancer cells. Many of these immune cells are T cells engineered to express chimeric antigen receptors (CARs). CARs are proteins that contain several distinct components that enable engineered T cells to recognize and kill cancer cells. These components include at least an extracellular portion and an intracellular portion. The extracellular portion contains a binding domain that specifically binds to a marker (e.g., an antigen) that is selectively present on the surface of the unwanted cells (e.g., STEAP1). Binding of the binding domain to such a marker transmits a signal from the intracellular portion to the T cell to destroy the bound cell. CARs can further contain a transmembrane domain that can link the extracellular portion to the intracellular portion.

[0009] Other components that can enhance the function of CARs can also be used. For example, spacers can often enhance the binding ability of the binding domain to the marker of the target cell by providing additional flexibility to the CAR's conformation, thereby enhancing the cytolytic effect. The appropriate length of the spacer for a particular CAR depends on many factors, including how close or far the target marker is located from the surface of the cell membrane of the unwanted cells. Thus, although the general structure of CARs is known, the ability of a particular CAR to induce cytolytic effect in vivo remains an area of ​​intense research and investigation. In addition, it remains difficult to induce cytolytic effect of CARs even at low antigen density.

[0010] Specifically regarding prostate cancer (PCa), the most common type of prostate cancer is usually classified as adenocarcinoma, which is a type of cancer that arises in the mucus-secreting glands of the organ. Prostate cancer arises from the prostate gland (the gland that produces seminal fluid) in the male reproductive system. Localized prostate cancer (PCa) is treatable, with a 5-year survival rate from diagnosis of nearly 100%. Some of the treatment options for prostate cancer include surgery, radiation, cryotherapy, or hormone therapy.

[0011] Because prostate cancer growth is often driven by male hormones called androgens, including testosterone, hormone therapy can be an effective treatment for prostate cancer. Such hormone therapy reduces the levels of androgens in the patient's body. Androgen levels can be reduced by surgical removal of the testicles, administration of drugs that block the production of androgens, and / or blocking the effects of androgens on the body. Unfortunately, the majority of hormone-dependent cancers become refractory to these types of treatments after one to three years and begin to regrow despite continued hormone therapy. When hormone therapy loses its therapeutic value against prostate cancer, such prostate cancer is called "castration-resistant" prostate cancer or CRPC. Unfortunately, when prostate cancer becomes CRPC, the five-year survival rate drops to 30%. Thus, there is a strong need for treatments that can treat CRPC, among other cancers with poor survival rates. Summary of the Invention [Means for solving the problem]

[0012] The present disclosure provides chimeric antigen receptors (CARs) that bind to STEAP1 for the treatment of STEAP1-expressing cancers. The CARs disclosed herein have the important advantage of being able to bind to STEAP1 and induce cytotoxicity even when the density of STEAP1 antigen is low, and being highly specific for STEAP1. These characteristics of the CARs disclosed herein allow for clinically significant advances to be achieved by using CARs early in cancer progression, before the antigen density increases due to cancer proliferation and growth, while minimizing off-target toxicity. Intervention at an early stage of cancer development often results in significantly better therapeutic outcomes. Potential toxicity is also reduced, making this a clinically significant step.

[0013] In certain embodiments, a CAR of the disclosure, when expressed by a cell, comprises: (i) an extracellular portion comprising an scFv binding domain derived from bundled tuzumab vedotin (DSTP3086S; a humanized variant of the murine monoclonal antibody mAb 120.545) and a long spacer comprising an IgG4 hinge-CH2-CH3 with 4 / 2-NQ mutations in the CH2 domain; (ii) an intracellular portion comprising a CD3z activation domain and a 4-1BB costimulatory domain; and (iii) a CD28 transmembrane domain linking the extracellular portion to the intracellular portion.

[0014] The CARs disclosed herein can be used to treat cancers that express STEAP1, such as prostate cancer, Ewing's sarcoma family of tumors (EFT), bladder cancer, ovarian cancer, rhabdomyosarcoma, etc. In certain embodiments, the CARs disclosed herein can be used to treat lethal metastatic castration-resistant prostate cancer. [Brief description of the drawings]

[0015] Some of the drawings submitted in this application may be more easily understood in color, and applicants hereby contemplate color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0016] [Fig. 1A-1E]Comparative analysis of six-transmembrane epithelial antigen of prostate 1 (STEAP1) and prostate-specific membrane antigen (PSMA) in lethal metastatic castration-resistant prostate cancer (mCRPC). (1A) Characterization of mCRPC tissue spotted onto tissue microarray 92 (UW TAN TMA92) generated at the University of Washington Tissue Acquisition Necropsy. (1B) Paired plots showing mean H-scores of STEAP1 and PSMA immunohistochemistry (IHC) staining for each mCRPC tissue core. The mCRPC cores are, from left to right in the graph, 10-013_BB, 10-013_Q, 10-013_R, 10-056_J, 10-056_K, 10-068_II, 10-068_PP, 11-028_DD, 11-028_H, 11-028_L, 12-005_H, 12-005_K, 12-005_QQ, 12-011_I, 12-011_J, 12-011_LL, 12 -021_H, 12-021_I, 13-012_I, 13-012_L, 13-012_OO, 13-042_JJ, 13-042_M, 13-042_O, 13-084_H, 13-084_I I, 13-099_H, 13-099_J, 13-101_K, 13-101_SS, 13-101_UU, 13-104_I, 13-104_K, 13-104_OO, 13-117_BB, 13 -117_H, 13-117_OO, 13-122_H, 13-122_II, 13-122_OO, 14-031_GG, 14-031_H, 14-031_K, 14-039_E, 14-039 _K, 14-043_CC, 14-043_H, 14-043_OO, 14-053_AA, 14-053_H, 14-053_O, 14-077_K, 14-077_N, 14-091_TT, 1 4-091_E, 14-096_FF, 14-096_II, 14-105_I, 14-105_J, 14-105_VV, 15-003_GG, 15-003_H, 15-010_H, 15-01 0_K, 15-019_CC, 15-019_HH, 15-023_I, 15-023_L, 15-023_LL, 15-069_I, 15-069_KK, 15-069_P, 15-090_H,15-090_I, 15-090_LL, 15-096_FF, 15-096_H, 15-096_L, 16-052_BB, 16-052_JJ, 16-071_H, 16-071_I, 16-071_KK, 16- 080_FF, 16-080_L, 16-080_LL, 16-101_EE, 16-101_J, 16-101_N, 17-007_AA, 17-007_EE, 17-007_H, 17-017_H, 17-017 (1C) Contingency table showing the frequency of mCRPC tissue cores with STEAP1 IHC staining or PSMA IHC staining above or below the H-score threshold of 30. (1D, 1E) Photomicrographs of selected mCRPC tissue cores after IHC staining for STEAP1 or PSMA are shown, highlighting (1D) areas of STEAP1 expression but not PSMA expression, and (1E) areas of intratumor heterogeneity with PSMA expression but not STEAP1 expression. Scale bar = 50 μm.

[0017] [Figures 2A-2D]Characterization of STEAP1 expression in lethal mCRPC tissues. (2A) Photomicrographs of selected mCRPC tissue cores after IHC staining for STEAP1 are shown, highlighting cell membrane staining consistent with staining intensity scores of 0, 1, 2 or 3. (2B) Plots of STEAP1 H-scores by metastatic site for mCRPC tissue cores. Dashed line indicates STEAP1 H-score of 30. ** indicates p<0.01. (2C) Plots of STEAP1 H-score vs. androgen receptor (AR) H-score or (2D) Plots of STEAP1 H-score vs. synaptophysin (SYP) H-score for each mCRPC tissue core. Pearson correlation coefficients (r) and p-values ​​are shown.

[0018] [Figures 3A-3I]Screening of second generation 4-1BB chimeric antigen receptors (CARs) to identify lead cells for STEAP1 CAR T cell therapy. (3A) Schematic of STEAP1 CAR lentiviral construct and variations with short, medium or long spacers. LTR = long terminal repeat; MNDU3 = Moloney murine leukemia virus U3 region; scFv = single chain variable region fragment; VL = light chain variable region; VH = heavy chain variable region; tm = transmembrane domain; EGFRt = truncated epidermal growth factor receptor; 4 / 2 NQ = CH2 domain mutation that prevents binding to Fcγ receptor. (3B) Immunoblot analysis showing expression of STEAP1 in 22Rv1 parental cells, 22Rv1 STEAP1 knockout (ko) cells, and 22Rv1 STEAP1 ko cells where expression from lentivirus rescued STEAP1 expression. GAPDH was used as a protein loading control. (3C) IFN-γ enzyme-linked immunosorbent assay (ELISA) results for non-transduced or STEAP1-BBζ CAR T cells co-cultured with each 22Rv1 cell derivative at a 1:1 ratio for 24 h. n=4 replicates per condition. Error bars indicate standard deviation (SD). (3D) Relative cell viability of 22Rv1 target cells over time, as measured by fluorescent live cell imaging, after co-culture of 22Rv1 target cells with (left) STEAP1-BBζ CAR T cells or (right) non-transduced T cells at various target:effector (E:T) cell ratios. (3E) Time course of relative cell viability of 22Rv1 STEAP1 ko target cells measured by fluorescent live cell imaging after co-culture with (left) STEAP1-BBζ CAR T cells or (right) non-transduced T cells at various E:T cell ratios. In Figures 3D and 3E, n=4 replicates were performed for each condition, and error bars indicate standard error of the mean (SEM). (3F) Immunoblot analysis showing that STEAP1 is expressed in androgen receptor (AR)-positive but not AR-negative human prostate cancer cell lines.GAPDH was used as a protein loading control. (3G) Quantification of IFN-γ by ELISA after 24 hours of co-culture of non-transduced T cells or STEAP1-BBζ CAR T cells with human prostate cancer cell lines shown in Figure 3F at a 1:1 ratio. n=4 replicates per condition. Error bars indicate SD. (3H) Long exposure immunoblot analysis to assess STEAP1 expression in 22Rv1, PC3 and PC3-derived PC3 STEAP1 ko lines. GAPDH was used as a protein loading control. (3I) Quantification of IFN-γ by ELISA after 24 hours of co-culture of non-transduced T cells or STEAP1-BBζ CAR T cells with human prostate cancer cell lines shown in Figure 3F at a 1:1 ratio. n=4 replicates per condition. Error bars indicate SD.

[0019] [Figure 4A-4D] Validation of antigen-specific activation and target cell lysis of STEAP1-BBζ CAR T cells. (4A) Representative flow cytometry plots showing immunophenotyping of CD4 and CD8 T cell products at day 10 of expansion in non-transduced control or STEAP1 CAR lentivirus-transduced conditions. (4B) Quantification of IFN-γ by ELISA in control culture conditions or (4C) non-transduced or STEAP1-BBζ CAR T cells co-cultured with DU145 or DU145 STEAP1 cell lines at a 1:1 ratio for 24 hours. n=4 replicates per condition. Error bars indicate SD. (4D) Relative cell viability of DU145 STEAP1 target cells over time measured by fluorescent live cell imaging after co-culture of DU145 STEAP1 target cells with STEAP1-BBζ CAR T cells or non-transduced T cells at a 1:1 ratio. Each condition had n=4 replicates, and error bars indicate SEM.

[0020] [Figure 5A-5G]Measurement of STEAP1 extracellular domain specificity of STEAP1-BBζ CAR T cells using mouse / human Steap1 chimeras. (5A) Immunoblot analysis confirming absence of human STEAP1 (hSTEAP1) and mouse Steap1 (mSteap1) expression in DU145 cell line and expression of hSTEAP1 and mSteap1 in lentivirally recombinant DU145 hSTEAP1 and DU145 mSteap1 lines. GAPDH was used as a protein loading control. (5B) Quantification of IFN-γ by ELISA is shown when STEAP1-BBζ CAR T cells were co-cultured with (5B) DU145, DU145 hSTEAP1 or DU145 mSteap1 cell lines at a 1:1 ratio for 24 hours or (5C) STEAP1-BBζ CAR T cells were co-cultured in control culture conditions for 24 hours. n=4 replicates were performed for each condition. Error bars indicate SD. (5D) Schematic diagram of mSteap1 protein with the highlighted extracellular domains (mECD, boxes without triangles) and mouse / human Steap1 chimeric proteins in which each mECD is replaced with the corresponding hSTEAP1 extracellular domain (hECD, boxes with triangles). (5E) Quantification of IFN-γ by ELISA after 24 h co-culture of STEAP1-BBζ CAR T cells with each DU145 line engineered to express hSTEAP1, mSteap1 or mouse / human Steap1 chimeric protein at a 1:1 ratio. n=4 replicates per condition. Error bars indicate SD. (5F) Time course of relative cell viability of each target cell shown in Fig. 5E measured by fluorescent live cell imaging after co-culture of each target cell at a 1:1 ratio with STEAP1-BBζ CAR T cells. n=4 replicates per condition. Error bars indicate SEM. (5G) shows that alignment of the amino acid sequences of hSTEAP1 ECD2 (SEQ ID NO: 178) and mSteap1 ECD2 (SEQ ID NO: 179) revealed the presence of non-conserved residues at positions 198 and 209.

[0021] [Figures 6A-6D] Evaluation of STEAP1-BBζ CAR T cell reactivity against STEAP1B isoforms. (6A) Amino acid sequence alignment of human STEAP1B isoform 1 (SEQ ID NO: 178), human STEAP1B isoform 2 (SEQ ID NO: 178), or human STEAP1B isoform 3 (SEQ ID NO: 178) with hSTEAP1 ECD2 (SEQ ID NO: 178) shows complete sequence conservation. (6B) Plot showing consensus membrane protein topology predictions by TOPCONS for hSTEAP1, mSteap1, and the three human STEAP1B isoforms, with their confidence scores. (6C) Plot of membrane protein topology predictions for human STEAP1B isoform 1 showing discordance between different algorithms. (6D) Quantification of IFN-γ by ELISA in STEAP1-BBζ CAR T cells co-cultured with DU145 lines engineered to express hSTEAP1, mSteap1 or human STEAP1B isoforms at a 1:1 ratio for 24 hours. n=4 replicates per condition. Error bars indicate standard deviation. The data show no reactivity of the disclosed CARs against the STEAP1B isoform, highlighting the specificity of the disclosed CARs and demonstrating extremely reduced or no potential off-target toxicity.

[0022] [Figures 7A-7H]7A shows the in vivo antitumor activity of STEAP1-BBζ CAR T cell therapy in a prostate cancer model naturally expressing STEAP1. (7A) Time course of 22Rv1 subcutaneous tumor volume in NSG mice treated with a single intratumoral injection of 5x106 untransduced T cells or STEAP1-BBζ CAR T cells with normal CD4 / CD8 ratio. Error bars indicate SD. * indicates p<0.05; **** indicates p<0.0001. (7B) Schematic diagram of tumor challenge experiments in 22Rv1 (top) and C4-2B (bottom) disseminated models. fLuc = firefly luciferase; BLI = bioluminescence imaging. (7C) Continuous bioluminescence live imaging of NSG mice implanted with 22Rv1-fLuc metastatic tumors and treated with a single intravenous injection of 5×106 untransduced T cells or STEAP1-BBζ CAR T cells at a normal CD4 / CD8 ratio on day 0. X indicates a dead mouse. Brightness scale is shown. (7D) Plot showing the time course of total flux quantified in bioluminescence live imaging of each mouse shown in FIG. 7C. (7E) Kaplan-Meier survival curves of the mice shown in FIG. 7C with statistical significance determined by log-rank test. (7F) Continuous bioluminescence live imaging of NSG mice implanted with C4-2B metastatic tumors and treated with a single intravenous injection of 5×106 untransduced T cells or STEAP1-BBζ CAR T cells at a normal CD4 / CD8 ratio (i.e., 1:1) on day 0. X indicates a dead mouse. Brightness scale is shown. (7G) Time course plot of total flux quantified from live bioluminescence imaging for each mouse shown in Figure 7F. (7H) CD3+EGFRt+ STEAP1-BBζ CAR T cells quantified by flow cytometric analysis of splenocytes from STEAP1-BBζ CAR T cell-treated mice on day 49, the final day of the experiment. All experiments shown in the figures were performed at least twice.

[0023] [Figure 8A-8D]Antitumor therapeutic activity of STEAP1-BBζ CAR T cell therapy in a xenograft model of a human prostate cancer cell line. (8A) Photomicrograph showing CD3 IHC staining of 22Rv1 subcutaneous tumors 25 days after intratumoral treatment with STEAP1-BBζ CAR T cells. Scale bar = 100 μm. (8B) Plot showing mean body weight over time of NSG mice implanted with 22Rv1-fLuc metastatic tumors and treated with non-transduced T cells or STEAP1-BBζ CAR T cells. Error bars indicate SD. (8C) Plot showing mean body weight over time of NSG mice implanted with C4-2B-fLuc metastatic tumors and treated with non-transduced T cells or STEAP1-BBζ CAR T cells. Error bars indicate SD. (8D) Ex vivo bioluminescence imaging of liver and lung tissues from NSG mice implanted with C4-2B-fLuc metastatic tumors and treated with (left) non-transduced T cells or (right) STEAP1-BBζ CAR T cells. Brightness scale is indicated.

[0024] [Figure 9A-9H]Construction of a mouse-in-mouse system using a newly created human STEAP1 knock-in mouse model (hSTEAP1-KI) and a murine STEAP1 CAR is shown. (9A) Schematic diagram showing the homologous recombination method using a targeting vector to knock-in exons 2 to 5 of human STEAP1 into the mouse Steap1 locus on a C57Bl / 6 background. FRT = flippase recognition target. (9B) Visualization of PCR products for genotyping using tail tips from wild-type (+ / +), heterozygous (KI / +), or homozygous (KI / KI) mice using primer pairs amplifying the wild-type allele or a portion of the hSTEAP1-KI allele. NTC = null template control. (9C) qPCR results of human STEAP1 expression normalized to 18S expression in a survey of tissues from hSTEAP1-KI / + mice. Organs specific to each sex: n = 3, and common organs: n = 6. Error bars indicate SD. (9D) Photomicrographs of STEAP1 IHC staining of prostate tissue from (left) + / + or (right) KI / + mice and (9E) adrenal gland from KI / + mice. Scale bar = 50 μm. (9F) Schematic of retroviral construct of murine STEAP1 CAR. MuLV = murine leukemia virus; mCD19t = murine truncated CD19. (9G) Quantification of retroviral transduction efficiency of activated murine T cells in three independent experiments based on the frequency of murine CD3+CD19t+ cells measured by flow cytometry. (9H) Time course of relative cell viability measured by fluorescent live cell imaging after co-culture of RM9 or RM9-hSTEAP1 target cells with murine STEAP1-mBBζ CAR T cells or non-transduced T cells at a 1:1 ratio. n = 4 replicates per condition. Error bars indicate SEM. *** indicates p < 0.001.

[0025] [Fig. 10A-10H]10A shows the efficacy and safety of murine STEAP1-mBBζ CAR T cells in hSTEAP1-KI mice with syngeneic disseminated prostate cancer. (10A) Schematic of tumor challenge experiment of RM9-hSTEAP1 disseminated model in hSTEAP1-KI / + mice. Cy = cyclophosphamide (for preconditioning). (10B) Serial bioluminescence live imaging of hSTEAP1-KI / + mice implanted with RM9-hSTEAP1-fLuc metastatic tumors and treated with a single intravenous injection of 5x106 murine non-transduced T cells or STEAP1-mBBζ CAR T cells on day 0. X indicates dead mouse. Luminance scale is shown. (10C) Plot of total flux quantified over time from bioluminescence live imaging of each mouse shown in FIG. 10B. (10D) Kaplan-Meier survival curves of the mice shown in FIG. 10B with statistical significance determined by log-rank test. (10E) Plots of body weight over time for each mouse (numbered in FIG. 10B) from mice treated with non-transduced T cells and (10F) from mice treated with STEAP1-mBBζ CAR T cells. (10G) Photomicrographs of STEAP1 IHC staining of RM9-hSTEAP1 tumors after treatment with non-transduced T cells show areas of strong and homogenous STEAP1 expression (left) and areas of heterogeneous STEAP1 expression (right). Scale bar=50 μm. (10H) Representative STEAP1 IHC staining of RM9-hSTEAP1 tumors after treatment with STEAP1-mBBζ CAR T cells shows no STEAP1 expression. Scale bar=50 μm.

[0026] [Figure 11A-11B]Figure 11 shows that histology was maintained and no increased T cell infiltration was observed in the prostate or adrenal glands of hSTEAP1-KI / + mice treated with murine STEAP1-mBBζ CAR T cells. Representative photomicrographs of hematoxylin & eosin (H&E) and CD3 IHC staining of hSTEAP1-KI / + prostates excised from mice treated with (11A) non-transduced T cells or (11B) STEAP1-mBBζ CAR T cells are shown. Arrowheads indicate the few CD3+ cells observed. Scale bar = 50 μm.

[0027] [Figures 12A-12G](12A) IFN-γ enzyme-linked immunosorbent assay (ELISA) results for 24 h co-culture of non-transduced T cells or STEAP1 CAR T cells with short or medium spacers with each 22Rv1 cell derivative. n=4 replicates per condition. Error bars indicate SD. (12B) Plot and table of absolute STEAP1 molecule per cell in 22Rv1, C4-2B, PC3 and DU145 prostate cancer cell lines as measured by flow cytometry using Quantum Simply Cellular Microspheres from Bangs Laboratories. (12C) Time course of relative cell viability of PC3 target cells as measured by fluorescent live cell imaging after co-culture of PC3 target cells with non-transduced T cells or STEAP1-BBζ CAR T cells at a 1:1 ratio. n=4 replicates per condition, error bars indicate SEM. (12D) Schematic of tumor challenge experiment in PC3 disseminated model. (12E) Serial bioluminescence live imaging (BLI) of NSG mice implanted with PC3-fLuc metastatic tumors and treated with a single intravenous injection of 5x106 untransduced T cells or STEAP1-BBζ CAR T cells at normal CD4 / CD8 ratios on day 0. Mice from both treatment groups were euthanized at week 4 due to the development of severe acute graft-versus-host disease (GVHD). Luminance scale is indicated. (12F) Plot of total flux quantified over time from bioluminescence live imaging of each mouse shown in Figure 12E. (12G) Time course of relative cell viability of Ewing sarcoma cell lines RD-ES or SK-ES-1 target cells after co-culture with untransduced or STEAP1-BBζ CAR T cells at a 1:1 ratio, as measured by fluorescent live cell imaging. n=4 replicates per condition, error bars indicate SEM.

[0028] [Figure 13] 1 shows sequences supporting the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] According to the World Health Organization, cancer is the second leading cause of death worldwide, with an estimated 9.6 million cancer deaths in 2018. For many years, the treatment of choice for cancer was surgery, chemotherapy and / or radiation therapy. More recently, more targeted therapies have been developed to specifically target cancer cells, primarily by identifying and exploiting specific molecular changes found in cancer cells. For example, many cancer cells selectively express certain markers (e.g., antigens) on their cell surface, and these markers are used as targets for antibody-based therapies.

[0030] One of the key aspects of successful targeted therapy is the selection of a target cancer cell marker that is immunogenic, plays a crucial role in proliferation and differentiation, is expressed exclusively on the surface of all malignant cells and malignant stem cells, and tests positive in all or at least the majority of patients (Cheever, et al., 2009. Clin. Cancer Res. 15(17): 5323-8337).

[0031] STEAP1 (also known as PRSS24, STEAP, six transmembrane epithelial antigen of the prostate 1, or STEAP family member 1) is a 339 amino acid long protein named for its six transmembrane domains, which is upregulated in various tumors, including prostate cancer, bladder cancer, ovarian cancer, rhabdomyosarcoma, and Ewing sarcoma family tumors (EFT) (Hubert et al., Proc Natl Acad Sci USA 96(25): 14523-8 (1999); Rodeberg et al., Clin Cancer Res 11(12): 4545-52 (2005)). Transcriptomic and proteomic analyses, as well as functional studies, have shown that STEAP1 expression correlates with oxidative stress response and increased reactive oxygen species.

[0032] Prostate cancer is the most commonly diagnosed cancer in men, after skin cancer, and the second leading cause of cancer death in men. In prostate cancer, STEAP1 is expressed in up to 88% of lethal metastatic prostate cancers, whereas prostate-specific membrane antigen (PSMA) is expressed in only up to 61% of lethal metastatic prostate cancers.

[0033] Ewing sarcoma family tumors (EFTs) are a family of small round blue cell tumors originating from bone or soft tissue. This family is the second most common malignant bone tumor in children and adolescents, with an incidence rate of 200 cases per year in the United States (Esiashvili et al., J Pediatr Hematol Oncol. 30(6): 425-30 (2008)). EFTs are characterized by a specific translocation of EWS (Ewing sarcoma gene) on chromosome 22 with one of the E26 transformation-specific transcription factor family genes. STEAP1 may be a useful immunohistological marker for EFT patients, and STEAP1 immunoreactivity was detected on the cell membrane in 71 of 114 EFT samples (62.3%) (Grunewald et al., Ann Oncol , 23(8): p. 2185-90 (2012)). Another genetic profiling study in EFT patients showed that the absence of STEAP1 transcripts in bone marrow strongly correlated with overall patient survival and survival without new metastases. STEAP1 expression was detected in more than 60% of EFT tumors but was rarely detected in normal tissues (bladder and prostate secretory tissues), making STEAP1 a potentially useful target for antibody- and immune cell-based strategies.

[0034] Human STEAP1 (NCBI Reference Sequence: NP 036581.1) has the following amino acid sequence: MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAlVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL (SEQ ID NO: 142).

[0035] Mouse STEAP1 (NCBI Reference Sequence: NP 081675.2) has the following amino acid sequence: MEISDDVTNPEQLWKMKPKGNLEDDSYSTKDSGETSMLKRPGLSHLQHAVHVDAFDCPSELQHTQEFFPNWRLPVKVAAIISSLTFLYTLLREIIYPLVTSREQYFYKIPILVINKVLPMVAITLLALVYLPGELAAVVQLRNGTKYKKFPPWLDRWMLARKQFGLLSFFFAVLHAVYSLSYPMRRSYRYKLLNWAYKQVQQNKEDAWVEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTVHALVFAWNKWVDVSQFVWYMPPTFMIAVFLPTLVLICKIALCLPCLRKKILKIRCGWEDVSKINRTEMASRL (SEQ ID NO: 143).

[0036] Canine STEAP1 (NCBI Reference Sequence: XP 013974694.1) has the following amino acid sequence: MESRQDITSQEELWTMKPRRNLEEDDYLDKDSGDTRVLKRPVLLHMHQTTHFDEFDCPAELKHKQELFPMWRWPVKIAAVISSLTFLYTLLREIIHPFVTSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAVVQLHNGTKYKKFPHWLDRWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVTLAILALLAVTSIPSVSDSLTWREFHYIQSKLGMVSLLLGTIHALIFAWNKWVDIKQFVWYTPPTFMIAVFLPIVVLICKAILFLPCLRKKILKIRHGWEDVTKINKTEMS (sequence number 144).

[0037] Great strides have been made in engineering immune system cells to target and kill unwanted cells, such as cancer cells. Many of these immune cells are T cells engineered to express chimeric antigen receptors (CARs). CARs are proteins that contain several distinct components that enable engineered immune cells to recognize and kill cancer cells. These components include at least an extracellular portion and an intracellular portion when expressed by a cell. The extracellular portion contains a binding domain that specifically binds to a marker (e.g., an antigen) that is selectively present on the surface of the unwanted cells (e.g., STEAP1). The binding domain is usually a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be based on other formats that specifically bind to the marker of interest.

[0038] When the binding domain binds to such a marker, a signal is transmitted from the intracellular portion to the immune cell to destroy the bound cell. The intracellular portion generates such an activation signal based on the effector domain incorporated as part of it. First generation CARs were developed using the cytoplasmic domain of CD3ζ as the effector domain. Second generation CARs were developed by combining the cytoplasmic domain of CD3ζ with the cluster of differentiation 28 (CD28) or 4-1BB (CD137) cytoplasmic domain. Third generation CARs were developed by combining the cytoplasmic domain of CD3ζ with the cytoplasmic domain of CD28 and the cytoplasmic domain of 4-1BB as the effector domain.

[0039] A CAR can further comprise a transmembrane domain that links the extracellular portion to the intracellular portion, although not all CARs require a transmembrane domain.

[0040] Other components can also be used that can enhance the function of the CAR. For example, spacers can provide additional flexibility to the CAR's conformation and often enhance the binding ability of the binding domain to the marker of the target cell. The appropriate length of the spacer for a particular CAR depends on many factors, including how close or far the target marker is located from the surface of the cell membrane of the undesired cell.

[0041] The present disclosure provides CARs for the treatment of STEAP1-related diseases, such as prostate cancer, Ewing's sarcoma family of tumors (EFT), bladder cancer, ovarian cancer, and rhabdomyosarcoma. In certain embodiments, the CARs disclosed herein can be used to treat lethal metastatic castration-resistant prostate cancer. In certain embodiments, the CARs disclosed herein can be used to treat Ewing's sarcoma. In certain embodiments, the CARs disclosed herein can confer cytolytic activity even at low antigen density and show little or no cross-reactivity to the closely related STEAP1B. For example, data presented herein shows significant cytolytic activity against PC3 cell lines, which express only 1,491 STEAP1 molecules per cell. This data is compared to the 22Rv1 cell line, which expresses 69,475 STEAP1 molecules per cell, and the C4-2B cell line, which expresses 67,462 STEAP1 molecules per cell. Furthermore, data presented herein show that the CARs described herein are highly reactive to STEAP1, but not to the STEAP1B isoform, the protein with the highest homology to STEAP1. This result indicates that the CARs described herein are not only able to confer cytolytic activity at low antigen density, but also have high specificity for STEAP1, thereby reducing clinical concerns regarding off-target reactivity. The ability to confer cytolytic activity at low antigen density provides important clinical utility as a treatment, and can be effectively administered before tumor burden increases to high antigen density.

[0042] As used herein, "low antigen density" means expression of fewer than 50,000 cancer molecules per lesional cell, fewer than 40,000 cancer molecules per lesional cell, fewer than 30,000 cancer molecules per lesional cell, fewer than 20,000 cancer molecules per lesional cell, fewer than 10,000 cancer molecules per lesional cell, fewer than 5,000 cancer molecules per lesional cell, fewer than 4,000 cancer molecules per lesional cell, fewer than 3,000 cancer molecules per lesional cell, fewer than 2,000 cancer molecules per lesional cell, or fewer than 1,500 cancer molecules per lesional cell.

[0043] As used herein, "low STEAP1 antigen density" means that fewer than 50,000 STEAP1 molecules per lesion cell, fewer than 40,000 STEAP1 molecules per lesion cell, fewer than 30,000 STEAP1 molecules per lesion cell, fewer than 20,000 STEAP1 molecules per lesion cell, fewer than 10,000 STEAP1 molecules per lesion cell, fewer than 5,000 STEAP1 molecules per lesion cell, fewer than 4,000 STEAP1 molecules per lesion cell, fewer than 3,000 STEAP1 molecules per lesion cell, fewer than 2,000 STEAP1 molecules per lesion cell, or fewer than 1,500 STEAP1 molecules per lesion cell are expressed.

[0044] A "STEAP1-associated disease" is a disease in which STEAP1 is expressed in diseased or infected cells in a subject's body, and in which STEAP1 provides an antigen for targeted delivery of a therapeutic treatment. In such diseases, STEAP1 must be selectively expressed by diseased or infected cells so that on-target / off-target side effects are minimized or eliminated.

[0045] STEAP1 expressing diseased cells are the cells targeted for destruction by the treatment described herein.STEAP1 expressing diseased cells include, for example, prostate cancer cells (e.g., castration-resistant prostate cancer cells), Ewing's sarcoma family tumor cells (including Ewing's sarcoma cells), bladder cancer cells, breast cancer cells, ovarian cancer cells, colon cancer cells, lung cancer cells and kidney cancer cells.

[0046] In certain embodiments, the CAR of the disclosure, when expressed by a cell, comprises: (i) an extracellular portion comprising an scFv binding domain in the VL-VH order derived from bundled tuzumab vedotin (DSTP3086S; a humanized variant of the murine monoclonal antibody mAb 120.545) and a long spacer comprising an IgG4 hinge-CH2-CH3 with 4 / 2-NQ mutations in the CH2 domain; (ii) an intracellular portion comprising a CD3z activation domain and a 4-1BB costimulatory domain; and (iii) a CD28 transmembrane domain linking the extracellular portion to the intracellular portion. In certain embodiments, the scFv binding domain in the VL-VH order derived from bundled tuzumab vedotin is as set forth in SEQ ID NO: 3. In certain embodiments, the long spacer comprising an IgG4 hinge-CH2-CH3 with 4 / 2-NQ mutations in the CH2 domain is as set forth in SEQ ID NO: 20 and is encoded by the sequence set forth in SEQ ID NO: 21. In certain embodiments, the CD3z activation domain is set forth in SEQ ID NO:24 and is encoded by the sequence set forth in SEQ ID NO:22. In certain embodiments, the 4-1BB costimulatory domain is set forth in SEQ ID NO:30 and is encoded by the sequence set forth in SEQ ID NO:27. In certain embodiments, the CD28 transmembrane domain is set forth in SEQ ID NO:37 and is encoded by the sequence set forth in SEQ ID NO:33. In certain embodiments, the CAR of the present disclosure has the sequence set forth in SEQ ID NO:2 and is encoded by the sequence set forth in SEQ ID NO:1. Additional sequences and coding sequences are shown in FIG.

[0047] In certain embodiments, the present disclosure provides a CAR comprising a single chain variable fragment (scFv) that binds to STEAP1, a spacer, a transmembrane domain, and an intracellular effector domain.

[0048] In certain embodiments, the present disclosure provides a CAR comprising a single chain variable fragment (scFv) that binds to STEAP1, an IgG4 hinge and CH2-CH3 spacer, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain. In certain embodiments, the CAR of the present disclosure comprises an scFv that binds to STEAP1, an IgG4 hinge and CH2-CH3 spacer, a CD28 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ signaling domain, a truncated domain of Thosea asigna virus 2A (T2A), and a truncated EGFR. In certain embodiments, the STEAP1 CAR is delivered to immune cells using a lentiviral vector. In certain embodiments, the cells transduced to express the STEAP1 CAR are cells sorted by expression of truncated EGFR. In certain embodiments, the scFV that binds to STEAP1 is derived from bundled tuzumab vedotin (DSTP3086S).

[0049] In certain embodiments, the disclosure provides a CAR with a long spacer. In certain embodiments, the long spacer includes the hinge region, CH2 domain, and CH3 domain of IgG4 (the total length is 282 amino acids long). The IgG4 domain utilized as a spacer may include a mutation that prevents binding to human Fc receptors. In certain embodiments, the mutation includes replacing the first six amino acids of the CH2 domain of IgG4 (APEFLG, SEQ ID NO: 145) with the first five amino acids of IgG2 (APPVA, SEQ ID NO: 146). In certain embodiments, the long spacer is engineered to have a 4 / 2-NQ mutation in the CH2 domain.

[0050] Various aspects of the present disclosure are described in further detail below. Various aspects of the present disclosure include the following: (i) immune cells; (ii) cell sample collection and cell enrichment; (iii) cell populations genetically engineered to express chimeric antigen receptors (CARs); (iii-a) genetic engineering techniques; (iii-b) CAR components; (iii-bi) binding domains; (iii-b-ii) spacers; (iii-biii) transmembrane domains; (iii-b-iv) intracellular effector domains; (iii-bv) linkers; and (iii-b-vi) tag cassettes. , control functions including transduction markers and / or suicide switches; (iii-b-vii) multimerization domains; (iv) characterization of engineered cells; (v) cell activation culture conditions; (vi) ex vivo produced cell preparations; (vii) targeted viral vectors and nanoparticles for in vivo cell engineering; (viii) methods of use; (ix) reference levels obtained from control populations; (x) kits; (xi) exemplary embodiments; (xii) experimental examples; and (xiii) conclusion. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the present disclosure.

[0051] (i) Immune cells The present disclosure describes cells that are genetically engineered to express a CAR. The genetically engineered cells may include T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or a mixture of HSCs and HPCs (i.e., HSPCs). In certain embodiments, the genetically engineered cells include T cells.

[0052] Several types of T cell subsets have been found so far, and each T cell subset has a characteristic function. For example, most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced by independent genes, the T cell receptor α gene and the T cell receptor β gene (TCRα and TCRβ), respectively, and are called the αTCR chain and the βTCR chain.

[0053] γδ T cells are a small subset of T cells that have a distinctive T cell receptor (TCR) on their surface. In γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is less common than αβ T cells (representing only 2% of all T cells).

[0054] CD3 is expressed on all mature T cells. Activated T cells express 4-1BB (CD137), CD69, and CD25. CD5 and the transferrin receptor are also expressed on T cells.

[0055] T cells can be further classified into helper T cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), the latter of which includes cytolytic T cells. Helper T cells are responsible for helping other white blood cells in immunological processes, such as maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages. Helper T cells are also known as CD4+ T cells, because they express the CD4 protein on their surface. Helper T cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells divide rapidly and secrete small proteins called cytokines that are responsible for regulating or supporting an active immune response.

[0056] Cytotoxic T cells can destroy virus-infected and tumor cells and are also involved in transplant rejection. Cytotoxic T cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. Cytotoxic T cells recognize their targets by binding to antigens bound to MHC class I molecules, which are present on the surface of almost all cells in the body.

[0057] As used herein, "central memory" T cells (or "TCM") refer to CTLs that have experienced an antigen and express CD62L or CCR-7 and CD45RO on their surface, but do not express CD45RA or have reduced expression of CD45RA, compared to naive cells. In a specific embodiment, central memory cells are positive for CD62L, CCR7, CD25, CD127, CD45RO and CD95, and have reduced expression of CD45RA, compared to naive cells.

[0058] As used herein, "effector memory" T cells (or "TEMs") refer to T cells that have experienced an antigen and do not express CD62L or have reduced expression of CD62L on their surface compared to central memory cells, and do not express CD45RA or have reduced expression of CD45RA compared to naive cells. In certain embodiments, effector memory cells are negative for CD62L and CCR7 and positive or negative for CD28 and CD45RA compared to naive or central memory cells. Effector T cells are also positive for granzyme B and perforin compared to memory or naive T cells.

[0059] As used herein, "naive" T cells refer to T cells that have not experienced an antigen and express CD62L and CD45RA, but not CD45RO, in comparison to central memory cells or effector memory cells. In certain embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD127, and CD45RA.

[0060] Natural killer cells (also known as NK cells, K cells and killer cells) are activated in response to interferon or macrophage-derived cytokines. NK cells play a role in combating viral infections, whereas adaptive immune responses produce antigen-specific cytotoxic T cells that can eliminate infections. NK cells express CD8, CD16 and CD56, but not CD3.

[0061] NK cells include NK-T cells. NK-T cells are a specialized T cell population that express the semi-invariant T cell receptor (TCRαβ) and surface antigens normally associated with natural killer cells. NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-associated immune surveillance or tumor-associated immune suppression. Like natural killer cells, NK-T cells can also induce cytotoxicity associated with perforin, Fas, and TNF. Activated NK-T cells can produce IFN-γ and IL-4. In certain embodiments, NK-T cells are CD3+ / CD56+.

[0062] Macrophages (and their precursor cells, monocytes) are present in all tissues of the body (possibly as microglia, Kupffer cells, and osteoclasts) and phagocytose apoptotic cells, pathogens, and other non-self components. Monocytes / macrophages express CD11b, F4 / 80; CD68; CD11c; IL-4Rα; and / or CD163.

[0063] Immature dendritic cells (i.e., preactivated dendritic cells) phagocytose peripheral antigens and other non-self components, become activated, migrate to T cell areas of lymphoid tissues, and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and DEC-205.

[0064] Hematopoietic stem / progenitor cells, or HSPCs, refer to the combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0065] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal in vivo or of self-renewal and proliferation in vitro, and that can differentiate into any other type of hematopoietic cell.

[0066] Hematopoietic progenitor cells are cells derived from hematopoietic stem cells or cells derived from fetal tissue that can be further differentiated into mature cells. In certain embodiments, hematopoietic progenitor cells are CD24 lo Lin - CD117 + Hematopoietic progenitor cells. HPCs can differentiate (i) into myeloid progenitor cells that can ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets or dendritic cells, or (ii) into lymphoid progenitor cells that can ultimately give rise to T cells, B cells and NK cells.

[0067] HSPCs may be positive for specific markers that are expressed in HSPCs in greater amounts than other types of hematopoietic cells. Such markers include, for example, CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or combinations thereof. In addition, HSPCs may be negative for markers that are expressed in other types of hematopoietic cells. Such markers include, for example, Lin, CD38, or combinations thereof. HSPCs are preferably CD34+ cells.

[0068] A statement that a cell or cell population is "positive" for or expresses a particular marker refers to the presence of the particular marker detectable on the surface or interior of the cell. When referring to a cell surface marker, "positive" refers to the presence of cell surface expression, as detected by, for example, flow cytometry using staining with an antibody that specifically binds to the cell surface marker and detecting the antibody, at a level substantially higher than that detected by the same procedure under identical conditions using an isotype-matched control in the flow cytometry, and / or at a level substantially similar to that of cells known to be positive for that marker, and / or at a level substantially higher than that of cells known to be negative for that marker.

[0069] The statement that a cell or cell population is "negative" for a particular marker or that a cell or cell population does not express the marker means that there is substantially no detectable presence of the particular marker on the surface of the cell or within the cell. When referring to a cell surface marker, "negative" means that there is no cell surface expression detected, for example, by flow cytometry using staining with an antibody that specifically binds to the cell surface marker and detecting the antibody, and that the staining is not detected in the flow cytometer at a level substantially higher than that detected by the same procedure under the same conditions using an isotype-matched control and / or is detected at a level substantially lower than that of cells known to be positive for that marker and / or is detected to the same extent as that of cells known to be negative for that marker.

[0070] Cells which are genetically modified in accordance with the teachings of the present disclosure may, where appropriate, be cells derived from the patient (autologous cells), cells allogeneic to the patient, in vivo cells or ex vivo cells.

[0071] (ii) Cell sample collection and cell enrichment Methods for collecting samples and concentrating samples are known to those skilled in the art. In some embodiments, the cells are derived from a cell line. In some embodiments, the cells are derived from a heterologous cell source, for example, from a mouse, a rat, a non-human primate, or a pig. In certain embodiments, the cells are derived from a human, for example, from a patient to be treated.

[0072] In some embodiments, the T cells are derived from or isolated from a sample such as whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organ, and / or cells derived from these organs. In certain embodiments, cells are obtained from the circulating blood of a subject, such as by apheresis or leukapheresis. In certain embodiments, the sample includes lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSCs, HPCs, HSPCs, red blood cells, and / or platelets, and in some aspects, the sample includes cells other than red blood cells and platelets and further processing is required. In certain embodiments, the T cells are derived from PBMCs.

[0073] In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and to suspend the cells in a suitable buffer or medium for the next processing step. In certain embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution does not contain calcium and / or magnesium, and / or does not contain a large proportion of divalent cations or does not contain any divalent cations at all. Washing can be performed using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In certain embodiments, the washed cells can be resuspended in various types of biocompatible buffers, such as Ca++ / Mg++-free PBS.

[0074] Isolation may include one or more of a variety of cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to a particular reagent, and / or affinity (e.g., immunoaffinity) for an antibody or other binding partner. In certain embodiments, isolation is performed sequentially and / or simultaneously in a single method using one device or one equipment. In certain embodiments, isolation, culture and / or recombination of various different populations begins with one starting composition or starting material, such as one sample.

[0075] In certain embodiments, samples can be enriched for T cells using density-based cell separation and related techniques. For example, white blood cells can be separated from other cell types in peripheral blood by lysis of red blood cells followed by centrifugation through a Percoll or Ficoll gradient.

[0076] In certain embodiments, a bulk T cell population that is not enriched for a particular type of T cell can be used. In certain embodiments, a selected type of T cell can be enriched and / or isolated by positive and / or negative selection using a cell marker. In positive selection, cells with a cell marker bound to a capture agent are obtained and used for further application. In negative selection, cells that did not bind to a capture agent, such as an antibody against a cell marker, are obtained and used for further application. In some examples, both the positively selected and negatively selected fractions can be obtained and used for further application. In certain embodiments, CD4+ and / or CD8+ T cells are enriched from PBMCs.

[0077] The above cell separation does not necessarily achieve 100% enrichment or removal of a particular cell population or cell that expresses a particular marker.For example, positive selection or enrichment of a particular type of cell means increasing the number or proportion of such cells, but does not necessarily completely remove cells that do not express that marker.Similarly, negative selection, removal or elimination of a particular type of cell means decreasing the number or proportion of such cells, but does not necessarily completely remove such cells.

[0078] In some instances, the separation step is performed multiple times, whereby the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.

[0079] In some embodiments, antibodies or binding domains against cell markers can be attached to solid supports or solid matrices, such as magnetic or paramagnetic beads, to separate cells by positive and / or negative selection. For example, in some embodiments, immunomagnetic separation techniques (or affinity magnetic separation techniques) are used to separate or isolate cells and cell populations (reviewed in "Methods" in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher (c) Humana Press Inc., Totowa, NJ). See also U.S. Patent Publication No. 4,452,773; U.S. Patent Publication No. 4,795,698; U.S. Patent Publication No. 5,200,084; and European Patent Publication No. 452342.

[0080] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The MACS system allows for high purity selection of magnetic particle-bound cells. In certain embodiments, MACS is operated in a mode in which non-target and target species are eluted sequentially after application of an external magnetic field, i.e., cells that are not bound to the magnetic particles are eluted while those that are bound to the magnetic particles are retained. Then, after this first elution step, the cells trapped by the magnetic field and prevented from elution are released in some manner that allows for their elution and recovery. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

[0081] In some embodiments, the cell populations described herein are collected and enriched (or removed) by flow cytometry, where cells stained for multiple cell surface markers are placed in a fluid stream for analysis. In some embodiments, the cell populations described herein are collected and enriched (or removed) by preparative (FACS) sorting. In some embodiments, the cell populations described herein are collected and enriched (or removed) by using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO2010 / 033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In either case, cells can be labeled with multiple markers to isolate well-defined subsets of cells with high purity.

[0082] Cell markers for various T cell subpopulations are described above. In certain embodiments, specific T cell subpopulations, e.g., T cells positive for or highly expressing one or more cell surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4 and / or CD45RA T cells, are isolated by positive or negative selection techniques.

[0083] CD3+CD28+ T cells can be positively selected and expanded using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0084] In certain embodiments, a CD8+ or CD4+ selection step is performed to separate CD4+ helper T cells from CD8+ cytotoxic T cells. Such CD8+ and CD4+ populations can be further sorted into various subpopulations by positive or negative selection for markers expressed on, or relatively highly expressed in, one or more naive, memory and / or effector T cell subpopulations.

[0085] In some embodiments, enrichment is performed to obtain central memory T (TCM) cells. In certain embodiments, memory T cells are contained in both the CD62L positive and CD62L negative subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted of CD62L fraction, CD8 fraction and / or CD62L+CD8+ fraction using, for example, anti-CD8 and anti-CD62L antibodies.

[0086] In some embodiments, central memory T (TCM) cells are enriched based on positive or high cell surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3 and / or CD127. In some aspects, TCM cells are enriched by negative selection of cells expressing or high expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by removing cells expressing CD4, CD14 and CD45RA, and positively selecting or enriching cells expressing CCR7, CD45RO and / or CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed by starting with a negative cell fraction selected based on CD4 expression, and subjecting this fraction to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L expression. Such selections may be performed simultaneously in some aspects, or sequentially in other aspects, and may be performed in any order. In some embodiments, a CD4+ cell population or subpopulation can be obtained by carrying out the same selection steps based on CD4 expression as those used to prepare a CD8+ cell population or subpopulation, where both the positive and negative fractions obtained by CD4-based separation can be retained and subjected to one or more further positive or negative selection steps.

[0087] Additionally, other cell types can be enriched based on known marker profiles and known techniques, for example, CD34+ HSCs, HSPs and HSPCs can be enriched using anti-CD34 antibodies directly or indirectly coupled to magnetic microparticles in combination with a magnetic cell separator (e.g., the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany)).

[0088] (iii) a cell population genetically engineered to express a chimeric antigen receptor (CAR); The cell population may be genetically engineered to express a chimeric antigen receptor (CAR) as described herein.

[0089] (iii-a) Genetic engineering techniques The desired gene encoding the CAR disclosed herein can be introduced into cells by methods known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate transfection, infection with a viral or bacteriophage vector containing the gene sequence, cell fusion, chromosomal gene transfer, microcell fusion gene transfer, spheroplast fusion, in vivo nanoparticle delivery, etc. A variety of techniques are known in the art for introducing foreign genes into cells (see, for example, Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92), and such techniques may be used to the extent that they do not unduly disrupt the developmental and physiological functions required by the recipient cells. These techniques can be used to stably transfer foreign genes into cells, such that the foreign genes are expressed in the cell, and in certain instances, preferably heritable and expressed in its progeny cells.

[0090] "Gene" refers to a nucleic acid sequence encoding a CAR comprising a STEAP1 binding domain as described herein. The definition of this term includes various sequence polymorphisms, mutations and / or sequence variants, where such changes do not substantially affect the function of the encoded CAR. The term "gene" may include not only the coding sequence, but also regulatory regions such as promoter regions, enhancer regions, and termination regions. The gene sequence encoding the molecule may be DNA or RNA that induces the expression of the CAR. These nucleic acid sequences may be DNA strand sequences that are transcribed into RNA or RNA sequences that are translated into protein. The nucleic acid sequence includes both full-length nucleic acid sequences and partial sequences derived from full-length proteins. These sequences may further include sequences that may be introduced to confer codon preference in a particular type of cell, or degenerate codons of the native sequence. Portions of the complete gene sequence are also referenced throughout this disclosure, as would be understood by one of skill in the art.

[0091] Genetic sequences encoding CAR are provided herein, which can be easily made by synthetic or recombinant methods from the relevant amino acid sequences based on other information provided herein. In some embodiments, the genetic sequences encoding any of these sequences may have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence, so that the genetic sequences encoding these sequences can be easily cleaved and replaced with another genetic sequence encoding another sequence. In some embodiments, the genetic sequences encoding the above sequences may be codon-optimized for expression in mammalian cells.

[0092] "Encode" refers to the property that a particular nucleotide sequence within a gene, such as a cDNA or mRNA, serves as a template for the synthesis of another macromolecule, such as a defined amino acid sequence. Thus, a particular gene encodes a protein when its corresponding mRNA is transcribed and translated to produce the protein in a cell or other biological system. A "gene sequence encoding a protein" includes any degenerate nucleotide sequence that encodes the same amino acid sequence or an amino acid sequence having substantially similar form and function.

[0093] Multiple polynucleotide gene sequences encoding two or more portions of the CAR to be expressed can be operably linked to each other and can also be linked to relevant regulatory sequences. For example, a regulatory sequence can be operably linked to an exogenous nucleic acid sequence, thereby expressing the exogenous nucleic acid sequence. In another example, a first nucleic acid sequence and a second nucleic acid sequence can be operably linked if they are arranged in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Usually, operably linked DNA sequences are contiguous, and if a coding region is necessary or useful, the coding region is linked in the same reading frame.

[0094] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, cosmid, virus, or phage. An "expression vector" is a vector that, when placed under the appropriate environment, can induce expression of a protein encoded by one or more genes incorporated into the expression vector.

[0095] "Lentivirus" refers to the virus of the retrovirus genus that can infect dividing and non-dividing cells.Some examples of lentivirus include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0096] Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, and in particular include self-inactivating lentiviral vectors such as those described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include the LENTIVECTOR® gene delivery technology available from Oxford BioMedica, LENTIMAX® available from Lentigen, and others. TM Vector systems and the like. In addition, non-clinical lentiviral vectors are also available and known to those skilled in the art. In certain embodiments, lentivirus or lentiviral vectors are used to genetically modify cells to express CAR.

[0097] A "retrovirus" is a virus with an RNA genome. A "gammaretrovirus" refers to a virus that belongs to the Retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0098] Retroviral vectors can also be used (see Miller, et al., 1993, Meth. Enzymol. 217:581-599). In such an embodiment, the gene to be expressed is cloned into the retroviral vector and delivered into the cell. In a particular embodiment, the retroviral vector contains all the cis-acting sequences required for packaging and integration of the viral genome, i.e., (a) long terminal repeats (LTRs) or parts thereof located at both ends of the vector, (b) primer binding sites for negative and positive strand DNA synthesis, and (c) packaging signals required for integration of genomic RNA into virions. Further details regarding retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV), and alphaviruses can also be used.Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991, Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. See Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods for gene delivery include the use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triple-helical DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0099] There are numerous viral vectors available that are suitable for the present disclosure, including those identified for human gene therapy (see Pfeifer and Verma, 2001, Ann. Rev. Genomics Hum. Genet. 2:177). Methods for using retroviral and lentiviral vectors and packaging them into cells to transduce mammalian host cells with viral particles containing CAR transgenes are described in, for example, U.S. Patent Publication No. 8,119,772; Walchli, et al., 2011, PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Ther. 14:1155; Frecha, et al., 2010, Mol. Ther. 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97.In addition, retroviral and lentiviral vector constructs and their expression systems are commercially available.

[0100] Targeted genetic engineering methods may be used. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is a recombinant nuclease system used in genetic engineering using bacterial systems. Information on the CRISPR-Cas system and its components can be found in, for example, U.S. Patent Publication No. 8,697,359, U.S. Patent Publication No. 8,771,945, U.S. Patent Publication No. 8,795,965, U.S. Patent Publication No. 8,865,406, U.S. Patent Publication No. 8,871,445, U.S. Patent Publication No. 8,889,356, U.S. Patent Publication No. 8,889,418, U.S. Patent Publication No. 8,895,308, U.S. Patent Publication No. 8,906,616, U.S. Patent Publication No. 8,103,131 ... No. 8,932,814, U.S. Patent Publication No. 8,945,839, U.S. Patent Publication No. 8,993,233, and U.S. Patent Publication No. 8,999,641 and related applications thereof; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724 , WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473, WO2015 / 089486, WO2016205711, WO2017 / 106657 and WO2017 / 127807 and related applications thereof.

[0101] In certain embodiments, zinc finger nucleases (ZFNs) are used as gene editing agents. ZFNs are a type of site-specific nuclease engineered to bind and cleave DNA at specific locations. ZFNs are used to introduce double-strand breaks (DSBs) at specific sites in DNA sequences, allowing the introduction of double-strand breaks to target unique sequences in the genome of various types of cells. Zinc fingers are 30 amino acid long domains in zinc finger binding domains, whose structure is stabilized by the coordination of zinc ions. Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. Designed zinc finger domains are domains that do not exist in nature, and their design / composition is mainly constructed based on rational criteria, such as the application of computer algorithms and substitution rules that process information in databases that store information on the design and binding data of existing ZFPs. A well-known example of a ZFN is a fusion of the FokI nuclease and a zinc finger DNA binding domain.For further information regarding ZFNs and ZFNs useful within the teachings of the present disclosure, see, e.g., U.S. Patent Publication Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013, No. 219; U.S. Patent Publication No. 7,030,215; U.S. Patent Publication No. 7,220,719; U.S. Patent Publication No. 7,241,573; U.S. Patent Publication No. 7,241,574; U.S. Patent Publication No. 7,585,849; U.S. Patent Publication No. 7,595,376; U.S. Patent Publication No. 6,903,185; U.S. Patent Publication No. 6,479,626; U.S. Patent Publication No. 2003 / 0232410 and U.S. Patent Publication No. 2009 / 0203140, as well as Gaj et al., Nat Methods, 2012, 9(8):805-7;Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8;Kim et al., Genome Res, 2012, 22(7): 1327-33;Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646;Miller, et al. Nature biotechnology 25, 778-785 (2007);Bibikova, et al. Science 300, 764 (2003);Bibikova, et al. Genetics 161, 1169-1175 (2002);Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000);Kim,et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0102] In certain embodiments, transcription activator-like effector nucleases (TALENs) can be used as gene editing agents. TALENs are fusion proteins that contain transcription activator-like effector (TALE) DNA binding proteins and DNA cleavage domains. TALENs are used to edit genes and genomes by inducing double-strand breaks in DNA to induce cell repair mechanisms. In general, two TALENs bind to both sides of a target DNA site, sandwiching the target DNA site, and dimerize the DNA cleavage domains to induce double-strand breaks. For further information regarding TALENs, see U.S. Patent Publication No. 8,440,431; U.S. Patent Publication No. 8,440,432; U.S. Patent Publication No. 8,450,471; U.S. Patent Publication No. 8,586,363; and U.S. Patent Publication No. 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0103] In certain embodiments, MegaTALs can be used as gene editing agents. MegaTALs have a nuclease structure that cuts a single strand at an extremely rare position, and a TALE is fused to the DNA cleavage domain of the meganuclease. Meganucleases (also called homing endonucleases) are single-chain peptides that combine DNA recognition and nuclease functions in the same domain. Unlike TALENs, megaTALs require the delivery of a single peptide chain for their functional activity.

[0104] In certain embodiments, a transposon-based system can be used as a gene editing agent to integrate the CAR construct into cells. In general, such methods involve introducing into cells (i) a first vector encoding a transposase (or a transposase polypeptide) and (ii) a second vector encoding a desired genetic element flanked by transposon repeat sequences. Transposons, or transposable elements, contain a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream thereof, and encode an enzyme that facilitates the cleavage of a target DNA sequence and the insertion of a nucleic acid into the target DNA sequence.

[0105] There are several transposon / transposase systems that have been engineered to accommodate the genetic insertion of heterologous DNA sequences. Examples of such transposases include sleeping beauty ("SB", e.g., from the salmonid genome), piggyback (e.g., from lepidopteran insect cells and / or Myotis bat), mariner (e.g., from Drosophila melanogaster), frog prince (e.g., from leopard frog), Tol1; Tol2 (e.g., from medaka), TcBuster (e.g., from Tribolium castaneum), Helraiser, Himar1, Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmar1, and spinON. Transposase and transposon systems are also described in U.S. Patent No. 6,489,458; U.S. Patent No. 7,148,203; U.S. Patent No. 8,227,432; and U.S. Patent No. 9,228,180.

[0106] (iii-b) Components of CAR As mentioned above, CAR molecules contain several distinctive components that allow genetically engineered T cells to recognize and kill unwanted cells, such as cancer cells. These components include at least an extracellular portion and an intracellular portion. The extracellular portion contains a binding domain that specifically binds to a marker that is selectively present on the surface of the unwanted cells. When the binding domain binds to such a marker, the intracellular portion activates the T cell to destroy the bound cell. CARs further contain a transmembrane domain that links the extracellular portion to the intracellular portion, and other components that can enhance the function of the CAR. For example, the incorporation of a spacer sequence and / or one or more linker sequences into the CAR can provide additional flexibility in the 3D structure of the CAR and often enhance the binding ability of the binding domain to the marker of the target cell.

[0107] (iii-bi) Binding domainIn a particular example, the present disclosure provides a binding domain for use in an antibody-based CAR that binds to STEAP1. Antibodies are generated from two genes, a heavy chain gene and a light chain gene. Antibodies usually contain two identical copies of a heavy chain and two identical copies of a light chain. In the heavy and light chain variable regions, segments called complementarity determining regions (CDRs) determine binding to an epitope. Each heavy chain has three CDRs (i.e., CDRH1, CDRH2, and CDRH3), and each light chain has three CDRs (i.e., CDRL1, CDRL2, and CDRL3). The CDR regions are flanked by framework residues (FRs). The exact amino acid sequence boundaries of a CDR or FR can be readily determined using any of a number of known numbering schemes. Known numbering schemes include those described in Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273: 927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262: 732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86: 9268-9272 (AbM numbering scheme); North et al. (2011) J. Mol. Biol. 406(2):228-56 (North numbering scheme); Lefranc MP et al. (2003) Dev Comp Immunol 27(1): 55-77 (IMGT numbering scheme); and Honegger and Pluckthun (2001) J Mol Biol 309(3): 657-670 ("Aho" numbering scheme).The boundaries of the CDRs or FRs may vary depending on the scheme used to identify them. For example, Kabat's scheme is based on structural alignment, while Chothia's scheme is based on structural information. The numbering in Kabat and Chothia schemes is based on the length of the most common antibody region sequences, with insertions indicated by letters, e.g., "30a", and deletions in some antibodies. The numbering in these two schemes differs due to the inclusion of insertions and deletions ("indels") at different positions. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In certain embodiments, the CDR sequences of the antibodies disclosed herein are shown according to Kabat's numbering. North's numbering uses longer sequences than the other schemes in the structural analysis of the three-dimensional structure of the CDR loops. CDR residues can be identified using software programs such as ABodyBuilder.

[0108] In some cases, another scFv based on the binding domain described herein for use in CAR can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be made by linking the VH and VL regions of an antibody using a flexible polypeptide linker. When a short polypeptide linker (e.g., 5-10 amino acids long) is used, intrachain folding is prevented. Interchain folding requires the combination of two variable regions to form a binding site for a functional epitope. For examples of linker orientation and size, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, US Patent Publication No. 2005 / 0100543, US Patent Publication No. 2005 / 0175606, US Patent Publication No. 2007 / 0014794, WO2006 / 020258, and WO2007 / 024715. More specifically, the linker sequence used to link the VL and VH of the scFv is usually 5 to 35 amino acids long. In certain embodiments, the VL-VH linker may be 5 to 35 amino acids long, 10 to 30 amino acids long, or 15 to 25 amino acids long. The activity may be maintained or enhanced by changing the length of the linker, which may provide superior efficacy in activity studies. In general, scFv is often used as the binding domain of CAR. In certain embodiments, the CAR of the present disclosure comprises a binding domain that binds to STEAP1. In certain embodiments, the binding domain that binds to STEAP1 is an scFv. In certain embodiments, the binding domain that binds to STEAP1 is an scFV derived from bundled tuzumab vedotin (DSTP3086S). In certain embodiments, the binding domain that binds to STEAP1 is a humanized variant of mouse monoclonal antibody mAb 120.545.In certain embodiments, the binding domain that binds to STEAP1 is encoded by the sequence set forth in SEQ ID NO: 6. In certain embodiments, the binding domain that binds to STEAP1 is set forth in SEQ ID NO: 3.

[0109] Other binding fragments such as Fv, Fab, Fab', F(ab')2 can also be used in the CARs disclosed herein. Other examples of antibody-based binding domain formats for use in CARs include grababodies made from scFvs and soluble VH domain antibodies. These antibodies use only the heavy chain variable region to form the binding region. See, e.g., Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0110] In certain embodiments, the binding domain comprises a humanized antibody or a recombinant fragment thereof. In certain embodiments, a non-human antibody is humanized, in which one or more amino acid residues of the antibody are modified to increase the similarity to an antibody or fragment thereof that is naturally produced in humans. Such non-human amino acid residues are often referred to as "import" residues, and are usually taken from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs and framework regions derived from a non-human immunoglobulin molecule, and the amino acid residues comprising the framework are derived exclusively or predominantly from human germline.Humanized antibodies can be made using a variety of techniques known in the art, such as CDR grafting (see, e.g., European Patent No. 239,400; WO 91 / 09967; and U.S. Patent Publication Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (see, e.g., European Patent Publication Nos. 592,106 and 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Patent Publication No. 5,565,332), as well as methods described in, e.g., U.S. Patent Publication No. 2005 / 0042664, U.S. Patent Publication No. 2005 / 0048617, U.S. Patent Publication No. 6,407,213, U.S. Patent Publication No. 5,766,886, WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994).Framework residues in the framework regions are often substituted with corresponding residues from a CDR donor antibody, for example to improve binding to STEAP1. Such substitutions of framework residues are identified by methods known in the art, for example by modeling the interactions of CDR and framework residues to identify framework residues important for binding to STEAP1, and by identifying framework residues that are not commonly found at particular positions by sequence comparison (see, for example, U.S. Patent Publication No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0111] A functional variant includes one or more residue additions or substitutions that do not substantially affect the physiological action of the protein. A functional fragment includes one or more deletions or truncations that do not substantially affect the physiological action of the protein. The lack of substantial effect can be confirmed by observing comparable experimental results in activation or binding assays. Functional variants and functional fragments of intracellular domains (e.g., intracellular signaling moieties) transmit activating or inhibitory signals to the same extent as the wild-type reference intracellular domain when in the activated state described in this disclosure. Functional variants and functional fragments of binding domains also bind to their cognate antigens or cognate ligands at a level comparable to the wild-type reference binding domain.

[0112] In certain embodiments, the VL region of a binding domain of the disclosure is derived from or generated based on the VL region of an antibody of the disclosure and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of these changes, compared to the VL region of an antibody of the disclosure. The insertions, deletions or substitutions may be made anywhere in the VL region, including at the amino or carboxy terminus or both, so long as each CDR contains no changes or at most one, two or three changes and the binding domain comprising the recombined VL region is capable of specifically binding to a target with an affinity similar to that of the wild-type binding domain.

[0113] In certain embodiments, the VH region of a binding domain of the disclosure is derived from or generated based on the VH region of an antibody of the disclosure and may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of these changes, compared to the VH region of an antibody of the disclosure. The insertions, deletions or substitutions may be made anywhere in the VH region, including at the amino or carboxy terminus or both, so long as each CDR contains no changes or at most one, two or three changes and the binding domain comprising the recombined VH region is capable of specifically binding to a target with an affinity similar to that of the wild-type binding domain.

[0114] In certain embodiments, the binding domain comprises or is a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to the light chain variable region (VL) or heavy chain variable region (VH) or both, and contains no, or at most one, two, or three changes in each CDR compared to an antibody or fragment or derivative thereof of the present disclosure that specifically binds to STEAP1.

[0115] (iii-b-ii) Spacer Spacers are used to provide appropriate distance from other components of CAR and / or flexibility relative to other components.As described herein, in certain embodiments, the length of the spacer is customized so that it can bind to STEAP1 expressing cells and induce cell destruction.In certain embodiments, the length of the spacer can be selected according to the location of the epitope of the cell marker, the affinity of the binding domain for the epitope, and / or the ability of the STEAP1 binding agent to induce cell destruction after binding to STEAP1.

[0116] Typical spacers include spacers of 10 to 250 amino acids in length, spacers of 10 to 200 amino acids in length, spacers of 10 to 150 amino acids in length, spacers of 10 to 100 amino acids in length, spacers of 10 to 50 amino acids in length, and spacers of 10 to 25 amino acids in length.

[0117] In certain embodiments, the spacer is 5, 8, 10, 12, 14, 20, 21, 26, 27, 45, 50 or 75 amino acids long. Spacers of such lengths are considered short spacers.

[0118] In certain embodiments, the spacer is 76, 90, 100, 110, 120, 125, 128, 131, 135, 140, 150, 160, 170, or 179 amino acids long. Spacers of such lengths qualify as medium length spacers.

[0119] In certain embodiments, the spacer is 180, 190, 200, 210, 220, 228, 230, 240, 250, 260, or 270 amino acids long. Spacers of such lengths qualify as long spacers.

[0120] Exemplary spacers include the entire length or a portion of the hinge region of an immunoglobulin. The hinge region of an immunoglobulin may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In certain embodiments, the hinge region of an immunoglobulin is a human immunoglobulin hinge region. As used herein, "wild-type immunoglobulin hinge region" refers to the amino acid sequence of the hinge located between and connecting the CH1 domain and the CH2 domain of the heavy chain in the upper and middle parts of a natural antibody (in the case of IgG, IgA and IgD), or the amino acid sequence of the hinge located between and connecting the CH1 domain and the CH3 domain of the heavy chain (in the case of IgE and IgM).

[0121] The immunoglobulin hinge region may be an IgG, IgA, IgD, IgE or IgM hinge region. The IgG hinge region may be an IgG1, IgG2, IgG3 or IgG4 hinge region. Sequences from IgG1, IgG2, IgGG3, IgGG4 or IgD may each be used alone, in combination with all or a portion of the CH2 region, in combination with all or a portion of the CH3 region, or in combination with all or a portion of the CH2 region and all or a portion of the CH3 region.

[0122] In certain embodiments, the spacer is a short spacer comprising an IgG4 hinge region. In certain embodiments, the short spacer is encoded by SEQ ID NO: 11, 12 or 13. In certain embodiments, the spacer is a medium-length spacer comprising an IgG4 hinge region and an IgG4 CH3 region. In certain embodiments, the medium-length spacer is encoded by SEQ ID NO: 15 or 18. In certain embodiments, the spacer is a long spacer comprising an IgG4 hinge region, an IgG4 CH2 region and an IgG4 CH3 region. In certain embodiments, the long spacer is encoded by SEQ ID NO: 21. In certain embodiments, the long spacer comprises a 4 / 2-N / Q mutation in the CH2 domain. The mutations can be used to block Fcγ receptor binding and activation-induced cell death.

[0123] Other examples of hinge regions that can be used in the CARs described herein include the hinge regions present in the extracellular domains of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be wild-type or variants thereof.

[0124] In certain embodiments, the spacer comprises the hinge region of the interdomain region (stalk region) of a type II C-type lectin or the hinge region of the stalk region of a cluster of differentiation (CD) molecule. The "stalk region" of a type II C-type lectin or CD molecule refers to the portion of the extracellular domain (ECD) that is located between the C-type lectin-like domain (CTLD) (e.g., similar to the CTLD of the natural killer cell receptor) and the hydrophobic portion (transmembrane domain) in a type II C-type lectin or CD molecule. For example, the extracellular domain of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, whereas the CTLD corresponds to amino acid residues 61-176, and the stalk region of the human CD94 molecule includes amino acid residues 34-60, and is therefore located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999; for a description of other stalk regions, see further Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). These type II C-type lectins or CD molecules may further have linking amino acids between the stalk region and the transmembrane domain or between the stalk region and the CTLD (discussed below). In another example, the 233 amino acid long human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) consisting of amino acids 71 ​​to 93 and an extracellular domain consisting of amino acids 94 to 233. The CTLD of the human NKG2A protein includes amino acids 119 to 231, and the stalk region includes amino acids 99 to 116, which may be flanked by additional linking amino acids.Other type II C-type lectins or CD molecules, or their extracellular ligand-binding domains, stalk regions and CTLDs, are also known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1; NP 001773.1; AAL65234.1; and CAA04925.1 for sequences and descriptions of human CD23, human CD69, human CD72, human NKG2A and human NKG2D, respectively).

[0125] (iii-b-iii) Transmembrane domain As described herein, the transmembrane domain in the CAR serves to link the extracellular portion to the intracellular portion via the cell membrane. The transmembrane domain can tether the expressed molecule to the cell membrane in the engineered cell.

[0126] The transmembrane domain may be of natural origin and / or synthetic origin. If of natural origin, the transmembrane domain may be derived from a membrane-bound or transmembrane protein. The transmembrane domain may comprise at least the transmembrane region of the α, β or ζ chain of the T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 or TLR9. In certain embodiments, the transmembrane domain comprises at least one of the following: e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, ITGAM, CDl lb, ITGAX, CDl It may include the transmembrane domain of lc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C. In certain embodiments, various human hinges can be used as well, such as human Ig (immunoglobulin) hinges (e.g., IgG4 hinges and IgD hinges), GS linkers (e.g., GS linkers described herein), KIR2DS2 hinges, CD8a hinges, etc.In certain embodiments, the CAR comprises a CD28 transmembrane domain, which has been shown to lower the antigen threshold for activation of second generation 4-1BB CAR T cells.

[0127] In certain embodiments, the transmembrane domain has a three-dimensional structure, typically 15-30 amino acids long, that is thermodynamically stable in the cell membrane. The structure of the transmembrane domain can include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof.

[0128] The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids in the extracellular region of the CAR (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids in the intracellular region of the CAR (e.g., up to 15 amino acids of the intracellular portion). In one embodiment, the transmembrane domain may be derived from the same protein from which the signaling domain, the costimulatory domain, or the hinge domain are derived. In another embodiment, the transmembrane domain is derived from a different protein from the protein from which the other domains of the CAR are derived. In some cases, the transmembrane domain may be selected or modified by amino acid substitutions that allow the chimeric receptor domain to avoid binding to transmembrane domains from the same or different surface membrane proteins, thereby minimizing interactions with other unintended members of the receptor complex. In certain embodiments, the transmembrane domain is encoded by a nucleic acid sequence encoding the CD28 transmembrane domain (SEQ ID NO: 33, 34, 35, or 36). In certain embodiments, the transmembrane domain comprises the amino acid sequence of the CD28 transmembrane domain (SEQ ID NO: 37, 38, or 39).

[0129] (iii-b-iv) Intracellular effector domain The intracellular effector domain of a CAR is responsible for the activation of the cell in which the CAR is expressed. Thus, the term "effector domain" is meant to include a portion of the intracellular domain sufficient to transduce an activation signal. The effector domain can directly or indirectly promote a biological or physiological response of a cell when it receives an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when the CAR binds to a target molecule, or it binds directly to the target molecule, resulting in a signal being induced from the effector domain. The effector domain may directly promote a cellular response if it contains one or more signaling domains or signaling motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs). In another embodiment, the effector domain promotes a cellular response indirectly by associating with one or more additional proteins that directly promote the cellular response, such as a costimulatory domain.

[0130] The effector domain can activate at least one function of the recombinant cell upon binding of the recombinant cell to a cell marker expressed by a cancer cell. Activation of the recombinant cell can include one or more of differentiation, proliferation, and / or activation or other effector functions. In certain embodiments, the effector domain can include an intracellular signaling moiety that includes a T cell receptor and a costimulatory domain, and the costimulatory domain can include a cytoplasmic sequence derived from a co-receptor or costimulatory molecule.

[0131] The effector domain may include one, two, or more intracellular signaling moieties (e.g., a signaling domain of a receptor or a cytoplasmic signaling sequence), a costimulatory domain, or a combination thereof. Exemplary effector domains include signaling and / or stimulatory domains selected from 4-1BB (CD137), CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ (FcεR1b), FcRγ, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, and any combination thereof.In certain embodiments, exemplary effector domains include CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. In certain embodiments, the effector domain comprises a CD3 zeta signaling domain.

[0132] The stimulatory intracellular signaling subsequence may comprise an iTAM. Examples of iTAMs that comprise a cytoplasmic major signaling sequence include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, common FcRγ (FCER1G), FcγRlla, FcRβ (Fcε Rib), DAP10 or DAP12. In certain embodiments, variants of CD3ζ retain at least one, two, three or all of the ITAM regions.

[0133] In certain embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, the cytoplasmic signaling protein being a lymphocyte receptor or signaling domain thereof, a protein containing multiple ITAMs, a costimulatory domain, or any combination thereof.

[0134] Further examples of intracellular signaling moieties include the intracytoplasmic sequences of the CD3 zeta chain and / or coreceptors which act in concert with this to initiate subsequent signaling upon engagement with the binding domain.

[0135] A costimulatory domain is a domain whose activation is required for efficient lymphocyte response to binding to a cell marker. Several molecules can be substituted as intracellular signaling moieties or costimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3, as well as ligands that specifically bind to CD83. For example, it has been demonstrated that costimulation of CD27 enhances the proliferation, effector function, and survival of human CAR T cells in vitro, and enhances the persistence and anticancer activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such costimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, and CDl lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In certain embodiments, the costimulatory domain comprises a 4-1BB signaling domain.

[0136] In certain embodiments, the nucleic acid sequence encoding the intracellular signaling moiety comprises a sequence encoding CD3ζ (SEQ ID NO: 22 or 23) and a sequence encoding a variant of the signaling moiety of 4-1BB (SEQ ID NO: 27, 28, or 29). In certain embodiments, the amino acid sequence of the intracellular signaling moiety comprises an intracellular signaling moiety that comprises a variant of CD3ζ (SEQ ID NO: 24, 25, or 26) and a portion of 4-1BB (SEQ ID NO: 30, 31, or 32).

[0137] In certain embodiments, the intracellular signaling moiety comprises (i) all or a portion of the signaling domain of CD3zeta, (ii) all or a portion of the signaling domain of 4-1BB, or (iii) all or a portion of the signaling domain of CD3zeta and 4-1BB. In certain embodiments, the intracellular signaling moiety comprises (i) all or a portion of the signaling domain of CD3zeta, (ii) all or a portion of the signaling domain of 4-1BB, (iii) all or a portion of the signaling domain of CD28, or (iv) all or a portion of the signaling domain of CD3zeta, 4-1BB, and CD28.

[0138] The intracellular portion may be a member of the Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), a NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), a Hedgehog signaling pathway (e.g., PTCH or SMO), a receptor tyrosine kinase (RTK) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomyosin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle-specific kinase (MuSK) receptor family); G protein-coupled receptors (GPCRs) (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0139] (iii-bv) Linker In this specification, a linker can be any part that connects two components in a CAR molecule.Some linkers only serve the purpose of connecting components, but many linkers serve other purposes besides connecting.For example, a linker can connect the VL and VH of an antibody-derived binding domain in scFv, and can function as a linking amino acid between the components of a CAR.

[0140] The linker may be flexible, rigid, or semi-rigid, depending on the desired function of the linker. The linker may further comprise a linking amino acid. For example, in certain embodiments, the linker provides flexibility and space for conformational movement between the various components of the CAR. A commonly used flexible linker is the Gly-Ser linker. In certain embodiments, the linker sequence comprises a repeat sequence of glycine and serine, for example, 1 to 10 (Gly x Ser y ) n In this case, x and y are independently an integer of 0 to 10, except when both x and y are 0, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. A specific example is a repeat sequence consisting of (Gly4Ser) n (SEQ ID NO: 147), (Gly3Ser) n (Gly4Ser) n (SEQ ID NO: 148), (Gly3Ser) n (Gly2Ser) n (SEQ ID NO: 149), or (Gly3Ser) nIn certain embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 151), (Gly4Ser)3 (SEQ ID NO: 152), (Gly4Ser)2 (SEQ ID NO: 153), (Gly4Ser)1 (SEQ ID NO: 154), (Gly3Ser)2 (SEQ ID NO: 155), (Gly3Ser)1 (SEQ ID NO: 156), (Gly2Ser)2 (SEQ ID NO: 157), (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 158), GGSGGGSGSG (SEQ ID NO: 159) or GGSGGGSG (SEQ ID NO: 160).

[0141] In certain embodiments, the linker region is (GGGGS) n (SEQ ID NO: 147), where n is an integer including 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In certain embodiments, the spacer is (EAAAK) n (SEQ ID NO: 161), where n is an integer including 1, 2, 3, 4, 5, 6, 7, 8, 9 or greater.

[0142] In some cases, flexible linkers may not be able to maintain the distance or position of the CAR required for a particular application. In such cases, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In certain embodiments, proline-rich linkers are peptide sequences that have more proline residues than would be expected to be included in the sequence by chance alone. In certain embodiments, proline-rich linkers are linkers whose sequence is at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50% or at least 51% full proline residues. Specific examples of proline-rich linkers include fragments of salivary proline-rich proteins (PRPs).

[0143] The linker may be susceptible to cleavage, such as acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, disulfide bond cleavage, etc. (cleavable linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker). In some embodiments, the linker is a charge-prone linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0144] The linking amino acid may be a linker that can be used to link sequences together when distance is not required and / or not desirable using a spacer. For example, the linking amino acid may be a short amino acid sequence that can be used to link intracellular costimulatory signaling moieties together. In certain embodiments, the linking amino acid is 9 amino acids or less in length (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length). In certain embodiments, a glycine-serine doublet can be used as a suitable linking amino acid linker. In certain embodiments, a single amino acid, such as, for example, alanine or glycine, can be used as a suitable linking amino acid.

[0145] (iii-b-vi) Control functions including tag cassettes, transduction markers and / or suicide switches In certain embodiments, the CAR construct may include one or more tag cassettes and / or transduction markers. The tag cassettes and / or transduction markers may be used to activate, promote growth, detect, enrich, isolate, track, remove and / or eliminate genetically modified cells in vitro, in vivo and / or ex vivo. A "tag cassette" refers to a unique synthetic peptide sequence that is attached, fused or incorporated as part of a CAR, and that can be used to activate, promote growth, detect, enrich, isolate, track, remove and / or eliminate tagged proteins and / or cells expressing the tagged proteins by specifically binding cognate binding molecules (e.g., ligands, antibodies or other binding partners). Transduction markers may also be used for the same purpose, but they are derived from natural molecules and are often expressed using skipping factors that can separate the transduction marker from other components in the CAR molecule.

[0146] Examples of tag cassettes that bind to cognate binding molecules include His tag (HHHHHH; SEQ ID NO: 162), Flag tag (DYKDDDDK; SEQ ID NO: 163), Xpress tag (DLYDDDDK; SEQ ID NO: 164), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 165), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 166), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 167), Myc tag (EQKLISEEDL; SEQ ID NO: 168), Strep tag (referring to the conventional STREP® tag (WRHPQFGG; SEQ ID NO: 169)), STREP® tag II (WSHPQFEK; SEQ ID NO: 170 (IBA Institut fur Bioanalytik, Germany); see, for example, U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 171), Softag 3 (TQDPSRVG; SEQ ID NO: 172), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 173).

[0147] Binding molecules that specifically bind to the tag cassette sequences disclosed herein to form complexes are commercially available. For example, His tag antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, GenScript, etc. Flag tag antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, Sigma-Aldrich, etc. Xpress tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, GenScript, etc. Avi tag antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, Genecopoeia, etc. Calmodulin tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, Pierce Antibodies, etc. HA tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, Abcam, etc. Myc tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, Cell Signal, etc. Strep tag antibodies are commercially available from manufacturers such as Abcam, Iba, Qiagen, etc.

[0148] The transduction marker may be selected from at least one of truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); the ECD of human CD34; and / or RQR8, which combines a target epitope from the CD34 antigen (see Fehse et al, Mol. Therapy 1(5 Pt 1);448-456, 2000) and a target epitope from the CD20 antigen (see Philip et al, Blood 124: 1277-1278). In certain embodiments, the cells are genetically engineered to express EGFRt.

[0149] In certain embodiments, the CAR construct can include a polynucleotide encoding a self-cleaving polypeptide, which is disposed between the polynucleotide encoding the CAR construct and the polynucleotide encoding the transduction marker (e.g., EGFRt). Exemplary self-cleaving polypeptides include the 2A peptide (P2A) from porcine teschovirus-1, the 2A peptide (T2A) from Thosea asigna virus, the 2A peptide (E2A) from equine rhinitis A virus, the 2A peptide (F2A) from foot and mouth disease virus, or variants thereof. In addition, exemplary nucleic acid and amino acid sequences of 2A peptides are also described, for example, in Kim et al. (PLOS One 6: e18556 (2011)). In certain embodiments, the cell is genetically modified to include the self-cleaving polypeptide. In certain embodiments, the self-cleaving polypeptide comprises T2A.

[0150] The control function may be included in the CAR in multiple copies or expressed as a separate molecule using skipping factors. For example, the CAR may have one, two, three, four or five tag cassettes and / or one, two, three, four or five transduction markers may be expressed. For example, embodiments of the present disclosure may include a CAR construct with two Myc tag cassettes, a cassette containing a His tag and an HA tag, a cassette containing a HA tag and a Softag1 tag, or a cassette containing a Myc tag and an SBP tag. Exemplary transduction markers and cognate pairs are described in US Patent Publication No. 13 / 463,247.

[0151] One advantage of incorporating at least one regulatory function into a CAR is that CAR-expressing cells administered to a subject can be expanded or removed by using a binding molecule cognate to the tag cassette. In certain embodiments, the present disclosure provides a method for removing recombinant cells expressing a CAR by using an antibody specific for the tag cassette, by using a cognate binding molecule specific for the regulatory function, or by using a second CAR-expressing recombinant cell with specificity for the regulatory function. Removal of recombinant cells may be performed using a removal agent specific for the regulatory function. For example, when using EGFRt, an anti-EGFRt binding domain (e.g., an antibody or scFv) fused or conjugated to a cytotoxic reagent (e.g., a toxin or a radioactive metal) may be used, or an anti-EGFRt / anti-CD3 bispecific scFv or anti-EGFRt CAR T cells may be used.

[0152] In certain embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into the CAR construct as a suicide switch.

[0153] In certain embodiments, recombinant cells expressing a CAR may be detected or tracked in vivo by using an antibody (e.g., an anti-tag antibody) that specifically binds to a regulatory function, or by other cognate binding molecules that specifically bind to a regulatory function, where the binding partner of the regulatory function is conjugated to a fluorescent dye, radioactive tracer, iron oxide nanoparticle, or other imaging agent known in the art such that it can be detected by x-ray, CT scan, MRI scan, PET scan, ultrasound, flow cytometry, near-infrared imaging systems, or other imaging methods (see, e.g., Yu, et al., Theranostics 2:3, 2012).

[0154] Thus, recombinant cells expressing at least one regulatory function and a CAR can, for example, be more easily identified, isolated, sorted, induced to grow, tracked, and / or removed than recombinant cells that do not contain the tag cassette.

[0155] (iii-b-vii) Multimerization domain

[0156] In certain embodiments, CAR may contain a multimerization domain. The biological activity of a protein depends on its tertiary and quaternary structure. Quaternary structure requires physical and chemical interaction with another protein subunit or polypeptide. A "multimerization domain" is a domain that allows two or more proteins (monomers) to interact with each other through covalent and / or non-covalent bonds. The presence of a multimerization domain in a protein allows protein interaction to occur to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0157] In certain embodiments, the multimerization domain is a dimerization domain that allows the formation of a dimer by binding two complementary monomers together. In certain embodiments, the complementary monomers include PRKAR1A and PRKAR1A (SEQ ID NO: 87 and SEQ ID NO: 88), PRKAR1B and PRKAR1B (SEQ ID NO: 89), PRKAR1R (SEQ ID NO: 90 and SEQ ID NO: 91), and PRKAR1E (SEQ ID NO: 92). In certain embodiments, the dimerization docking domain (DDD) may be derived from a cAMP-dependent protein kinase (PKA) regulatory subunit and may pair with an anchor domain (AD). The anchor domain may be derived from a specific region of various A-kinase anchor proteins (AKAPs) that mediate the association with the R subunit of PKA. In certain embodiments, the complementary monomers include DDD (SEQ ID NO: 93 and SEQ ID NO: 94) and AD (SEQ ID NO: 95 and SEQ ID NO: 96). Those skilled in the art will recognize that other DDDs and ADs are known and can be used, including the four-helix bundle DDD domain from p53 (Newlon, et al. EMBO J. 2001; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227), DCoH (pterin-4-α-carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1α (TCF1)), and HNF-1 (hepatocyte nuclear factor 1) (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). Other AD sequences that may be used include those described in U.S. Patent Publication No. 2003 / 0232420(A1).

[0158] In certain embodiments, the complementary binding domains can dimerize. In certain embodiments, the binding domain is a transmembrane polypeptide derived from the FcεRI chain. In certain embodiments, one CAR comprises a portion of the FcεRIα chain and the other CAR comprises a portion of the FcεRIβ chain, so that the FcεRI chains can spontaneously dimerize to form a dimeric CAR. In certain embodiments, the CAR comprises a portion of the FcεRIα chain and a portion of the FcεRIγ chain, so that the FcεRI chains can spontaneously trimerize to form a trimeric CAR. In another embodiment, the multi-chain CAR comprises a portion of the FcεRIα chain, a portion of the FcεRIβ chain, and a portion of the FcεRIγ chain, so that the FcεRI chains can spontaneously tetramerize to form a tetrameric CAR.

[0159] In certain embodiments, the complementary binding domains may be derived from a binding event such as the binding between an enzyme and its substrate / inhibitor, for example, between a cutinase and a phosphonate (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052). Such complementary binding domains may be used to generate two associating moieties (the "docking" step) which may then be stabilized by covalent bonds (the "locking" step).

[0160] In certain embodiments, the binding domain may be derived from binding events such as binding between dimeric pairs of receptors, including interleukin 8 receptor (IL-8R); integrin heterodimers such as LFA-I and GPIIIb / IIIa; dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin 3 (NT-3), interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, members of the PDGFs, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993); and affinity-modified variants of these domains, such as those described in WO2012001647(A2).

[0161] In certain embodiments, the dimerization domain may comprise a protein sequence motif, such as a coiled coil, an acid patch, a zinc finger, a calcium hand, a CH1-CL pair, a recombinantly generated "interface" with "knobs" and / or "protrusions" (U.S. Patent Publication No. 5,821,333), a leucine zipper (U.S. Patent Publication No. 5,932,448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336-44, 2004), PTB (Zhou et al., Nature, 378:584-592, 1995), WW (Sudol Prog Biochys MoL Bio, 65:113-132, 1996), PDZ (Kim et al., Nature, 378: 85-88, 1995; Komau et al., Science, 269:1737-1740, 1995), and WD40 (Hu et al., J. Biol. Chem., 273:33489-33494, 1998).

[0162] In certain embodiments, sequences corresponding to the dimerization domain include the leucine zipper domain of Jun (SEQ ID NO: 97), the dimerization domain of Fos (SEQ ID NO: 98), the consensus sequence of the WW motif (SEQ ID NO: 99), the dimerization domain of the SH2B adaptor protein available under GenBank Accession No. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621, 2005; SEQ ID NO: 100), the SH3 domain of IB1 available under GenBank Accession No. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006; SEQ ID NO: 101), the PTB domain of human DOK-7 available under GenBank Accession No. NP_005535.1 (Wagner et al., Cold Spring Harb Perspect Biol. 5: a008987, 2013; SEQ ID NO: 102), the PDZ-like domain of SATB1 available under UniProt accession number Q01826 (Galande et al., Mol Cell Biol. Aug; 21: 5591-5604, 2001; SEQ ID NO: 103), the WD40 repeats of APAF available under UniProt accession number O14727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463; SEQ ID NO: 104), the PAS motif of the dioxin receptor available under UniProt accession number I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079-4088, 1998; SEQ ID NO: 105), and the EF-hand structure of parvalbumin available under UniProt accession number P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014; sequence number 106).

[0163] In certain embodiments, the complementary binding domains can be induced using a third molecule or chemical inducer. This dimerization method requires that the first CAR contains the dimerization binding domain 1 (CBD1) of a chemical inducer, and the second CAR contains the second dimerization binding domain (CBD2) of said chemical inducer, and these CBD1 and CBD2 can bind simultaneously to a chemical inducer (CID) that induces dimerization. CBD1 can contain the rapamycin binding domain (SEQ ID NO: 107) of FK binding protein 12 (FKBP12), and CBD2 can contain the FKBP12-rapamycin binding (FRB) domain (SEQ ID NO: 108) of mTOR. In this case, the CID can contain rapamycin or a derivative thereof, and CBD1 and CBD2 can be heterodimerized by rapamycin or a derivative thereof. When CBD1 and CBD2 are the FK506 (tacrolimus) binding domain of FKBP12 and the cyclosporine binding domain of cyclophilin A, the CID may comprise an FK506 / cyclosporine fusion protein. When CBD1 and CBD2 are the FKBP12 binding domain containing the F36V mutation, the CID may be AP1903. When CBD1 and CBD2 are the estrogen binding domain (EBD) and the streptavidin binding domain, the CID may be an estrone / biotin fusion protein. When CBD1 and CBD2 are the glucocorticoid binding domain (GBD) and the dihydrofolate reductase (DHFR) binding domain, the CID may be a dexamethasone / methotrexate fusion molecule. When CBD1 and CBD2 are the O 6 -alkylguanine-DNA alkyltransferase (AGT) binding domain and DHFR binding domain, the CID is 6-benzylguanine derivative / methotrexate fusion molecule. When CBD1 and CBD2 are retinoic acid receptor domain and ecdysone receptor domain, CID may contain RSL1. Also, affinity to CID can be modified using CID binding domain. For example, binding to rapamycin can be modified by modifying amino acids 2095, 2098 and 2101 of FRB (Bayle et al, Chemistry & Biology 13, 99-107, 2006).

[0164] Furthermore, a C4b multimerization domain can also be used. Specific C4b multimerization domains that can be used in the present disclosure are shown in SEQ ID NOs: 109-141. In certain embodiments, the C4b multimerization domain is a multimerization domain that includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucine at positions 29, 34, 36, and / or 41; (iv) tyrosine at position 32; (v) lysine at position 33; and / or (vi) cysteines at positions 6 and 18. In certain embodiments, the C4b multimerization domain is a multimerization domain that includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucine at positions 29, 34, 36, and 41; (iv) tyrosine at position 32; (v) lysine at position 33; and (vi) cysteines at positions 6 and 18.

[0165] The C4b multimerization domain may comprise any of SEQ ID NOs: 109-141 with a deletion of at least one consecutive amino acid residue at the N-terminus (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive residues). Further embodiments may comprise a deletion of at least one consecutive amino acid residue at the C-terminus (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive residues).

[0166] Particular embodiments of the C4b multimerization domain include, or are modified to include, at least one of residues A6; E11; A13; D21; C22; P25; A27; E28; L29; R30; T31; L32; L33; E34; I35; K37; L38; L40; E41; I42; Q43; K44; L45; E48; L49; and Q50. In further embodiments, it comprises or is modified to comprise residues A6; E11; A13; D21; C22; P25; A27; E28; L29; R30; T31; L32; L33; E34; I35; K37; L38; L40; E41; I42; Q43; K44; L45; E48; L49; and Q50. A particular C4b multimerization domain embodiment comprises the amino acid sequence "AELR".

[0167] In certain embodiments, ferritin-based dextramer multimerization can be utilized. Exemplary ferritin fusion sequences are described in PMID 26279189.

[0168] In certain embodiments, further methods of causing dimerization can be utilized. Further modifications to form dimerization domains in CARs may include the introduction of a second cysteine ​​residue in both CARs to form a second interchain disulfide bond in the C-terminal domain; exchange of interacting residues in the C-terminal domain of each CAR construct ("knob-in-hole"); and direct fusion of the variable domain of CAR to CD3ζ (CD3ζ fusion) (Schmitt et al., Hum. Gene Ther. 2009. 20:1240-1248).

[0169] (iv) Characterization of genetically modified cells In certain embodiments, the genetically modified cells can be assessed for cell surface expression of the CAR, in certain embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the genetically modified cells express detectable levels of the CAR.

[0170] Cell surface protein expression can be measured by flow cytometry using methods known in the art, in which a cell population is labeled with an agent (e.g., an antibody) that is tagged with a fluorescent molecule and targets the desired cell surface marker, thereby quantifying the cell population positive for that cell surface marker and quantifying the amount of expression of that cell surface marker.

[0171] The integration of CAR into the genome of the engineered cells can be measured by digital droplet PCR (ddPCR). Digital PCR can quantify the DNA concentration in a sample. In digital PCR, a PCR reaction mixture (e.g., containing a nucleic acid molecule sample and multiple copies of a PCR probe) is divided into reaction compartments, some of which contain no PCR probes and some contain one or more copies of the PCR probe. Each compartment is amplified by PCR and analyzed by a PCR reaction. Compartments containing one or more copies of the probe and one or more copies of the target DNA molecule yield positive endpoints, whereas compartments containing no PCR probe yield negative endpoints. The positive reaction compartments are then fitted to a Poisson distribution to determine the absolute copy number of the target DNA molecule per volume of the sample before division (i.e., copies per μl of sample) (see Hindson, B. et al., (2011) Anal Chem. 83:8604-8610). Digital droplet PCR is a variant of digital PCR characterized by the partitioning of nucleic acid samples into droplets using a water-in-oil emulsion. PCR amplification is performed on the droplets in bulk, and a fluidics system is used to dispense and analyze the droplets individually. Those skilled in the art can use ddPCR to perform absolute quantification of DNA in a sample to perform copy number variation analysis or to evaluate the efficiency of genome editing.

[0172] The recombinant cells can also be assessed for cytokine-independent growth. The recombinant cells are expected to grow only in the presence of stimulatory cytokines (e.g., IL-2 and IL-7). Growth in the absence of cytokines is indicative of tumorigenicity. In certain embodiments, the recombinant cells are grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days in the presence or absence of one or more stimulatory cytokines (e.g., IL-2 and IL-7). In certain embodiments, growth is assessed by cell number and viability using conventional methods (e.g., flow cytometry, microscopy, optical density, metabolic activity). In certain embodiments, growth is assessed beginning on day 1, 2, 3, 4, 5, or 6. In certain embodiments, growth is evaluated every 1, 2, 3, 4, 5, 6, 7, or 8 days. In certain embodiments, growth is evaluated in the absence of cytokines at the end of the growth phase. In some specific embodiments, no tumorigenicity is defined when no growth of recombinant cells is observed in the absence of cytokines. In certain embodiments, no growth is defined when the cell population at the end of the growth phase grows less than 0.1-fold, less than 0.2-fold, less than 0.3-fold, less than 0.4-fold, less than 0.5-fold, less than 0.6-fold, less than 0.7-fold, less than 0.8-fold, less than 0.9-fold, less than 1.0-fold, less than 1.1-fold, less than 1.2-fold, less than 1.3-fold, less than 1.4-fold, or less than 1.5-fold at the beginning of the growth phase. In certain embodiments, recombinant cells do not grow in the absence of cytokine stimulation, growth factor stimulation, and antigen stimulation.

[0173] (v)Cell activation culture conditions A population of cells can be expanded by incubating in a culture starter composition. Incubation can be performed in a culture vessel, such as a bag, cell culture plate, flask, chamber, chromatography column, crosslinked gel, crosslinked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tube set, well, vial, or other culture or cell culture vessel.

[0174] In certain embodiments, the cell population can be incubated in the culture initiation composition before or after genetic recombination. In certain embodiments, the incubation of the cell population can be performed for 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, or 1 day before genetic recombination. In certain embodiments, the incubation of the cell population can be performed for 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, or 1 day after genetic recombination. In certain embodiments, the incubation of the cell population can be performed simultaneously with genetic recombination.

[0175] Culture conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, and agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors (e.g., cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and other agents designed to activate cells)).

[0176] In some embodiments, the incubation is carried out according to techniques such as those described in U.S. Patent Publication No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0177] Exemplary culture media for culturing T cells include: (i) RPMI supplemented with nonessential amino acids, sodium pyruvate, and penicillin / streptomycin; (ii) HEPES, 5–15% human serum, 1–3% L-glutamine, 0.5–1.5% penicillin / streptomycin, and 0.25 × 10 -4 ~0.75×10 -4 M RPMI supplemented with β-mercaptoethanol; (iii) RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin, and 100 m / ml streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U / ml penicillin, and 100 μg / mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum (Gemcell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% penicillin / streptomycin (Gibco), 1% GlutaMax (Gibco), and 2% N-acetylcysteine ​​(Sigma-Aldrich, St. Louis, MO). Examples include: Additionally, T cell culture media is also available commercially from HyClone (Logan, Utah). Additional T cell activation components that can be added to the above culture media are described in more detail below.

[0178] In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starter composition (e.g., at a ratio of at least 5, 10, 20, or 40 or more PBMC feeder cells per T lymphocyte in the cell population prior to expansion) and then incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some embodiments, the non-dividing feeder cells may comprise γ-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with 3000-3600 rads of γ-irradiation to prevent cell division. In some embodiments, the feeder cells are added to the culture medium prior to adding the T cell population.

[0179] The incubation of T cells may further include a step of adding EBV-transformed non-dividing lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with 6000 to 10,000 rad of gamma rays. In some embodiments, the LCL feeder cells are provided in an appropriate amount, for example, such that the ratio of LCL feeder cells to T lymphocytes at the start of proliferation is at least 10:1.

[0180] In some embodiments, the stimulatory conditions include a temperature suitable for proliferation of human T lymphocytes, which may be, for example, at least 25°C, at least 30°C, or at least 37°C.

[0181] The T cell activation culture conditions include conditions under which the T cells in the culture initiation composition proliferate or expand. The T cell activation conditions may include one or more cytokines, for example, interleukin (IL)-2, IL-7, IL-15, and / or IL-21. The IL-2 content may be in the range of 10 to 100 ng / ml (e.g., 40 ng / ml, 50 ng / ml, or 60 ng / ml). The IL-7, IL-15, and / or IL-21 may each be in the range of 0.1 to 50 ng / ml (e.g., 5 ng / ml, 10 ng / ml, or 15 ng / ml). In a particular embodiment, 50 ng / ml of IL-2 is used. In a particular embodiment, 10 ng / ml of IL-7, IL-15, and IL-21 are each used.

[0182] In certain embodiments, the T cell activating culture conditions may include epitopes that stimulate T cells, including CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1BB (CD 137), B7-H3, CTLA-4, Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1R, LAT, LFA-1, LIGHT, MHCI, MHCII, NKG2D, OX40, ROR2, and RTK.

[0183] CD3 is the main signal transduction factor of the T cell receptor. As mentioned above, CD3 is expressed on all mature T cells. In a specific embodiment, the molecule that stimulates CD3 (i.e., the CD3 binding domain) is the OKT3 antibody (U.S. Patent Publication No. 5,929,212; U.S. Patent Publication No. 4,361,549; ATCC® CRL-8001). TM; and Arakawa et al., J. Biochem. 120, 657-662 (1996)), 20G6-F3 antibody, 4B4-D7 antibody, 4E7-C9 antibody, or 18F5-H10 antibody.

[0184] In certain embodiments, the CD3-stimulating molecule may be included in the culture medium at a concentration of at least 0.25 ng / ml or 0.5 ng / ml, or at a concentration of 2.5-10 μg / ml. In certain embodiments, the CD3-stimulating molecule (e.g., OKT3) is used at a concentration of 5 μg / ml.

[0185] In certain embodiments, activation molecules associated with an avi tag can be biotinylated and bound to streptavidin beads, for example, to construct a removable T cell epitope stimulated activation system.

[0186] Exemplary domains that bind to CD28 include TGN1412, CD80, CD86 or 9D7 antibodies, or derivatives thereof. Antibodies that bind to CD28 further include 9.3, KOLT-2, 15E8, 248.23.2, EX5.3D10, and CD28.3 antibodies (deposited as synthetic single-chain Fv constructs under GenBank accession number: AF451974.1; see also Vanhove et al., BLOOD, 15 Jul. 2003, Vol. 102, No. 2, pages 564-570). In addition, there is 1YJD, which is the crystal structure of human CD28 complexed with the Fab fragment of mitogenic antibody (5.11A1). In certain embodiments, an antibody that does not compete with 9D7 antibody is selected.

[0187] The 4-1BB binding domain can be derived from LOB12, IgG2a, LOB12.3 or IgG1, as described in Taraban et al. Eur J Immunol. 2002 December; 32(12):3617-27. In certain embodiments, the 4-1BB binding domain is derived from a monoclonal antibody described in U.S. Patent Publication No. 9,382,328. Other 4-1BB binding domains are described in U.S. Patent Publication No. 6,569,997, U.S. Patent Publication No. 6,303,121, and Mittler et al. Immunol Res. 2004; 29(1-3):197-208.

[0188] OX40 (CD134) and / or ICOS activation may be utilized.OX40 binding domains are described in US Patent Publication No. 20100196359, US Patent Publication No. 20150307617, WO 2015 / 153513, WO2013 / 038191, and Melero et al. Clin Cancer Res. 2013 Mar. 1; 19(5):1044-53.Exemplary binding domains that can bind and activate ICOS are described, for example, in US Patent Publication No. 20080279851, and Deng et al. Hybrid Hybridomics. 2004 June; 23(3):176-82.

[0189] If the T cell activator is soluble, it can be conjugated to another molecule, such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used. Typically, PEG molecules with a molecular weight of 1000 Da or less can be dissolved in water or culture medium. In some cases, such PEG-based reagents can be prepared using commercially available activated PEG molecules (e.g., PEG-NHS derivatives available from NOF North America Corporation, Irvine, Calif., USA, or activated PEG derivatives available from Creative PEGWorks, Chapel Hill, North Carolina, USA).

[0190] In certain embodiments, the cell stimulant is immobilized on a solid phase in the culture medium. In certain embodiments, the solid phase is a surface of a culture vessel (e.g., a bag, a cell culture plate, a chamber, a chromatography column, a cross-linked gel, a cross-linked polymer, a column, a culture dish, a hollow fiber, a microtiter plate, a silica-coated glass plate, a tube, a tube set, a well, a vial, or the surface of other structures or vessels for cells or cell culture).

[0191] In certain embodiments, solid phases can be added to the culture medium, such as, for example, beads, hollow fibers, resins, membranes, and polymers.

[0192] Exemplary beads include magnetic, polymeric and resin beads (e.g., Strep-Tactin® Sepharose, Strep-Tactin Superflow, and Strep-Tactin MacroPrep (IBA, Gottingen)). Anti-CD3 / anti-CD28 beads are commercially available as T cell proliferation reagents (Invitrogen). Anti-CD3 / anti-CD28 beads are 4.5 μm uniformly sized superparamagnetic non-pyrogenic sterile polystyrene beads coated with a mixture of affinity purified monoclonal antibodies against the CD3 and CD28 cell surface molecules on human T cells. Hollow fibers are available from TerumoBCT (Lakewood, Colorado, USA). Resins include resins for metal affinity chromatography (IMAC), such as TALON® resins (Westburg, Leusden). Membranes include papers and membrane substrates of chromatography matrices, such as nitrocellulose membranes or polyvinylidene fluoride (PVDF) membranes.

[0193] Exemplary polymers include polysaccharides, such as polysaccharide matrices. Such matrices include agarose gels (e.g., Superflow TM Agarose or Sepharose® materials, such as Superflow, which are commercially available in a variety of bead sizes and pore sizes. TM Sepharose), or cross-linked dextran gels. Further examples include particulate cross-linked agarose matrices to which dextran is covalently attached, which are commercially available (in various bead sizes and with various pore sizes) and are available from GE Healthcare as Sephadex® or Superdex®.

[0194] Synthetic polymers that may be used include polyacrylamides, polymethacrylates, copolymers of polysaccharides and agarose (e.g., polyacrylamide / agarose complexes), or copolymers of polysaccharides and N,N'-methylenebisacrylamide. Examples of copolymers of dextran and N,N'-methylenebisacrylamide include the Sephacryl® series of materials (Pharmacia Fine Chemicals, Piscataway, NJ).

[0195] In certain embodiments, particulate silica bound to synthetic polymers or particulate silica bound to natural polymers may be used, such as polysaccharide-grafted silica, polyvinylpyrrolidone-grafted silica, polyethylene oxide-grafted silica, poly(2-hydroxyethylaspartamide) silica, and poly(N-isopropylacrylamide)-grafted silica.

[0196] The cell activator can be immobilized on the solid phase via a covalent bond or can be reversibly immobilized on the solid phase via a non-covalent bond.

[0197] In a specific embodiment, the T cell activation culture medium contains HEPES, 5-15% human serum, 1-3% L-glutamine, 0.5-1.5% penicillin / streptomycin, 0.25×10 -4 ~0.75×10 -4 The T cell populations are cultured in RPMI supplemented with 0.1-0.5×10 β-mercaptoethanol, and IL-7, IL-15, and IL-21 at concentrations of 5-15 ng / ml (e.g., 10 ng / ml), respectively. Cultures are performed in flat-bottom well plates with 0.1-0.5×10 6 Cells are seeded at a density of 100 / well. On day 3 after activation, cells are transferred to tissue culture treated plates.

[0198] In a specific embodiment, the T cell activation culture medium contains HEPES, 10% human serum, 2% L-glutamine, 1% penicillin / streptomycin, 0.5×10 -4 The CD8+ T cell populations were cultured in RPMI supplemented with M β-mercaptoethanol, and IL-7, IL-15, and IL-21 at concentrations of 5-15 ng / ml (e.g., 10 ng / ml) each. Cultures were performed in non-tissue culture treated 96 / 48 well plates with 0.1-0.5 × 10 6 Cells are seeded at a density of 100 / well. On day 3 after activation, cells are transferred to tissue culture treated plates. Culture conditions for HSC / HSP may include proliferation with the addition of a Notch agonist (see, e.g., U.S. Patent Publication Nos. 7,399,633; 5,780,300; 5,648,464; 5,849,869; and 5,856,441), in which the growth factors are 25-300 ng / ml SCF, 25-300 ng / ml Flt-3L, 25-100 ng / ml TPO, 25-100 ng / ml IL-6, and 10 ng / ml IL-3. In more specific embodiments, 50ng / ml, 100ng / ml or 200ng / ml of SCF; 50ng / ml, 100ng / ml or 200ng / ml of Flt-3L; 50ng / ml or 100ng / ml of TPO; 50ng / ml or 100ng / ml of IL-6; and 10ng / ml of IL-3 may be used.

[0199] (vi) Ex vivo produced cell preparations In certain embodiments, the genetically modified cells can be harvested from the culture medium, washed, concentrated, and mixed with a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, saline, water, Hank's solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Morton Grove, Ill.), glycerol, ethanol, and combinations thereof.

[0200] In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum can be added to the carrier. In certain embodiments, the carrier for infusion comprises buffered saline with 5% HSA or dextrose. Other tonicity agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, and the like.

[0201] The carrier may contain a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer and / or a trimethylamine salt.

[0202] Stabilizers refer to a wide variety of additives that can have various functions, from additives that can prevent cell adhesion to the walls of a container to bulking agents. Typical stabilizers include polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, and cyclitols (e.g., inositol); PEG; amino acid polymers; urea, glutathione, thioctophosphate, and the like. sulfur-containing reducing agents such as acids, sodium thioglycolate, thioglycerol, α-monothioglycerol, sodium thiosulfate; low molecular weight polypeptides (i.e., polypeptides having less than 10 residues); proteins such as HSA, bovine serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran.

[0203] Where necessary or beneficial, the formulation may include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0204] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides, hexamethonium chloride, alkylparabens (e.g., methylparaben and propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0205] The therapeutically effective amount of cells contained in the formulation is 10 2 More than 10 3 More than 10 4 More than 10 5 More than 10 6 More than 10 7 More than 10 8 More than 10 9 More than 10 10 More than 10 11 The number may be more than one.

[0206] In the formulations disclosed herein, the cells are typically contained in a volume of 1 L or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the density of cells administered is typically less than 10 4 Density >10 7 Density greater than 10 8 The density exceeds 50 / ml.

[0207] As discussed above, the preparation may include at least one type of genetically modified cell (e.g., a recombinant T cell, a recombinant NK cell, or a recombinant stem cell), or the preparation may include different types of genetically modified cells (e.g., a combination of T cells, NK cells, and / or stem cells).

[0208] Different types of genetically modified cells or cell subsets (e.g., recombinant T cells, recombinant NK cells and / or recombinant stem cells) can be provided in various ratios, for example, 1:1:1 ratio, 2:1:1 ratio, 1:2:1 ratio, 1:1:2 ratio, 5:1:1 ratio, 1:5:1 ratio, 1:1:5 ratio, 10:1:1 ratio, 1:10:1 ratio, 1:1:10 ratio, 2:2:1 ratio, 1:2:2 ratio, 2:1:2 ratio, 5:5:1 ratio, 1:5:5 ratio, 5:1:5 ratio, 10:10:1 ratio, 1:10:10 ratio, 10:1:10 ratio, etc. These ratios can also be applied to the number of cells expressing the same or different CAR components. When only two types of cells are combined, or when only two types of CAR components are included and expressed in the formulation, these ratios may be any combination of two numbers that can be made from the combination of the three numbers above. In some embodiments, the combined cell populations are tested in vitro, in vivo, and / or ex vivo for efficacy and / or cell proliferation, and a ratio of cells that provides efficacy and / or cell proliferation is selected. Particular embodiments include a 1:1 ratio of CD4 T cells to CD8 T cells.

[0209] The cell-based formulations disclosed herein can be prepared for administration, for example, by injection, infusion, perfusion or lavage. The formulations can be further formulated for bone marrow injection, intravenous injection, intradermal injection, intraarterial injection, intralymph node injection, intralymphatic injection, intraperitoneal injection, intralesional injection, intratumoral injection, intravesical injection and / or subcutaneous injection.

[0210] (vii) Targeted viral vectors and nanoparticles for modifying cells in vivo Targeted viral vectors and / or nanoparticles can also be used to genetically modify immune cells in vivo or ex vivo. Viral vectors that can be used to deliver a gene encoding a CAR to cells are described elsewhere herein, and many targeted viral vectors (e.g., pseudotyped viral vectors) are known in the art.

[0211] Exemplary cell-targeting nanoparticles include nanoparticles with cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) attached to their surface that are selectively taken up by a selected cell type due to the cell-targeting ligand attached to their surface, and then the nanoparticles deliver the genetically engineered components to express the CAR.

[0212] Exemplary nanoparticles include liposomes (tiny vesicles with at least one lipid bilayer surrounding an aqueous core forming a concentric sphere), liposomal nanoparticles (liposomal structures used to encapsulate smaller nanoparticles within their cores), and lipid nanoparticles (liposome-like structures that do not have the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles can also be used, as well as porous nanoparticles composed of materials capable of forming a porous network. Exemplary materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0213] Nanoparticles for in vivo delivery and cellular uptake may have an uncharged or negatively charged coating, and the size of the nanoparticles may be 130 nm or less. The size of the nanoparticles may be measured using conventional techniques, such as, for example, dynamic light scattering and / or electron microscopy. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110 and WO201822672.

[0214] The therapeutically effective amount of the vector and / or nanoparticles contained in the formulation may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dose may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg or 0.5-1 mg / kg. In another example, the dose may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0215] (viii) How to use The methods disclosed herein include treating a subject (human, companion animal (dog, cat, reptile, bird, etc.), livestock (horse, cow, goat, pig, chicken, etc.), or research animal (monkey, rat, mouse, fish, etc.)) with the formulations disclosed herein. Treating a subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes an amount that can provide an effective amount, prophylactic treatment, and / or therapeutic treatment.

[0216] "Effective amount" refers to the amount of formulation required to cause a desired physiological change in a subject. For example, an effective amount can provide an immunogenic anti-cancer effect. Effective amounts are often administered for research purposes. The effective amount disclosed herein is an amount that can cause a statistically significant effect in an animal model or in vitro assay related to the evaluation of the development or progression of cancer. The immunogenic formulation can be provided in an effective amount, which stimulates an immune response.

[0217] "Preventive treatment" includes treatment for subjects who do not show signs or symptoms of cancer or who show only early signs or symptoms of cancer, with the aim of reducing or reducing the risk of the cancer developing further. Thus, preventive treatment serves as a treatment to prevent STEAP1 expressing cancer. In certain embodiments, preventive treatment inhibits, delays or prevents the development of metastasis from the primary cancer tumor site.

[0218] "Therapeutic treatment" includes treatment administered to a subject exhibiting symptoms or signs of cancer with the intent of reducing or eliminating the signs or symptoms of cancer. Therapeutic treatment may prevent, control, or eliminate the presence or activity of cancer and / or may prevent, control, or eliminate the side effects of cancer.

[0219] The terms effective amount, functioning as a prophylactic treatment or a therapeutic treatment are not mutually exclusive, and in certain embodiments, an administered dose may accomplish more than one treatment.

[0220] In certain embodiments, the therapeutically effective amount can provide anti-cancer effects. Anti-cancer effects include reducing the number of cancer cells, reducing the number of metastases, reducing tumor volume, increasing life expectancy, inducing chemotherapy sensitivity or radiosensitivity in cancer cells, inhibiting angiogenesis in the vicinity of cancer cells, inhibiting the proliferation of cancer cells, inhibiting the proliferation of tumors, preventing or reducing metastasis, increasing the life span of subjects, inhibiting cancer-related pain, and / or reducing relapse or recurrence of cancer after treatment. In certain embodiments, the therapeutically effective amount can provide anti-cancer effects in the presence of low antigen density.

[0221] A "tumor" is a swelling or lesion formed by the abnormal proliferation of cells (called neoplastic or neoplastic cells). "Tumor cells" are abnormal cells that grow by rapid and unregulated cellular proliferation and continue to grow even after the stimuli that initiated new proliferation have ceased. A tumor is one in which the structural organization and functional coordination of normal tissue is partially or completely lost, usually forming a identifiable mass of tissue, which may be benign, premalignant, or malignant.

[0222] In certain embodiments, the therapeutically effective amount induces an immune response, which may be directed against cancer cells that express STEAP1.

[0223] "STEAP1 positive cells" refer to cells that express STEAP1 on their surface. "STEAP1 positive cancer cells" refer to cancer cells that express STEAP1 on their surface. In some embodiments, the expression of STEAP1 on the cell surface is measured by using an antibody against STEAP1 in a method such as, for example, immunohistochemical analysis or FACS. Alternatively, the expression of STEAP1 mRNA is believed to correlate with the expression of STEAP1 on the cell surface, and the expression of STEAP1 mRNA can be measured, for example, by in situ hybridization and / or RT-PCR (including quantitative RT-PCR).

[0224] Examples of STEAP1-associated diseases that can be treated with the CARs disclosed herein include prostate cancer (e.g., castration-resistant prostate cancer), Ewing's sarcoma family of tumors (including Ewing's sarcoma), bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, and kidney cancer.

[0225] As mentioned above, the CAR disclosed herein can be used to treat a subject with a cancer that has low antigen density. In a particular example, the STEAP1 antigen expression level of the cancer in the subject is evaluated, and the CAR disclosed herein is selected for use in treating the subject based on the low antigen density.

[0226] CAR disclosed herein is not limited to the treatment of the subject with cancer in low antigen density, but can also be used for the subject with cancer in high antigen density.High antigen density includes the subject with STEAP1 molecule per lesion cell more than 50,000; the subject with STEAP1 molecule per lesion cell more than 60,000; the subject with STEAP1 molecule per lesion cell more than 70,000; the subject with STEAP1 molecule per lesion cell more than 80,000; the subject with STEAP1 molecule per lesion cell more than 90,000; or the subject with STEAP1 molecule per lesion cell more than 100,000.

[0227] When administered, the therapeutically effective amount (also referred to herein as "dosage") can be estimated initially based on the results of in vitro assays and / or animal model studies. Such information can be used to more accurately determine the dosage that is useful for the intended subject. The actual dosage administered to a particular subject can be determined by a physician, veterinarian or researcher, taking into account parameters such as physical and physiological factors, such as target, body weight, disease severity, cancer type, cancer stage, previous or concurrent therapeutic interventions, subject's idiopathic disease and route of administration.

[0228] The therapeutically effective dose of the cell-based formulation is 10 4 ~10 9 pcs / kg body weight or 10 3 ~10 11 The therapeutically effective amount to be administered may be 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells or11 Examples include cells having more than one cell.

[0229] The therapeutically effective amount of the vector and / or nanoparticles contained in the formulation may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dose may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg or 0.5-1 mg / kg. In another example, the dose may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0230] The therapeutically effective amount can be achieved by one or multiple administrations during the course of the treatment regimen (e.g., daily, every other day, every 3rd day, every 4th day, every 5th day, every 6th day, once a week, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or once a year). In certain embodiments, the treatment protocol may be determined according to a clinical trial protocol or an FDA-approved treatment protocol.

[0231] The therapeutically effective amount can be administered, for example, by injection, infusion, perfusion or lavage. Routes of administration include intravenous bolus, intradermal, intraarterial, intraperitoneal, intralymphatic, intralymphatic, intraperitoneal, intralesional, intraprostatic, intrathecal, intratumoral, intravesical and / or subcutaneous administration.

[0232] In certain embodiments, the cells of the present disclosure are administered to patients in combination with (e.g., before, at the same time, or after) related therapeutic methods, and the number of related therapeutic methods is not particularly limited.In certain embodiments, the cells of the present disclosure can be used in combination with chemotherapy; photoirradiation; immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506; antibodies; other immunoablative agents such as CAM PATH; anti-CD3 antibodies; other antibody treatments; cytotoxins; fludarabine; cyclosporine; FK506; rapamycin; mycophenolic acid; steroids; FR901228; cytokines; or radiation.

[0233] In certain embodiments, the formulation comprising the effector immune cells expressing the CAR disclosed herein may be administered in combination with a chemotherapeutic agent, and the number of chemotherapeutic agents used in combination is not particularly limited. Examples of chemotherapeutic agents include thiotepa and cyclophosphamide (CYTOXAN). TMalkylating agents such as busulfan, improsulfan, and piposulfan; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines, including regimens with altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, or trimethylolmelamine; chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, and mechlorethamine , mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard and other nitrogen mustards; carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and other nitrosoureas; alacinomysin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin Antibiotics such as cyclosporine, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; methotrexate and 5-fluorouracil; antimetabolites such as 5-FU; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone;Adrenal suppressants such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; f Enammet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (Taxol; TM , Bristol-Myers Squibb), and docetaxel (Taxotere®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum preparations; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); targretin TM (Bexarotene) and Panretin TM Retinoic acid derivatives such as (abtretinoin); ONTAK TM(denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above chemotherapeutic agents. The definition of chemotherapeutic agents further includes antihormonal agents that control or suppress the action of hormones on tumors, such as antiestrogens such as tamoxifen, raloxifene, aromatase inhibitor 4,5-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, toremifene (Fareston); antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above antihormonal agents. Additionally, where deemed appropriate, a combination of chemotherapy agents may be administered, including CHOP therapy, a combination of cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0234] In some embodiments, the chemotherapeutic agent is administered simultaneously with administration of the recombinant cells or nucleic acids of the present disclosure, or within one week of administration of the recombinant cells or nucleic acids of the present disclosure. In another embodiment, the chemotherapeutic agent is administered 1-4 weeks, 1 week-1 month, 1 week-2 months, 1 week-3 months, 1 week-6 months, 1 week-9 months, or 1 week-12 months after administration of the recombinant cells or nucleic acids of the present disclosure. In another embodiment, the chemotherapeutic agent is administered at least one month prior to administration of the cells or nucleic acids of the present disclosure. In some embodiments, the methods of the present disclosure further include administering two or more chemotherapeutic agents.

[0235] Various additional therapeutic agents may be used in combination with the formulations described herein. For example, additional therapeutic agents that may be useful include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab; and CTLA-4 inhibitors such as ipilimumab (Yervoy®).

[0236] Additional therapeutic agents suitable for use in combination with the present disclosure include abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), degarelix, docetaxel, enzalutamide, Erleada® (apalutamide), flutamide, goserelin acetate, Jevtana® (cabazitaxel), leuprolide acetate, Lupron® (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron These include Depot-Ped (leuprolide acetate), mitoxantrone hydrochloride, Nilandron® (nilutamide), nilutamide, Provenge® (sipuleucel-T), radium-223 dichloride, sipuleucel-T, Taxotere (docetaxel), Viadur (leuprolide acetate), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate), or Zytiga (abiraterone acetate).

[0237] In further embodiments, the preparation comprising immune cells comprising CAR can be administered with an anti-inflammatory agent.Anti-inflammatory agents or anti-inflammatory drugs include steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone); and non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate.Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and salicylates.Exemplary analgesics include acetaminophen, oxycodone, tramadol, and propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers, such as CD4 and CD5; cytokine inhibitors, such as TNF antagonists, such as etanercept (Enbrel®), adalimumab (Humira®), and infliximab (Remicade®); chemokine inhibitors; and adhesion molecule inhibitors. Biological response modifiers also include monoclonal antibodies and recombinant molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral formulation (auranofin) and intramuscular injection), and minocycline.

[0238] In certain embodiments, the formulations described herein are administered in combination with a cytokine. As used herein, "cytokine" refers to a protein released by one cell population to act on another cell as an intercellular mediator. Examples of cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, human N-methionyl growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-β; platelet-derived growth factor; transactivator (TGF), such as TGF-α and TGF-β. Transforming growth factors (TGFs); insulin-like growth factors I and II; erythropoietin (EPO); bone morphogenetic factors; interferons, such as interferon alpha, interferon beta, and interferon gamma; colony stimulating factors (CSFs), such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF); granulocyte CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, and IL-15; tumor necrosis factors, such as TNF-alpha and TNF-beta; and other polypeptide factors, such as LIF and kit ligand (KL). As used herein, "cytokine" also includes naturally occurring proteins or proteins from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

[0239] (ix) Reference levels obtained from a control population The values ​​obtained for the parameters related to the treatment described herein can be compared with reference levels obtained from a control population, and this comparison can indicate whether the treatment described herein is effective for a subject in need of treatment. The reference levels can be obtained from one or more relevant datasets obtained from a control population. As used herein, a "dataset" is a set of values ​​obtained by evaluating a sample (or a sample population) under desired conditions. The values ​​of the dataset can be obtained, for example, by obtaining measurements from the samples through an experiment and then constructing a dataset from these measurements. As can be understood by those skilled in the art, the reference levels can be based on mathematical or statistical formulas known in the art that are useful for obtaining meaningful aggregate reference levels (e.g., mean, median, median of mean, etc.) obtained, for example, from a collection of individual data points. Alternatively, the reference levels or the datasets for generating them can be obtained from a service provider such as a laboratory, or from a database or server on which the datasets are stored.

[0240] The reference levels obtained from the dataset may be derived from measurements previously obtained from a control population. A "control population" is a grouping of subjects or samples with similar specified characteristics. The grouping may be according to, for example, clinical parameters, clinical assessments, treatment regimens, disease status, severity of the condition, etc. In certain embodiments, the grouping is based on age groups (e.g., 60-65 years) and cancer status. In certain embodiments, the normal control population includes individuals who are age-matched to the test subjects and do not have cancer. In certain embodiments, age-matched individuals may include individuals aged 0-10 years, 30-40 years, 60-65 years, 70-85 years, etc., depending on the context and clinical relevance. In certain embodiments, the control population may include individuals who have a STEAP1-associated disease but have not been administered a therapeutically effective amount.

[0241] In certain embodiments, a reference level associated with the numerical value of a particular parameter associated with a treatment described herein is obtained based on the numerical value of the particular corresponding parameter associated with the treatment in a control population to determine whether the treatment disclosed herein is therapeutically effective for a subject in need of treatment.

[0242] In certain embodiments, conclusions are drawn based on whether the sample value is statistically significantly different from the reference level. If the difference is at a level that would be expected to occur based on chance alone, the measured value is not statistically significantly different. In contrast, a statistically significant difference or increase is a difference or increase that is greater than would be expected to occur by chance alone. Statistical significance or lack of statistical significance may be determined using any of a variety of methods known in the art. One commonly used indicator of statistical significance is the p-value. The p-value indicates the probability of obtaining a result equal to a particular data point, where the data point is a result obtained by chance alone. A p-value of 0.05 or less is often considered to be a significant (not a coincidence) result. In certain embodiments, if there is no statistically significant difference between the sample value and the reference level, the sample value is "comparable" to the reference level obtained from a normal control population.

[0243] (x) Kit The present disclosure further includes kits. The kits may contain various components for carrying out the methods disclosed herein. For example, the kit may include, depending on the embodiment of the method to be performed, one or more nucleic acids encoding a CAR disclosed herein; a protein shown in FIG. 13 or a coding sequence thereof; a nucleic acid encoding a second generation 4-1BB chimeric antigen receptor (CAR); a lentiviral STEAP1 CAR construct with a short spacer, a medium length spacer or a long spacer; a nucleic acid encoding a scFv; a nucleic acid encoding a VL; a nucleic acid encoding a VH; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an EGFRt; a cell (e.g., immune cell, T cell, CD4 T cell, CD8 T cell, B cell, natural killer (NK) cell, NK-T cell, monocyte / macrophage, lymphocyte, hematopoietic stem cell (HSC), hematopoietic progenitor cell (HPC), and / or a mixture of HSC and HPC (i.e., HSPC), untransduced T cell, STEAP1-BBζ CAR T cell, 22Rv1 STEAP1 knockout (ko) cell, 22Rv1 STEAP1 in which expression of STEAP1 has been rescued by expression from a lentivirus, ko cells); cell lines (e.g., androgen receptor (AR) positive human prostate cancer cell lines, AR negative prostate cancer cell lines, DU145 cell lines, DU145 STEAP1 cell lines, lentivirally modified DU145 hSTEAP1 cell lines, lentivirally modified DU145 mSteap1 cell line, C4-2B prostate cancer cell line, PC3 prostate cancer cell line; tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy samples, tumors, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils or other organs, and / or cells derived from these organs); gene expression components (e.g., genes for expression provided by vectors (e.g., lentiviral and retroviral vectors), CRISPR components, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or targeted nanoparticles);Cell formulation or cell activation components (e.g., saline, buffered saline, phosphate buffered saline (PBS)); biocompatible buffers (Ca++ / Mg++-free PBS, saline, water, Hank's solution, Ringer's solution); T cell stimulatory epitopes (e.g., anti-CD3 / anti-CD28 conjugated beads, OKT3, TGN1412); compositions for initiating culture (RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, EBV-transformed non-dividing lymphoblastoid cells (LCL), IL-21, human serum albumin (HSA) or other human serum components or fetal bovine serum, dextrose, stabilizers, preservatives); combination therapy components (e.g., local anesthetics, chemotherapeutic agents, immunosuppressants, anti-inflammatory agents); fluorescently tagged antibodies; sequences for PCR amplification; cytokines (e.g., IL-2, IL-7, IL-15, IL-21); culture vessels; reference levels; hSTEAP1-KI mice with syngeneic disseminated prostate cancer; primer pairs for amplifying wild-type alleles or portions of hSTEAP1-KI alleles; GAPDH; enzyme-linked immunosorbent assay (ELISA) for IFN-γ; culture plates, etc.;

[0244] The following exemplary embodiments and examples are provided to illustrate certain non-limiting embodiments of the present disclosure. Those skilled in the art having reference to this disclosure will appreciate that various modifications can be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit and scope of the present disclosure.

[0245] (xi) Exemplary embodiments 1. A chimeric antigen receptor (CAR) that, when expressed by a cell, (i) a. a STEAP1-binding domain having a set of complementarity determining regions (CDRs) from the DSTP3086S antibody according to the North, IMGT, Kabat, or Chothia numbering system; b. IgG4 hinge-CH2-CH3 spacer with 4 / 2-NQ mutation in the CH2 domain and The extracellular portion contains; (ii) an intracellular portion that includes the CD3ζ signaling domain and the 4-1BB signaling domain; and (iii) a CD28 transmembrane domain linking the extracellular portion to the intracellular portion. Including, CAR. 2. Use of a CAR described in embodiment 1 for the treatment of a subject in need of treatment, wherein the subject has a condition with low STEAP1 antigen density. 3. A chimeric antigen receptor (CAR) that, when expressed by a cell, the extracellular portion containing the STEAP1-binding domain; an intracellular portion that includes an effector domain; and A transmembrane domain connecting the extracellular portion to the intracellular portion Including, CAR. 4. The CAR of embodiment 3, wherein the STEAP1 binding domain has a set of complementarity determining regions (CDRs) from the DSTP3086S antibody based on the North, IMGT, Kabat or Chothia numbering. 5. The CAR of embodiment 3 or 4, wherein the STEAP1 binding domain comprises a single chain variable fragment (scFv). 6. The CAR of embodiment 5, wherein the scFv has a heavy chain variable region having at least 90% sequence identity to the sequence set forth in SEQ ID NO:5, and a light chain variable region having at least 90% sequence identity to the sequence set forth in SEQ ID NO:4. 7. The CAR of embodiment 5, wherein the scFv has a heavy chain variable region having the sequence set forth in SEQ ID NO:5 and a light chain variable region having the sequence set forth in SEQ ID NO:4. 8. The CAR of embodiment 5, wherein the scFv has at least 90% sequence identity to the sequence shown in SEQ ID NO:3. 9. The CAR of embodiment 5, wherein the scFv has a sequence as set forth in SEQ ID NO:3. 10. The CAR according to any one of embodiments 5 to 9, wherein the scFv has a heavy chain variable region encoded by a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 9, and a light chain variable region encoded by a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 7. 11. The CAR of any one of embodiments 5 to 9, wherein the scFv has a heavy chain variable region encoded by the sequence shown in SEQ ID NO: 9 and a light chain variable region encoded by the sequence shown in SEQ ID NO: 7. 12. A CAR according to any one of embodiments 5 to 11, wherein the scFv is encoded by a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO:6. 13. The CAR of any one of embodiments 5 to 11, wherein the scFv is encoded by the sequence shown in SEQ ID NO:6. 14. The CAR of any one of embodiments 3 to 13, wherein the extracellular portion further comprises a spacer. 15. The CAR of embodiment 14, wherein the spacer is 230 amino acids or less in length. 16. The CAR of embodiment 14 or 15, wherein said spacer consists of the hinge region, the CH2 domain and the CH3 domain of IgG4; consists of the hinge region and the CH2 domain of IgG4; or consists of the hinge region of IgG4. 17. The CAR of embodiment 16, wherein said IgG4 is human IgG4. 18. A CAR described in any one of embodiments 14 to 17, wherein the spacer has at least 90% sequence identity with the sequence shown in SEQ ID NO: 20. 19. A CAR described in any one of embodiments 14 to 18, wherein the spacer has the sequence shown in SEQ ID NO: 20. 20. A CAR described in any one of embodiments 14 to 18, wherein the spacer is encoded by a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO: 21. 21. A CAR described in any one of embodiments 14 to 18, wherein the spacer is encoded by the sequence shown in SEQ ID NO: 21. 22. A CAR according to any one of embodiments 3 to 21, wherein the effector domain comprises the entire CD3ζ signaling domain or a portion thereof; the entire 4-1BB signaling domain or a portion thereof, the entire CD28 signaling domain or a portion thereof, the entire CD3ζ signaling domain or a portion thereof, and a 4-1BB signaling domain; the entire CD3ζ signaling domain or a portion thereof, and the entire CD28 signaling domain or a portion thereof; or the entire CD3ζ signaling domain or a portion thereof, the entire 4-1BB signaling domain or a portion thereof, and the entire CD28 signaling domain or a portion thereof. 23. The CAR of embodiment 22, wherein the effector domain comprises all or a portion of the CD3 zeta signaling domain and all or a portion of the 4-1BB signaling domain. 24. The CAR of embodiment 22 or 23, wherein the CD3 zeta signaling domain has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, 25 and / or 26. 25. The CAR of embodiment 22 or 23, wherein the CD3 zeta signaling domain has a sequence as set forth in SEQ ID NO: 24, 25 or 26. 26. A CAR described in any one of embodiments 23 to 25, wherein the CD3 zeta signaling domain is encoded by a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO: 22 and / or 23. 27. A CAR described in any one of embodiments 23 to 25, wherein the CD3 zeta signaling domain is encoded by the sequence shown in SEQ ID NO: 22 or 23. 28. A CAR according to any one of embodiments 23 to 27, wherein the 4-1BB signaling domain has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30, 31 and / or 32. 29. A CAR described in any one of embodiments 23 to 28, wherein the 4-1BB signaling domain has a sequence as set forth in SEQ ID NO: 30, 31 or 32. 30. A CAR according to any one of embodiments 23 to 28, wherein the 4-1BB signaling domain is encoded by a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, 28 and / or 29. 31. A CAR according to any one of embodiments 23 to 28, wherein the 4-1BB signaling domain is encoded by a sequence as set forth in SEQ ID NO: 27, 28 or 29. 32. A CAR described in any one of embodiments 3 to 31, wherein the transmembrane domain comprises a CD28 transmembrane domain. 33. The CAR of embodiment 32, wherein the CD28 transmembrane domain has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 37, 38 and / or 39. 34. The CAR of embodiment 32, wherein the CD28 transmembrane domain has a sequence as set forth in SEQ ID NO: 37, 38 or 39. 35. A CAR according to any one of embodiments 32 to 34, wherein the CD28 transmembrane domain is encoded by a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 33, 34, 35 and / or 36. 36. A CAR according to any one of embodiments 32 to 34, wherein the CD28 transmembrane domain is encoded by the sequence set forth in SEQ ID NO: 33, 34, 35 or 36. 37. A CAR according to any one of embodiments 3 to 36, wherein the STEAP1 binding domain comprises a STEAP1 scFv, the intracellular portion comprises a CD3ζ signaling domain and a 4-1BB signaling domain, and the transmembrane domain comprises a CD28 transmembrane domain. 38. A CAR described in any one of embodiments 3 to 37, having at least 90% sequence identity with the sequence shown in SEQ ID NO:2. 39. A CAR according to any one of embodiments 3 to 37, having the sequence shown in SEQ ID NO:2. 40. A CAR described in any one of embodiments 3 to 39, encoded by a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO:1. 41. A CAR described in any one of embodiments 3 to 40, encoded by the sequence shown in SEQ ID NO:1. 42. A CAR described in any one of embodiments 3 to 41, further comprising a tag cassette or a suicide switch. 43. A CAR according to any one of embodiments 3 to 42, further comprising a multimerization domain. 44. A CAR described in any one of embodiments 3 to 43, further comprising a self-cleaving polypeptide. 45. The CAR of embodiment 44, wherein the self-cleaving polypeptide is a self-cleaving polypeptide (P2A) from porcine teschovirus-1, a self-cleaving polypeptide (T2A) from Thosea asigna virus, a self-cleaving polypeptide (E2A) from equine rhinitis A virus, a self-cleaving polypeptide (F2A) from foot-and-mouth disease virus, or a variant thereof. 46. ​​The CAR of embodiment 44, wherein the self-cleaving polypeptide is a T2A self-cleaving polypeptide. 47. A CAR according to any one of embodiments 3 to 46, further comprising a transduction marker. 48. The CAR of embodiment 47, wherein the transduction marker is truncated epidermal growth factor receptor (EGFRt). 49. The CAR of embodiment 47 or 48, wherein the EGFRt has at least 90% sequence identity with the sequence set forth in SEQ ID NO: 41. 50. The CAR of embodiment 48 or 49, wherein the EGFRt has the sequence set forth in SEQ ID NO: 41. 51. A CAR according to any one of embodiments 48 to 50, wherein the EGFRt is encoded by a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 40. 52. A CAR according to any one of embodiments 48 to 50, wherein the EGFRt is encoded by the sequence shown in SEQ ID NO: 40. 53. A CAR according to any one of embodiments 1 to 52, wherein immune cells expressing the CAR are not reactive to cells expressing Steap1b. 54. A genetic construct encoding a CAR according to any one of embodiments 1, 3-53, 88 and 89. 55. The genetic construct of embodiment 54, having at least 90% sequence identity with the sequence shown in SEQ ID NO:2. 56. The genetic construct of embodiment 54, having the sequence shown in SEQ ID NO:2. 57. Nanoparticles encapsulating a genetic construct according to any one of embodiments 54 to 56. 58. A cell genetically engineered to express a CAR according to any one of embodiments 1, 3-53, 88 and 89. 59. The cell of embodiment 58, which is an autologous cell obtained from the subject or a cell allogeneic to the subject. 60. The cell according to embodiment 58 or 59, which is an in vivo cell or an ex vivo cell. 61. The cell according to any one of embodiments 58 to 60, which is a T cell, a B cell, a natural killer (NK) cell, an NK-T cell, a monocyte / macrophage, a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC). 62. The cell according to any one of embodiments 58 to 61, which is a T cell selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells and / or naive T cells. 63. A cell according to any one of embodiments 58 to 62, which is a CD8+ T cell. 64. A cell according to any one of embodiments 58 to 62, which is a CD4+ T cell. 65. A cell population genetically engineered to express a CAR according to any one of embodiments 1, 3-53, 88 and 89. 66. The cell population of embodiment 65, comprising autologous cells obtained from the subject or cells allogeneic to the subject. 67. The cell population of embodiment 65 or 66, which is an in vivo cell population or an ex vivo cell population. 68. The cell population of any one of embodiments 65 to 67, comprising T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs). 69. A cell population according to any one of embodiments 65 to 68, comprising CD4+ T cells and CD8+ T cells. 70. The cell population of embodiment 69, comprising CD4+ T cells and CD8+ T cells in a 1:1 ratio. 71. A formulation comprising (i) a cell genetically engineered to express a CAR described in any one of embodiments 1, 3-53, 88 and 89, and (ii) a pharma- ceutically acceptable carrier. 72. A method for treating a subject having a STEAP1-associated disease, comprising administering to the subject a therapeutically effective amount of the formulation of embodiment 71, thereby treating the subject having a STEAP1-associated disease. 73. The method of embodiment 72, wherein the STEAP1-associated disease in the subject is based on the presence of diseased cells expressing STEAP1 at a low density of STEAP1 antigen. 74. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of less than 50,000 STEAP1 molecules per diseased cell. 75. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of less than 30,000 STEAP1 molecules per diseased cell. 76. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of less than 15,000 STEAP1 molecules per diseased cell. 77. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of less than 10,000 STEAP1 molecules per diseased cell. 78. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of less than 5,000 STEAP1 molecules per diseased cell. 79. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of fewer than 2,000 STEAP1 molecules per diseased cell. 80. The method of embodiment 73, wherein the state of low density of STEAP1 antigen is a state of fewer than 1,500 STEAP1 molecules per diseased cell. 81. The method of embodiment 73, further comprising the steps of obtaining a sample of said diseased cells and measuring the density of STEAP1 antigen in said diseased cells. 82. The method of any one of embodiments 73 to 81, wherein the STEAP1-related disease comprises prostate cancer, Ewing's sarcoma family of tumors (EFT), bladder cancer, ovarian cancer or rhabdomyosarcoma. 83. The method of any one of embodiments 73-81, wherein the STEAP1-associated disease comprises fatal metastatic castration-resistant prostate cancer. 84. A method for providing an immune response against STEAP1-expressing cells in a subject in need thereof, comprising: A method comprising the step of providing an immune response against STEAP1 expressing cells to the subject by administering to the subject a therapeutically effective amount of the formulation of embodiment 71. 85. The method of embodiment 84, wherein the STEAP1-expressing cells comprise prostate cancer cells, Ewing's Sarcoma Family Tumor (EFT) cells, bladder cancer cells, ovarian cancer cells or rhabdomyosarcoma cells. 86. The method of embodiment 84, wherein the STEAP1-expressing cells comprise prostate cancer cells. 87. The method of embodiment 86, wherein the prostate cancer cells comprise lethal metastatic castration-resistant prostate cancer cells. 88.CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, common FcRγ (FCER1G), FcγRlla, FcRβ (Fcε Rib), DAP10, DAP12, CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, N Kp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDl 54. The CAR of any one of embodiments 1 and 3-53, comprising an intracellular signaling domain of lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and / or CD19a. 89.CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The CAR of any one of embodiments 1, 3-31, 38-53 and 88, comprising a transmembrane domain of AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C.

[0246] (xii) Experimental Examples Targeting STEAP1 with chimeric antigen receptor T cell therapy in advanced prostate cancer

[0247] method: Cell line: 22Rv1 (CRL-2505), LNCaP (CRL-1740), PC3 (CRL-1435), DU145 (HTB-81), NCI-H660 (CRL - 5813), C4-2B (CRL-3315), RM9 (RL-3312) and Myc-CaP (CRL-3255) were obtained from the American Type Culture Collection. LNCaP95 cells were a gift from Stephen R. Plymate (University of Washington, Seattle). MSKCC EF1 was derived from the MSKCC PCa4 organoid line, a gift from Yu Chen (Memorial Sloan Kettering Cancer Center), as previously reported (Lee, JK, et al., PNAS 115, E4473-e4482 (2018)). Cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin, and 4 mmol / L GlutaMAX (Thermo Fisher). 22Rv1 STEAP1 ko cells and PC3 STEAP1 ko cells were generated by transiently transfecting 22Rv1 cells with a pool of PX458 plasmids (Addgene, #48138) each expressing one of four sgRNAs targeting sequences predicted by the sgRNA Designer of the Broad Institute Genetic Perturbation Platform (Doench, JG, et al., Nat. Biotechnol. 34, 184-191 (2016)). The sequences of these sgRNAs are shown below. 1) 5'-ATAGTCTGTCTTACCCAATG-3' (SEQ ID NO: 174) 2) 5'-CCTTTGTAGCATAAGGACAC-3' (SEQ ID NO: 175) 3) 5'-ATCCACTTATCCAACCAATG-3' (SEQ ID NO: 176) 4) 5'-CATCAACAAAGTCTTGCCAA-3' (SEQ ID NO: 177) 48-72 hours after transfection, GFP-positive cells were sorted individually using a Sony SH800 cell sorter, seeded in 96-well plates, and clonally expanded. Human STEAP cDNA, mouse Steap1 cDNA, mouse Steap1 cDNA reconstructed with the extracellular domain of human STEAP1, or human STEAP1B cDNA was cloned into the EcoRI site of the third-generation lentiviral vector FU-CGW by Gibson assembly (Xin, L., et al., PNAS 103, 7789-7794 (2006)). Lentiviruses were produced and titered as previously reported (Xin, L., et al., PNAS 100, 11896-11903 (2003)), and then used to transduce 22Rv1 STEAP1 ko cells or DU145 cells.

[0248] result STEAP1 is widely expressed in lethal metastatic castration-resistant prostate cancer (mCRPC) tissues First, we evaluated the expression pattern and abundance of STEAP1 in comparison with prostate-specific membrane antigen (PSMA) in advanced prostate cancer. Immunohistochemistry (IHC) was performed on duplicate spots of a tissue microarray composed of 121 cores from metastatic tissues obtained by rapid biopsy from 45 patients with lethal mCRPC at the Tumor Acquisition Necropsy Program at the University of Washington between 2010 and 2017 (Roudier, MP, et al. Human pathology 34, 646-653 (2003)) (Figure 1A). A study pathologist assessed the cell membrane staining score of STEAP1 and PSMA for each core, and the staining intensity (0, 1, 2, or 3; Figure 2A) was multiplied by the proportion of cancer cell staining of each intensity to obtain a semiquantitative H-score (Figure 1B). Based on this result, a generalized linear mixed statistical model was used to determine that the odds of a nonzero staining result for STEAP1 were 7.7 times higher than for PSMA (95% CI: 2.8–20.8, p<0.001). Applying a minimum staining threshold with an H-score cutoff of 30, we found that 87.7% (100 of 114) of evaluable cores that met the threshold were stained for STEAP1, whereas only 60.5% (69 of 114) were stained for PSMA (Figure 1C). Furthermore, 28.1% (32 of 114) of cores showed staining for STEAP1 but not for PSMA (Figure 1D), compared with only 0.9% (1 of 114) cores that were stained for PSMA but not for STEAP1. Several cases showed heterogeneous expression of PSMA within the core (Figure 1E), consistent with a recent report of intratumoral PSMA heterogeneity in mCRPC biopsies (Paschalis, A., et al. European urology (2019)).

[0249] STEAP1 staining based on the minimum staining threshold was identified in 96% (48 of 50) of bone metastases, 95% (19 of 20) of lymph node metastases, and 76.6% (36 of 47) of visceral metastases (Figure 2B). No difference in staining intensity of STEAP1 was observed between bone and lymph node metastases, or between lymph node and visceral metastases. However, bone metastases showed a higher H-score for STEAP1 than visceral metastases (bone metastases 183.6 vs. visceral metastases 121.9, p = 0.0018). In cases spotted on tissue microarrays, Pearson's correlation coefficient was calculated and a positive correlation was identified between STEAP1 expression and androgen receptor (AR) expression (r = 0.3057, 95% CI: 0.1314-0.4616, p < 0.001) (Figure 2C). This result was expected, since STEAP1 is an androgen-regulated gene (Gomes, IM, et al., The Prostate 73, 605-613 (2013); and Sharp, A., et al., The Journal of clinical investigation 129, 192-208 (2019)). On the other hand, we found a negative correlation between STEAP1 expression and the expression of synaptophysin (SYP), a neuroendocrine differentiation marker (r=-0.2172, 95% CI:-0.3843~-0.03628, p=0.0192) (Figure 2D). These results suggest that, similar to PSMA (Bakht, MK, et al., Endocrine-related cancer 26, 131-146 (2018)), STEAP1 expression may be lost upon neuroendocrine transdifferentiation of prostate cancer.

[0250] Development of potent and antigen-specific STEAP1 CAR Given that STEAP1 is broadly expressed in late-stage mCRPC, providing a rationale for targeting STEAP1 in late-stage mCRPC, we generated lentiviruses expressing STEAP1-specific second-generation CARs. The costimulatory domain of 4-1BB was deemed suitable, as it is associated with T cell memory formation and long-term persistence (Salter, AI, et al., Science signaling 11(2018)), and the transmembrane domain of CD28 was introduced, as it has been shown to reduce the antigen threshold for activating second-generation 4-1BB CAR T cells (Majzner, RG, et al., Cancer discovery 10, 702-723 (2020)). In addition, we incorporated the full-length humanized single-chain variable fragment (scFv) derived from bundled tuzumab vedotin (DSTP3086S). Bundled tuzumab vedotin (DSTP3086S) is an antibody-drug conjugate targeting STEAP that was discontinued after Phase I / II clinical trials due to payload shedding, which caused a therapeutic window problem. This scFv is a humanized variant of a mouse monoclonal antibody (mAb 120.545) originally developed by Agensys, and has been shown to have an affinity of 1 nM in cell-based binding assays (Challita-Eid, PM, et al., Cancer research 67, 5798-5805 (2007)). To investigate whether the activity of the CAR can be regulated, three different hinge / spacer lengths were incorporated: a short spacer (IgG4 hinge), a medium-length spacer (IgG4 hinge-CH3), and a long spacer (IgG4 hinge-CH2-CH3). The long spacer was engineered to contain a 4 / 2-NQ mutation in the CH2 domain, as previously reported (Hudecek, M., et al., Cancer immunology research 3, 125-135 (2015)), which prevents Fcγ receptor binding and activation-induced cell death when long spacer CAR T cells are adoptively transferred into immunodeficient mice.The three CAR candidates were cloned into lentiviral vectors co-expressing truncated epidermal growth factor receptor (EGFRt) as a transduction marker (Figure 3A). Human CD4 and CD8 T cells enriched from peripheral blood mononuclear cells (PBMCs) from human donors collected by pheresis were transduced with the generated lentiviruses. Expanded CD4 and CD8 CAR T cells were immunophenotyped (Figure 4A) and reconstituted into a cell product of defined composition with normal CD4 / CD8 ratios to assess the functional activity of CAR T cells.

[0251] To control STEAP1 expression in a uniform genetic background, we focused on the human prostate cancer cell line 22Rv1, which naturally expresses STEAP1. We performed knockout (ko) of STEAP1 by CRISPR / Cas9 genome editing. We then transduced a lentivirus expressing STEAP1 into the 22Rv1 STEAP1 ko line to generate a STEAP1 rescue line (Figure 3B). We then used this cell line to first perform co-culture assays with three types of STEAP1 CAR T cells with short, medium or long spacers and screened the three types of STEAP1 CAR T cells by measuring the release of interferon-γ (IFN-γ) as an indicator of T cell activation. Only the STEAP1 CAR T cells with the long spacer (referred to as "STEAP1-BBζ CAR T cells") showed an antigen-specific IFN-γ release pattern (Figure 3C and Figure 4B). Moreover, STEAP1-BBζ CAR T cells lysed 22Rv1 cells in a substantial dose-dependent manner compared to non-transduced T cells (Figure 3D) and relatively improved viability of 22Rv1 STEAP1 ko cells (Figure 3E). We next performed a similar test in a human prostate cancer cell line DU145 that naturally lacks STEAP1 expression and was engineered to express STEAP1 by lentiviral transduction (DU145 STEAP1). In this experimental setting, activation of STEAP1-BBζ CAR T cells was observed only in coculture with DU145 STEAP1 cells, but not in coculture with DU145 parental cells (Figure 4C). Furthermore, cytolytic activity in coculture with DU145 STEAP1 cells was observed only in STEAP1-BBζ CAR T cells, but not in non-transduced T cells (Figure 4D).

[0252] Next, a larger panel of human prostate cancer cell lines was analyzed to characterize their native STEAP1 expression by immunoblot analysis. Cell lines known to have androgen receptor (AR) expression / activity (LNCaP, 22Rv1, VCaP, and LNCaP95) showed various levels of STEAP1 expression, whereas AR-null cell lines (PC3, DU145, MSKCC EF1, and NCI-H660) showed no detectable levels of STEAP1 expression (Figure 3F). Coculture of STEAP1-BBζ CAR T cells with these cell lines was further performed to further validate antigen-specific activation of STEAP1-BBζ CAR T cells based on IFN-γ release (Figure 3G). However, discrepancies were observed in the PC3 line, which induced significant activation of STEAP1-BBζ CAR T cells despite apparently failing to show STEAP1 expression (Figure 3F). Previous literature suggests that STEAP1 is expressed at low levels in PC3 cell lines (Gomes, IM, et al., Genes & cancer 5, 142-151 (2014)). Indeed, a band was observed in the immunoblot at significantly longer exposure times, suggesting the presence of very low STEAP1 expression (Figure 3H). To confirm whether the activation of STEAP1-BBζ CAR T cells was due to such low levels of STEAP1 expression in PC3 lines, we generated three STEAP1 ko lines derived from PC3 lines (Figure 3G) and re-cultured them with STEAP1-BBζ CAR T cells. Knocking out STEAP1 in PC3 lines abolished the activation of STEAP1-BBζ CAR T cells (Figure 3I), further confirming the specificity and providing evidence that STEAP1-BBζ CAR T cells are sensitive even at low antigen density.

[0253] Functional measurements of cross-reactivity of STEAP1-BBζ CAR with mouse Steap1 and human STEAP1B Consistent with the anti-human specificity of the scFv derived from mAb120.545, STEAP1-BBζ CAR T cells showed no cross-reactivity with mouse Steap1 (Figure 5A-5C). However, this prompted us to individually reconstitute the three human STEAP1 extracellular domains (ECDs) in mouse Steap1 to determine which ECDs are important for epitope recognition by STEAP1-BBζ CAR T cells (Figure 5D). We performed co-culture experiments of STEAP1-BBζ CAR T cells with DU145 cells engineered to express mouse Steap1 in which each mouse ECD was replaced with a human ECD. Human STEAP1 ECD2 was associated with STEAP1-BBζ CAR T cell activation (Figure 5E) and lysed target cells (Figure 5F), whereas human STEAP1 ECD1 and human STEAP1 ECD3 did not. Interestingly, human STEAP1 showed 93.9% (31 of 33 amino acids) homology with mouse Steap1 ECD2 ( Figure 5G ), indicating that Q198 and / or I209 of human STEAP1 are crucial for recognition by STEAP1-BBζ CAR T cells and induction of IFN-γ production.

[0254] Among the human STEAP family proteins, STEAP1B has the highest homology with STEAP1 (Gomes, IM, et al., Genes & cancer 5, 142-151 (2014)). Three STEAP1B transcripts were identified, all of which had completely conserved amino acid sequences of human STEAP1 ECD2 (Figure 6A). The consensus membrane protein topology prediction algorithm TOPCONS (Bernsel, A., et al., Nucleic acids research 37, W465-W468 (2009)) predicted that these ECD2 sequences are located extracellularly in the three STEAP1B protein isoforms (Figure 6B), but the confidence score was low due to the lack of consensus among the models (Figure 6C). Preliminary analyses using hidden Markov models also suggested that these ECD2 sequences are intracellular, rather than extracellular, in STEAP1B protein isoform 1 and isoform 2 (Gomes, IM, et al., Genes & cancer 5, 142-151 (2014)). However, these predictions need to be directly verified by solving the crystal structure of STEAP1B.

[0255] Potent antitumor effects of STEAP-1BBζ CAR T cells in a disseminated prostate cancer model naturally expressing STEAP1 in immunodeficient mice As an initial screen for antitumor activity in vivo, 22Rv1 cells were implanted into male NOD scidγ (NSG) mice to generate subcutaneous xenograft tumors. 3 When it reaches 5×10 6Mice were treated with a single intratumoral injection of untransduced T cells or STEAP1-BBζ CAR T cells. Intratumoral treatment with STEAP1-BBζ CAR T cells significantly inhibited tumor growth, reaching statistical significance by day 16 after treatment (Figure 7A). Mice were sacrificed on day 25, and residual tumors were removed from mice treated with STEAP1-BBζ CAR T cells, revealing extensive areas of necrotic debris and infiltration of CD3+ STEAP1-BBζ CAR T cells in viable tumor areas (Figure 8A).

[0256] Lentiviruses were transduced into 22Rv1 cells to express firefly luciferase (fLuc) and were then cultured for 10 min. 6 22Rv1-fLuc cells were injected into the tail vein of male NSG mice. Two weeks later, bioluminescence live imaging (BLI) was performed to visualize metastatic establishment, at which point 5 × 10 6 Mice were treated with a single intravenous injection of non-transduced T cells or STEAP1-BBζ CAR T cells (Figure 7B). Serial bioluminescence imaging showed that mice treated with non-transduced T cells rapidly progressed to disease, whereas mice treated with STEAP1-BBζ CAR T cells had significantly delayed tumor progression (Figure 7C,D) and prolonged survival (mice treated with STEAP1-BBζ CAR T cells survived 97 days compared with 31 days for non-transduced T cells; p=0.0018 by log-rank test; Figure 7E). There was no significant difference in mouse weight between the treatment groups (Figure 8B).

[0257] Additionally, C4-2B-fLuc cells were inoculated into male NSG mice by tail vein injection. C4-2B is a castration-resistant line derived from the LNCaP line (Chen, ME, et al., The Journal of biological chemistry 273, 17618-17625 (1998)) and shows growth kinetics consistent with typical prostate cancer. Four weeks after injection of C4-2B-fLuc cells, establishment of metastatic tumors was confirmed by bioluminescence imaging, and 5 × 10 6 Mice were treated with a single intravenous injection of non-transduced T cells or STEAP1-BBζ CAR T cells (Figure 7B). Within 5 weeks of treatment, serial bioluminescence imaging showed complete responses in all mice receiving STEAP1-BBζ CAR T cells (Figure 7F, Figure 7G). A trend toward enhanced weight loss was observed in the non-transduced T cell treatment group (Figure 8C), but this was not statistically significant, likely due to the small sample size. Necropsy of mice treated with STEAP1-BBζ CAR T cells revealed no gross lesions and no signal was detected in organs assessed by ex vivo bioluminescence imaging, suggesting that the mice appeared to be cured of tumors (Figure 8D). At the end of the experiment, CD3+EGFRt+ splenocytes were detected, identifying the peripheral persistence of STEAP1-BBζ CAR T cells (Figure 7H).

[0258] Safety and efficacy demonstrated in mouse-in-mouse STEAP1 CAR T cell trials The activation and cytolytic activity of STEAP1-BBζ CAR T cells was observed at very low STEAP1 antigen density in the PC3 cell line, raising concerns about on-target and off-target tumor toxicity. To evaluate potential toxicity in a genetically tractable model organism, human STEAP1 knock-in mice (hSTEAP1-KI) were generated by knocking in the human STEAP1 gene into the mouse Steap1 gene in a C57Bl / 6 background (Figure 9A). A mouse colony was established by genotyping by polymerase chain reaction (PCR) of tail DNA (Figure 9B). Both homozygous and heterozygous hSTEAP1-KI mice appeared to have no phenotypic or reproductive abnormalities compared to their wild-type littermates. A tissue survey of human STEAP1 expression in heterozygous male and female hSTEAP1-KI (hSTEAP1-KI / +) mice using quantitative reverse transcription PCR (qRT-PCR) showed the highest relative expression in the prostate, followed by the uterus and adrenal glands (Fig. 9C). Furthermore, in situ analysis of the prostate and adrenal glands of male hSTEAP1-KI / + mice by STEAP1 IHC staining revealed that human STEAP1 expression was restricted to the luminal epithelial cells of the prostate (Fig. 9D), and was also found in the adrenal cortex (Fig. 9E).

[0259] We generated a murine form of STEAP1 CAR (termed “STEAP1-mBBζ CAR”) in which the transmembrane domain of CD28, the costimulatory domain of 4-1BB, and the activation domain of CD3ζ were replaced by their corresponding mouse orthologues while retaining the scFv and IgG4 hinge-CH2-CH3 spacer in the STEAP1-BBζ CAR, and cloned it into a gammaretroviral construct (Figure 9F). In addition, we replaced the transduction marker EGFRt with a truncated mouse CD19 (mCD19t) to minimize potential immunogenicity. We confirmed that the retrovirus could efficiently transduce T cells enriched from mouse splenocytes (Figure 9G), and that the resulting mouse STEAP1-mBBζ CAR T cells could induce cytolysis of the RM9 mouse prostate cancer cell line (RM9-hSTEAP1) engineered to express human STEAP1 by lentiviral transduction (Figure 9H).

[0260] To investigate the safety and efficacy of STEAP1-mBBζ CAR T cell therapy, heterozygous male hSTEAP1-KI mice were inoculated with syngeneic RM9-STEAP1-fLuc cells via tail vein injection (Figure 10A). One week later, after confirming metastatic tumor establishment by bioluminescence imaging, mice were administered 100 mg / kg of cyclophosphamide via intraperitoneal injection for preconditioning. The next day, mice were randomized and administered 5 × 10 6Mice were treated with either non-transduced murine T cells or murine STEAP1-mBBζ CAR T cells. Both mice treated with murine STEAP1-mBBζ CAR T cells showed a reduction in tumor burden within 1 week of treatment, as analyzed by bioluminescence imaging (Figure 10B, 10C). This response was observed only for a short period of time, but was associated with a slight increase in survival (mice treated with murine STEAP1-mBBζ CAR T cells survived 21 days compared with 12 days for mice treated with non-transduced murine T cells; p=0.0138 by log-rank test; Figure 10D). Importantly, there was clear evidence of antitumor efficacy, as there was no overall toxicity or early death specifically associated with murine STEAP1-mBBζ CAR T cell therapy at this dose level. Weight loss associated with increasing tumor burden was common to both treatment groups (Figure 10E, 10F). Residual tumor foci excised at the end of the experiment from mice treated with non-transduced murine T cells showed expression of human STEAP1 with minimal regional heterogeneity (Fig. S10G). In contrast, all tumors excised from mice treated with murine STEAP1-mBBζ CAR T cells showed a notable absence of human STEAP1 expression (Fig. S10H). These findings suggest either an intrinsic resistance that predates STEAP1-RM9 tumor cells or adaptive resistance due to dynamic STEAP1 antigen loss. Importantly, heterozygous hSTEAP1-KI mice treated with STEAP1-mBBζ CAR T cells did not show obvious tissue destruction or increased infiltration of CD3+ T cells into the prostate compared to heterozygous hSTEAP1-KI mice treated with non-transduced T cells (Fig. S11A, S11B).

[0261] (xiii) Conclusion The nucleic acid and amino acid sequences provided herein are represented by the abbreviations used for nucleotide bases and amino acid residues as set forth in 37 CFR 1.831-1.835 and as set forth in WIPO Standard ST.26, effective July 1, 2022. Although only one strand is shown for each nucleic acid sequence, the complementary strand, if appropriate, is also included in the embodiments.

[0262] To the extent not explicitly stated herein, the coding sequences for the proteins disclosed herein, and the protein sequences encoded by the coding sequences disclosed herein, can be readily prepared by one of ordinary skill in the art.

[0263] Variants of the sequences disclosed and referenced herein are also included herein. Guidelines for determining which amino acid residues can be substituted, inserted or deleted without losing biological activity can be determined using computer programs well known in the art, such as DNASTAR. TM Software (Madison, WI, USA) can be used to find the amino acid changes in the protein variants disclosed herein. The amino acid changes are preferably conservative amino acid changes, i.e., substitutions of similarly charged amino acids with each other or of uncharged amino acids with each other. Conservative amino acid changes include substitutions with members of a family of amino acids whose side chains are related.

[0264] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can be made without generally altering the biological activity of the resulting molecule.Those skilled in the art will be familiar with the fact that generally, a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter the biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu), and ketone (K). Group 7 (polar uncharged): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp) and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu and Ile; Group 10 (small aliphatic residues that are non-polar or slightly polar): Ala, Ser, Thr, Pro and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0265] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is widely understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity index of each amino acid is Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamic acid (-3.5); Gln (-3.5); aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0266] It is well known in the art that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity degree can also result in a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such changes, substitution of amino acids with hydrophobicity indices within ±2 is preferred, substitution of amino acids with hydrophobicity indices within ±1 is particularly preferred, and substitution of amino acids with hydrophobicity indices within ±0.5 is even more particularly preferred. Furthermore, it is well known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity.

[0267] As detailed in U.S. Patent No. 4,554,101, each amino acid residue is assigned a hydrophilicity value, which is as follows: Arg (+3.0); Lys (+3.0); Aspartic acid (+3.0±1); Glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is well known that certain amino acids can be substituted with other amino acids having a similar hydrophilicity value, and that such substitutions will result in biologically equivalent proteins, and in particular immunologically equivalent proteins. When making such changes, substitutions between amino acids whose hydrophilicity values ​​are within the range of ±2 are preferred, substitutions between amino acids whose hydrophilicity values ​​are within the range of ±1 are particularly preferred, and substitutions between amino acids whose hydrophilicity values ​​are within the range of ±0.5 are even more particularly preferred.

[0268] As outlined above, amino acid substitutions may be made on the basis of the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0269] Additionally, as described elsewhere herein, variants of a gene sequence include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that have no statistically significant effect on the function of the encoded product.

[0270] Variants of the protein, nucleic acid and gene sequences disclosed herein also include sequences having at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to the protein, nucleic acid or gene sequences disclosed herein.

[0271] "Percent sequence identity" refers to the relatedness of two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein, nucleic acid or gene sequences, as determined by the matching between strings of protein, nucleic acid or gene sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods for determining identity are preferably designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal format alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)). Related programs further include the GCG suite of programs (Wisconsin package version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the "default values" that are the basis of the program. In this specification, "default values" refers to a set of numerical values ​​or parameters that are preregistered in the software at the time of initialization of the software.

[0272] Variants also include nucleic acid molecules that hybridize to sequences disclosed herein under stringent hybridization conditions and have the same function as reference sequences.Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate and 20μg / ml denatured salmon sperm DNA fragmented, followed by washing the filter with 0.1xSSC at 50°C.The stringency of hybridization and signal detection are mainly changed by adjusting the concentration of formamide (lower the percentage of formamide, lower the stringency), salt conditions or temperature. For example, moderately stringent conditions include overnight incubation at 37° C. in 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml blocking salmon sperm DNA, followed by washing with 1×SSPE and 0.1% SDS at 50° C. Even lower stringency is achieved by performing stringent post-hybridization washes at high salt concentrations (e.g., 5×SSC). The above conditions can be varied by adding and / or substituting other blocking reagents used to reduce the background of hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary preparations. The addition of certain blocking reagents may require some modification of the hybridization conditions described above due to compatibility issues.

[0273] "Specifically binds" means that it does not bind significantly to other molecules or components in the relevant environmental sample, but does bind 10 5 M -1"High affinity" refers to binding between a binding domain (e.g., a binding domain of a CAR) and its cognate binding molecule with an affinity or Ka (i.e., the equilibrium binding constant of a particular binding interaction, expressed in units of 1 / M) equal to or greater than 10. Binding domains may also be classified as "high affinity" or "low affinity." In certain embodiments, a "high affinity" binding domain has an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 Or at least 10 13 M -1 In certain embodiments, a "low affinity" binding domain refers to a binding domain with a Ka of 10 7 M -1 Below, 10 6 M -1 Less than or equal to 10 5 M -1 Alternatively, affinity refers to the equilibrium dissociation constant (Kd) (units: M) of a particular binding interaction (e.g., 10 -5 M~10 -13In certain embodiments, a binding domain may have an "enhanced affinity," which refers to a selected or engineered binding domain that exhibits stronger binding to its cognate binding molecule than the wild-type (or parent) binding domain. For example, the enhanced affinity may be due to a higher Ka (equilibrium binding constant) for the cognate binding molecule than the reference binding domain, a lower Kd (dissociation constant) for the cognate binding molecule than the reference binding domain, or a lower dissociation rate (Koff) for the cognate binding molecule than the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind to a particular cognate binding molecule and for measuring binding affinity, such as Western blots, ELISAs, and BIACORE analyses (see, e.g., Scatchard, et al., 1949, Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or similar publications).

[0274] Unless otherwise stated, the present disclosure can be carried out using conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); FM Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and RI Freshney, ed. Animal Cell Culture (1987).

[0275] As will be appreciated by those of skill in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the particular components, steps, materials, or ingredients described. Thus, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprise" means, but is not limited to, the inclusion of any unrecited components, steps, materials, or ingredients, even if the amount is greater. The transitional phrase "consisting of" excludes any unrecited components, steps, materials, or ingredients. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the recited components, steps, materials, or ingredients and those components, steps, materials, or ingredients that do not materially affect the embodiment. A significant effect refers to an effect that statistically significantly reduces lysis by STEAP1-expressing cells in an in vitro cytotoxicity assay, as described herein.

[0276] Unless otherwise indicated, all numerical values ​​expressing quantities or properties of materials, such as molecular weight and reaction conditions, in the specification and claims are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding procedures. For clarity, the term "about," when used in conjunction with a stated value or range, has a meaning that would be reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0277] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation associated with their respective testing measurements.

[0278] In the description of the present invention (particularly in the description of the claims below), the terms "a", "an", "the" and similar modifiers are intended to include both the singular and the plural unless otherwise indicated or the context clearly indicates otherwise. Numerical ranges described herein are intended to be a shorthand way of referring to each numerical value falling within the range individually. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any method described herein can be performed in any suitable order unless otherwise indicated or the context clearly indicates otherwise. The use of any examples or language of examples (e.g., "etc.") provided herein is intended to be for the purpose of illustrating the invention only and does not limit the scope of the invention as described in the claims. No term described herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0279] Groupings of other elements of the invention disclosed herein or of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0280] Specific embodiments of the present invention are described herein, including those embodiments known to the inventors to be the best mode for carrying out the invention. Of course, those skilled in the art will readily appreciate that the embodiments described herein may be modified in various ways upon review of the above detailed description. The inventors anticipate that such modifications may be adopted by those skilled in the art, and intend that the present invention may be practiced in other ways than as specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter of the present invention to the extent permitted within the scope of applicable law. Moreover, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless otherwise indicated or the context clearly dictates otherwise.

[0281] Additionally, throughout this specification, various patents, publications, journal articles and other documents are cited (references herein). Each reference cited herein is individually incorporated herein by reference for the teachings thereof as if it were a part of this specification.

[0282] Finally, the embodiments of the invention disclosed herein are to be considered as illustrative of the principles of the invention. Other modifications may be adopted within the scope of the invention. Thus, by way of example, but not by way of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not to be limited to what has been precisely shown and described herein.

[0283] The details described herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the present invention, to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no structural details of the present invention are described in more detail than is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by reading the description of the present invention in conjunction with the drawings and / or examples.

[0284] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clear and unambiguous changes are made in the examples, or the meaning of the terms makes the interpretation meaningless or substantially meaningless. If the definition of a term does not make sense or substantially meaningless from the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. A nucleic acid encoding a chimeric antigen receptor (CAR), The CAR, when expressed by a cell, an extracellular portion comprising the STEAP1-binding domain from the DSTP3086S antibody and an IgG4 hinge-CH2-CH3 spacer; an intracellular portion comprising an effector domain; and a transmembrane domain connecting the extracellular portion to the intracellular portion A nucleic acid comprising:

2. the binding domain derived from the DSTP3086S antibody, a heavy chain variable region comprising the sequence set forth in SEQ ID NO:5 and a light chain variable region comprising the sequence set forth in SEQ ID NO:4; or a heavy chain variable region having at least 95% sequence identity to the sequence set forth in SEQ ID NO:5 and a light chain variable region having at least 95% sequence identity to the sequence set forth in SEQ ID NO:4; The nucleic acid of claim 1.

3. the binding domain derived from the DSTP3086S antibody, a heavy chain variable region encoded by the sequence set forth in SEQ ID NO:9 and a light chain variable region encoded by the sequence set forth in SEQ ID NO:7; or a heavy chain variable region encoded by a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO:9, and a light chain variable region encoded by a sequence having at least 95% sequence identity with the sequence set forth in SEQ ID NO:7; The nucleic acid of claim 1.

4. The nucleic acid of claim 1 , wherein the binding domain derived from the DSTP3086S antibody comprises a single-chain variable fragment (scFv).

5. The nucleic acid of claim 4, wherein the scFv comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

3.

6. The nucleic acid of claim 4, wherein the scFv is encoded by the sequence shown in SEQ ID NO: 6 or a sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO:

6.

7. The nucleic acid of claim 1 , wherein the STEAP1 binding domain derived from the DSTP3086S antibody does not bind to Steap1b.

8. 2. The nucleic acid of claim 1, wherein the IgG4 hinge-CH2-CH3 spacer comprises a 4 / 2-NQ mutation in the CH2 domain.

9. 2. The nucleic acid of claim 1, wherein the IgG4 hinge-CH2-CH3 spacer comprises the sequence set forth in SEQ ID NO: 20 or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

20.

10. 2. The nucleic acid of claim 1, wherein the IgG4 hinge-CH2-CH3 spacer is encoded by the sequence set forth in SEQ ID NO: 21 or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:

21.

11. the effector domain is Contains all or part of the CD3ζ signaling domain and the 4-1BB signaling domain; Contains all or part of the CD3ζ signaling domain; Contains all or part of the 4-1BB signaling domain; Contains all or part of the CD28 signaling domain; comprising all or part of the CD3ζ signaling domain and all or part of the CD28 signaling domain; or The nucleic acid of claim 1, comprising all or part of the CD3ζ signaling domain, all or part of the 4-1BB signaling domain, and all or part of the CD28 signaling domain.

12. The nucleic acid of claim 1, wherein the transmembrane domain comprises a CD28 transmembrane domain.

13. The nucleic acid of claim 1, wherein the CAR has a sequence set forth in SEQ ID NO:2 or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

2.

14. 10. The nucleic acid of claim 1, further comprising a tag cassette, a suicide switch, a multimerization domain, a self-cleaving polypeptide, or a transduction marker.

15. 2. The nucleic acid of claim 1, comprising the sequence set forth in SEQ ID NO: 1 or a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:

1.

16. 1. An immune cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR), The CAR, when expressed by a cell, an extracellular portion comprising the STEAP1-binding domain from the DSTP3086S antibody and an IgG4 hinge-CH2-CH3 spacer; an intracellular portion comprising an effector domain; and a transmembrane domain connecting the extracellular portion to the intracellular portion An immune cell comprising:

17. 17. The immune cell of claim 16, which is a T cell, a B cell, a natural killer (NK) cell, an NK-T cell, a monocyte / macrophage, a hematopoietic stem cell (HSC), or a hematopoietic progenitor cell (HPC).

18. The immune cell of claim 16, which is a T cell selected from the group consisting of CD8+ T cells, CD4+ T cells, CD3+ T cells, central memory T cells, effector memory T cells, and naive T cells.

19. A formulation for the treatment of a STEAP1-associated disease, comprising a nucleic acid encoding a chimeric antigen receptor (CAR), The CAR, when expressed by a cell, an extracellular portion comprising the STEAP1-binding domain from the DSTP3086S antibody and an IgG4 hinge-CH2-CH3 spacer; an intracellular portion comprising an effector domain; and a transmembrane domain connecting the extracellular portion to the intracellular portion A formulation comprising:

20. 20. The formulation of claim 19, wherein the STEAP1-associated disease comprises prostate cancer, Ewing's sarcoma family of tumors (EFT), bladder cancer, ovarian cancer, or rhabdomyosarcoma.

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