Neuroendocrine Cancer Treatment

Abstract

Provided herein are compositions and methods for treating neuroendocrine cancers and preventing or treating metastases from neuroendocrine cancers.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 144,337, filed February 1, 2021, which is incorporated by reference herein in its entirety and for all purposes.

[0002] Sequence Listing The sequence listing set forth in file 048537-640001WO SEQUENCE LISTING_ST25.TXT, 69,632 bytes, machine format IBM-PC, MS Windows Operating System, created on Mar. 23, 2020, is incorporated herein by reference. [Background technology]

[0003] Neuroendocrine tumors (NETs) are a rare type of tumor that arises from neuroendocrine cells. These cells have the characteristics of both nerve cells and hormone-producing cells, and release hormones into the blood in response to signals from the nervous system. Since neuroendocrine tumors arise from cells that produce hormones, the tumors can also produce hormones. Most neuroendocrine tumors arise in the digestive tract, pancreas, rectum, lung, appendix, and prostate. Neuroendocrine tumors include, but are not limited to, carcinoid tumors, pancreatic islet cell tumors, medullary thyroid carcinoma, pheochromocytoma, neuroendocrine carcinoma of the skin (Merkel cell carcinoma), small cell lung carcinoma, large cell neuroendocrine carcinoma, and neuroendocrine prostate carcinoma (NEPC).

[0004] NEPC is an aggressive histological subtype of prostate cancer, most commonly occurring in the later stages of prostate cancer as a treatment-resistant cancer. The poor prognosis of NEPC is due in part to late diagnosis and lack of effective therapeutic agents. Prostate cancer is an androgen-regulated disease, and androgen deprivation therapy (ADT) in combination with drugs that target androgen receptor (AR) signaling is the standard of care for metastatic prostate cancer. Inevitably, however, the cancer becomes resistant to ADT and develops into lethal castration-resistant prostate cancer (CRPC). CRPC is a fatal disease and the second leading cause of cancer death in men in the United States. One in six men will eventually be diagnosed with prostate cancer (PCa), making it a major health problem in the United States. One-quarter of men diagnosed with this cancer will develop advanced PCa, which is associated with poor medical prognosis and significantly lower 5-year survival rates. Furthermore, PCa often metastasizes to bone, where it typically becomes resistant to treatment and is incurable at this stage of development. A particularly aggressive form of CRPC, called neuroendocrine prostate cancer (NEPC), has emerged with increasing frequency in patients treated with ADT. Despite the recent development of several novel therapies that have improved patient survival, nearly all patients with CRPC develop resistance to therapy and disease progression. Recent genome-wide next-generation sequencing studies have identified recurrent molecular alterations in prostate cancer, elucidating mechanisms of treatment resistance and potential treatment vulnerability.

[0005] Prostate cancer can be targeted by CAR-T cells or other forms of immunotherapy. With early indications of clinical activity of CAR-T cells, many large-scale multicenter clinical trials are underway targeting either prostate-specific membrane antigen (PMSA) (NCT04249947, NCT04227275) or plasma membrane calcium ATPase (PSCA) (NCT04227275). However, there are aggressive CRPC and NEPC that do not express PSMA or PSCA, and thus there is an urgent unmet need to identify new prostate cancer antigens that can be targeted by CAR T cells.

[0006] The tyrosine-protein kinase transmembrane receptor ROR1, also known as neurotrophic tyrosine kinase receptor-related 1, is an enzyme encoded by the ROR1 gene in humans. ROR1 is a member of the receptor tyrosine kinase-like orphan receptor family. ROR1 is expressed on CRPC and NEPC, but not on normal postnatal and adult tissues. Furthermore, ROR1 appears to play a functional role in the tumorigenesis of PCa, including the proliferation of cancer-specific stem cells. ROR1 is expressed on parental tumors as well as tumor and metastasis-promoting stem cells, making it an attractive therapeutic target.

[0007] WNT signaling is known to regulate many important cellular processes, and aberrant Wnt signaling and mutations in the pathway have been associated with tumorigenesis, progression, and metastasis in many cancers, including prostate cancer (5, 6). Similarly, deregulated expression of the WNT coreceptors ROR1 and ROR2 has been associated with several cellular features that promote malignancy, namely, cell proliferation, survival, migration / invasion, and stemness. Wnt signaling was originally discovered as a group of signaling pathways important for normal development and physiology (5, 6). Aberrant Wnt signaling, consisting of canonical (β-catenin-dependent) and noncanonical pathways, is frequently altered in prostate cancer. Comprehensive sequencing studies in patients with castration-resistant prostate cancer (CRPC) have identified recurrent molecular alterations in Wnt signaling pathway components in approximately 20% of patients with advanced prostate cancer (4). Analysis of circulating tumor cells (CTCs) from CRPC patients demonstrates expression of Wnt5A, a prototypic noncanonical Wnt ligand, in >60% of patients with refractory disease ( 7 , 8 ).

[0008] The Wnt signaling pathway is complex, and the context-dependent activity of Wnt signaling is mediated through crosstalk between canonical and non-canonical Wnt signaling. The Wnt pathway interacts with androgen receptor (AR) signaling, a key pathway in the pathogenesis of prostate cancer (9). Non-canonical Wnt signaling is mediated in part through ROR1 tyrosine kinase-like orphan receptor activation by the Wnt5A ligand (Figure 1). ROR1 is a conserved oncofetal surface protein expressed in prostate cancer cells (10). ROR1 expression can enhance epithelial-mesenchymal transition, cancer cell proliferation, migration, and metastasis (11-13). Non-canonical Wnt signaling mediated through ROR1 promotes bone metastasis through induction of osteoblast activity (14). Furthermore, ROR1 is overexpressed in chemotherapy-resistant tumors (15, 16). ROR1 expression is associated with adverse outcomes in patients, such as poor therapeutic response and tumor recurrence (17-21). ROR1 regulates the stability and transcription of the drug efflux pump ABCB1, which is overexpressed in chemotherapy-resistant tumors (22). ROR1 inhibition sensitizes cancer cells to chemotherapeutic agents and directly correlates with reduced efflux of chemotherapeutic agents from tumor cells (22). Furthermore, the interaction between ROR1 and cortactin plays an important role in cancer cell migration and metastasis (23, 24).

[0009] In preclinical models, Wnt5A stimulates and accelerates prostate cancer tumor growth, which is reduced by Wnt5A knockdown or haploinsufficiency (12). In addition to playing a role in tumorigenesis, non-canonical Wnt ligands have been found to induce drug resistance to enzalutamide in vitro (25). Several clinical studies in advanced prostate cancer patients have found that the non-canonical Wnt pathway is activated in advanced prostate cancer and correlates with poor prognosis in CRPC (8, 26-31). Whole-transcriptome RNA-seq of single CTCs demonstrates significant enrichment of non-canonical Wnt signaling in patients with resistance to enzalutamide (7). Furthermore, Wnt5A expression in CTCs using multiplex qPCR has found that Wnt5A independently predicts worse overall survival (8, 26).

[0010] To target this proto-oncogene, a first-in-class ROR1-binding monoclonal antibody (mAb), cirumutuzumab, was developed at the University of California, San Diego (UCSD) (32). This human mAb has high affinity for human ROR1 and no obvious off-target activity in in vitro or in vivo test systems. In clinical trials, the anti-ROR1 mAb was well tolerated, with no antibody-related serious adverse events and an extended half-life, suggesting limited or no off-tumor binding and early indications of anti-tumor activity (NCT02222688) (33). Due to its favorable therapeutic index, cirumutuzumab has subsequently been used in several clinical studies targeting lymphoid and solid tumor cancers as the targeting moiety of an antibody-drug conjugate (ADC) (NCT03833180) in combination with standard chemotherapy (NCT02776917, 02860676 and 03088878), and clinical studies with this mAb are planned for patients with CRPC.

[0011] Elevated expression of ROR1 has been demonstrated in highly malignant taxane-resistant breast cancer cells. Sirmutuzumab has demonstrated substantial therapeutic activity against these chemotherapy-resistant tumors in preclinical models, as this mAb blocks ROR1 signaling by blocking Wnt5A-mediated activation. Based on these studies, a Phase Ib clinical trial combining cirumutuzumab with paclitaxel is being tested in patients with advanced breast cancer (NCT02776917) (34). Similar to breast cancer, taxane (docetaxel)-based chemotherapy regimens are the mainstay of treatment for patients with advanced CRPC (35). However, when combined with abiraterone and enzalutamide, this docetaxel-containing chemotherapy cocktail has minimal activity in the treatment of CPRC, with a median time to progression of 3 months and limited impact on reducing PSA levels (less than 30% of patients) (36). Furthermore, non-canonical Wnt signaling has been hypothesized to be a mechanism of resistance to enzalutamide (7) and taxane chemotherapy (16). Available data suggest that chemotherapy-mediated Wnt blockade may be the most effective method to effect Wnt pathway modulation (6).

[0012] There is an urgent need to achieve potent and specific therapies for the treatment of progressive, malignant and fatal neuroendocrine tumors. The methods provided herein address these and other needs in the art. Summary of the Invention

[0013] Described herein are compositions and methods for treating neuroendocrine cancer, including, inter alia, administration of cells expressing a chimeric antigen receptor targeting human ROR-1, as well as methods for preventing or treating metastasis from neuroendocrine cancer. The chimeric antigen receptors described herein can be expressed by T lymphocytes or natural killer cells isolated from an individual suffering from neuroendocrine cancer and re-administered to the individual. Administration of T cells expressing a CAR described herein can serve as an effective therapeutic treatment for neuroendocrine cancer expressing ROR-1. The function and efficacy of anti-ROR-1 CAR-T cells can be improved by optimized intracellular signaling domains and membrane spacers, as described herein.

[0014] In an aspect, the chimeric antigen receptor comprises: i) an antigen binding region, the antigen binding region specifically binds to ROR-1, the antigen binding region comprising a light chain variable domain and a heavy chain variable domain; ii) a spacer domain; iii) a transmembrane domain; and iv) an intracellular domain. In some embodiments, the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48. Alternatively, the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54.

[0015] In embodiments, the light chain variable domain is covalently linked to the heavy chain variable domain via a polypeptide linker. In embodiments, the polypeptide linker comprises the amino acid sequence of SEQ ID NO:24.

[0016] In embodiments, the spacer domain comprises an antibody domain, hi embodiments, the antibody domain comprises an immunoglobulin hinge domain, an immunoglobulin constant heavy 3 (CH3) domain, an immunoglobulin constant heavy 2 (CH2) domain, or a combination thereof.

[0017] In embodiments, the spacer domain comprises an amino acid sequence represented by SEQ ID NO:29, SEQ ID NO:41 or SEQ ID NO:42.

[0018] In embodiments, the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:21.

[0019] In embodiments, the heavy chain variable domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:27.

[0020] In embodiments, the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof, hi embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0021] In embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain, an intracellular T cell signaling domain, or a combination thereof. The intracellular costimulatory signaling domain is a 4-1BB intracellular costimulatory signaling domain, a CD28 intracellular costimulatory signaling domain, an ICOS intracellular costimulatory signaling domain, an OX-40 intracellular costimulatory signaling domain, or any combination thereof. In embodiments, the intracellular costimulatory signaling domain comprises a 41-BB intracellular costimulatory signaling domain. In embodiments, the intracellular costimulatory signaling domain comprises a CD28 intracellular costimulatory signaling domain and a 41-BB intracellular costimulatory signaling domain.

[0022] In embodiments, the intracellular costimulatory signaling domain further comprises an intracellular T cell signaling domain, hi embodiments, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0023] In embodiments, the chimeric antigen receptor binds to a cell expressing ROR-1. In embodiments, the cell expressing ROR-1 is a neuroendocrine cancer cell.

[0024] In an embodiment, the cell is a T lymphocyte. In an embodiment, the T lymphocyte is a CD4+ T lymphocyte or a CD8+ T lymphocyte.

[0025] In embodiments, the cells are natural killer cells. In embodiments, the natural killer cells are autologous to the subject. In embodiments, the natural killer cells are xenogeneic to the subject. In embodiments, the natural killer cells are allogeneic to the subject.

[0026] Aspects disclosed herein provide a nucleic acid encoding a chimeric antigen receptor as described herein. In embodiments, the nucleic acid is a viral vector. In embodiments, the viral vector is a lentiviral vector.

[0027] Aspects disclosed herein provide a cell comprising a nucleic acid as described herein. Aspects disclosed herein also provide a cell expressing a chimeric antigen receptor as described herein. In embodiments, the cell is a T lymphocyte. In embodiments, the T lymphocyte is a CD4+ T lymphocyte or a CD8+ T lymphocyte. In embodiments, the cell is a natural killer cell, a genetically engineered natural killer cell, or a CD56+ cell. In embodiments, the cell is a natural killer cell. In embodiments, the natural killer cell is autologous to the subject. In embodiments, the natural killer cell is xenogeneic to the subject. In embodiments, the natural killer cell is allogeneic to the subject.

[0028] Aspects disclosed herein provide a pharmaceutical composition comprising a therapeutically effective amount of the cells described herein and a pharma- ceutical acceptable diluent, carrier, or excipient. In embodiments, the composition is formulated for intravenous injection.

[0029] Aspects disclosed herein provide methods of treating neuroendocrine cancer in a subject in need thereof, and methods of preventing or treating metastasis in a subject having neuroendocrine cancer, comprising administering to the subject the cells described herein, as well as pharmaceutical compositions comprising the cells and excipients, additives, etc. provided herein. In embodiments, the neuroendocrine cancer is a carcinoid tumor, a pancreatic islet cell tumor, a medullary thyroid carcinoma, a pheochromocytoma, a neuroendocrine carcinoma of the skin, a small cell lung cancer, a large cell neuroendocrine carcinoma, a neuroendocrine prostate cancer (NEPC), or a metastatic castration-resistant prostate cancer (CRPC). In embodiments, the neuroendocrine cancer is a metastatic castration-resistant prostate cancer (CRPC).

[0030] In embodiments, the neuroendocrine cancerous cells express ROR-1. In embodiments, the subject has not responded to androgen deprivation therapy. In embodiments, the neuroendocrine cancer has metastasized to bone.

[0031] In embodiments, the methods provided herein further comprise administering cirumutuzumab to the subject. In embodiments, cirumutuzumab and the cells expressing a disclosed chimeric antigen receptor are administered separately. In embodiments, cirumutuzumab and the cells are administered together.

[0032] In embodiments, the methods provided herein further comprise administering a platinum-based chemotherapy to the subject. In embodiments, the platinum-based chemotherapy comprises carboplatin, cisplatin, etoposide, a taxane, or any combination thereof. In embodiments, the platinum-based chemotherapy comprises administering an antibody-drug conjugate to the subject. [Brief description of the drawings]

[0033] [Figure 1]Schematic diagram showing that signaling through the non-canonical Wnt pathway is mediated by binding of Wnt5A to its receptors ROR1 and ROR2. Sirmutuzumab is a monoclonal antibody that targets ROR1. [Diagram 2] FIG. 1 is a schematic diagram showing cirumutuzumab-based chimeric antigen receptor (CAR) T cells targeting ROR-1. [Diagram 3] Figure 1 shows graphs depicting in vitro cell killing activity of ROR1 CAR T cells derived from T cells of two healthy donors transduced with ROR1 in a 4-hour chromium release assay (left panel) and a 120-hour ACEA impedance assay (right panel), where T cells were transduced against leukemic Mec1 ROR1-positive cells at the indicated effect-to-target ratios (E:T) in the left panel, and against MB 231 ROR1-positive breast cancer cells in the ACEA impedance assay in the right panel. Anti-ROR1 CAR T cells showed high and specific cytotoxicity without significant killing of ROR1-negative target cells. [Figure 4-1] Bioluminescence imaging of mice inoculated with MEC1-ROR1 cells and ROR1 CAR T cells are shown. Animals treated with CAR-T cells had reduced disease burden compared to controls. The highest dose (3x106 CAR-T cells) cohort showed reduced leukemic burden to background levels by day 30 and had minimal disease over the duration of the study. Animals in the control group (untreated, mock transduced) had to be sacrificed on day 20. The right panel shows the total bioluminescence product harvested from the mice. Blue squares and circles are controls and green triangles are ROR1-CAR T treated mice. [Figure 4-2]Bioluminescence imaging of mice inoculated with MEC1-ROR1 cells and ROR1 CAR T cells are shown. Animals treated with CAR-T cells had reduced disease burden compared to controls. The highest dose (3x106 CAR-T cells) cohort showed reduced leukemic burden to background levels by day 30 and had minimal disease over the duration of the study. Animals in the control group (untreated, mock transduced) had to be sacrificed on day 20. The right panel shows the total bioluminescence product harvested from the mice. Blue squares and circles are controls and green triangles are ROR1-CAR T treated mice. [Figure 5-1] Figure 1 shows ROR-1 CAR T cell expression in mouse tissues at various time points. After ROR-1 CAR-T administration, animals were sacrificed on days 11 (upper panel) and 25 (lower panel). Blood and organs were collected and subjected to flow analysis for CAR expression and confirmed ROR1 binding activity. ROR-1 CAR-T cell numbers were substantially higher in mice carrying MEC-1 ROR1 cells (CAR+MEC1-ROR1) versus controls (CAR only), indicating elevated proliferation of ROR-1 CAR-T cells in tumor-burdened animals. Bars represent the mean of five mice per group, and error bars represent SE of the mean. [Figure 5-2] Figure 1 shows ROR-1 CAR T cell expression in mouse tissues at various time points. After ROR-1 CAR-T administration, animals were sacrificed on days 11 (upper panel) and 25 (lower panel). Blood and organs were collected and subjected to flow analysis for CAR expression and confirmed ROR1 binding activity. ROR-1 CAR-T cell numbers were substantially higher in mice carrying MEC-1 ROR1 cells (CAR+MEC1-ROR1) versus controls (CAR only), indicating elevated proliferation of ROR-1 CAR-T cells in tumor-burdened animals. Bars represent the mean of five mice per group, and error bars represent SE of the mean. [Figure 6]1 shows results from an analysis of tumor RNA sequences from 66 prostate cancer samples, expressed as single sample gene set enrichment analysis profiles of non-canonical WNT and stem cell gene sets in association with expression of ROR1 (mRNA). The analysis shows significant enrichment of WNT non-canonical and stem cell gene sets for ROR1 mRNA expression. [Figure 7A] Xenograft models obtained from prostate cancer patients are shown. Figure 7A shows hierarchical clustering of RNA-seq analysis for PCSD1 and PCSD13. Figure 7B shows flow cytometry (FACS) of ROR-1 protein on the cell surface of PCSD13 cells. Figure 7C shows that ROR1 is expressed on the cell surface of prostate cancer cell lines. Antibody isotype controls are in grey. Negative control cell lines: breast cancer line, MCF7, and ROR-1 overexpressing line, MCF7_ROR1. Prostate cancer lines: PC3 and DU145. [Figure 7B] Xenograft models obtained from prostate cancer patients are shown. Figure 7A shows hierarchical clustering of RNA-seq analysis for PCSD1 and PCSD13. Figure 7B shows flow cytometry (FACS) of ROR-1 protein on the cell surface of PCSD13 cells. Figure 7C shows that ROR1 is expressed on the cell surface of prostate cancer cell lines. Antibody isotype controls are in grey. Negative control cell lines: breast cancer line, MCF7, and ROR-1 overexpressing line, MCF7_ROR1. Prostate cancer lines: PC3 and DU145. [Figure 7C-1] Xenograft models obtained from prostate cancer patients are shown. Figure 7A shows hierarchical clustering of RNA-seq analysis for PCSD1 and PCSD13. Figure 7B shows flow cytometry (FACS) of ROR-1 protein on the cell surface of PCSD13 cells. Figure 7C shows that ROR1 is expressed on the cell surface of prostate cancer cell lines. Antibody isotype controls are in grey. Negative control cell lines: breast cancer line, MCF7, and ROR-1 overexpressing line, MCF7_ROR1. Prostate cancer lines: PC3 and DU145. [Figure 7C-2]Xenograft models obtained from prostate cancer patients are shown. Figure 7A shows hierarchical clustering of RNA-seq analysis for PCSD1 and PCSD13. Figure 7B shows flow cytometry (FACS) of ROR-1 protein on the cell surface of PCSD13 cells. Figure 7C shows that ROR1 is expressed on the cell surface of prostate cancer cell lines. Antibody isotype controls are in grey. Negative control cell lines: breast cancer line, MCF7, and ROR-1 overexpressing line, MCF7_ROR1. Prostate cancer lines: PC3 and DU145. [Figure 8] PCSD1 PDX tumor cells derived from 3D organoids replicate the tissue morphology of xenografts growing in femurs. Three-dimensional organoid cell cultures were established from subjects with prostate cancer. The 3D cultures consisted of two major cell masses: spheroids and epithelial cysts that resembled the gland-like structures seen in sections from xenografts growing in femurs and in patients replacing bone marrow. The spheroids were resistant to androgen deprivation-induced death. The spheroid masses contained cyst-like structures surrounded by tumor cells that resembled gland-like structures. Black arrows point to the cystic structures. The organoids are GFP+. PDXs were obtained from prostate cancer bone metastases. PCSD1 expressing GFP-luciferase formed distinct morphologies under 3D culture conditions. Enzalutamide treatment of PCSD1 reduced the luminal size and the number of epithelial cysts. Transcriptome analysis showed that AR-responsive genes such as PSA (KLK3) and TMPRSS2 were downregulated under ADT, while stem cell transcription factors, steroidogenic and neurogenic pathway genes were upregulated. Thus, although antiandrogens were still able to suppress canonical AR-responsive genes, tumor and organoid growth was resistant to ADT, including the antiandrogen enzalutamide. [Figure 9-1]Graph showing that ROR1 CAR-T cells kill ROR1-expressing prostate cancer cell lines. ROR1 CAR-T cells showed significant cytotoxic killing of PCa cell lines, PC3 or DU145 cells in culture. Increasing effect (E=T cells) to target (T=PCa cells) ratios showed increased killing compared to control T cells in an Incucyte assay. CAR-Ts cytotoxic T cell killing is effect:target (E:T) dose dependent. [Figure 9-2] Graph showing that ROR1 CAR-T cells kill ROR1-expressing prostate cancer cell lines. ROR1 CAR-T cells showed significant cytotoxic killing of PCa cell lines, PC3 or DU145 cells in culture. Increasing effect (E=T cells) to target (T=PCa cells) ratios showed increased killing compared to control T cells in an Incucyte assay. CAR-Ts cytotoxic T cell killing is effect:target (E:T) dose dependent. [Figure 10A] Graphs are presented showing that anti-ROR1 CAR-T cells specifically killed PDX pCSD13 cells in an effector:target (E:T) dose-dependent manner in culture in an Incucyte cytotoxicity assay. Left panel (A): Control T cells + PCSD13 (target) cells freshly isolated from xenografts and cultured for 48 hours. Right panel (B): Anti-ROR1 CAR-T (effector). The highest dose of anti-ROR1 CAR-T cells completely killed PCSD13 cells (blue line, E:T 3) compared to no CAR-T (black line, E:T 0) [Figure 10B] Graphs are presented showing that anti-ROR1 CAR-T cells specifically killed PDX pCSD13 cells in an effector:target (E:T) dose-dependent manner in culture in an Incucyte cytotoxicity assay. Left panel (A): Control T cells + PCSD13 (target) cells freshly isolated from xenografts and cultured for 48 hours. Right panel (B): Anti-ROR1 CAR-T (effector). The highest dose of anti-ROR1 CAR-T cells completely killed PCSD13 cells (blue line, E:T 3) compared to no CAR-T (black line, E:T 0) [Figure 11] Bioluminescence imaging of mice implanted subcutaneously with PC3 PCa cells and treated with ROR1 CAR-T cells. Animals were implanted with GFP luciferase expressing PC3 cells on day 1 and treated with a single injection of 3e7 CAR-T cells IV (CAR IV) or activated mock transduced cells (control) or 1e7 cells administered intratumorally (CAR IT). As shown, CAR-T treated mice that received a single intravenous or intratumoral injection had reduced disease burden when compared to control animals and had to be sacrificed by week 5. The CAR-T IV treated cohort had minimal disease at the end of the experiment. [Figure 12] 11 is a graph showing tumor volume measurement of transplanted PC3 cells by caliper measurement. Tumor volume was measured by weekly caliper measurement for mice treated as described in FIG. 11. The results are consistent with bioluminescence measurement and better demonstrate the activity of intravenously administered CAR product. Tumor volume was determined using the formula (mm3)=(L×w2) / 2. The value w (width) is the smaller of the two orthogonal tumor axes, and the value L (length) is the larger of the two orthogonal axes. Error bars represent the mean SO of triplicate measurements. [Figure 13] Optimized IHC staining analysis of ROR1 protein expression patterns in formalin-fixed paraffin-embedded (FFPE) breast adenocarcinoma tissues compared to negative controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] definition While various embodiments and aspects of the present invention have been shown and described herein, it will be understood by those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention.

[0035] The section headings used herein are for organizational purposes and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application (including, but not limited to, patents, patent applications, articles, books, manuals, and papers) are expressly incorporated by reference in their entirety for any purpose.

[0036] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known to the chemical arts.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.See, for example, Singleton et al., Dictionary Of Microbiology And Molecular Biology 2nd ed., J.Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention.The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0038] As used herein, the term "about" refers to a range of values ​​that includes the particular value and that one of ordinary skill in the art would consider to be reasonably similar to the particular value. In embodiments, the term "about" refers to within a standard deviation using generally accepted measurements in the art. In embodiments, about refers to a range that covers + / - 10% of the particular value. In embodiments, about refers to the specified value.

[0039] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their complements in either single-stranded or double-stranded form. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Nucleic acid, as used herein, also refers to nucleic acids that have the same basic chemical structure as naturally occurring nucleic acids. Such analogs have modified sugars and / or modified peptide backbones, but retain the same basic chemical structure as naturally occurring nucleic acids. Nucleic acid mimetics refer to compounds that have a structure that differs from the general chemical structure of nucleic acids, but function in a similar manner to naturally occurring nucleic acids. Examples of such analogs include, but are not limited to, phosphorothiolates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0040] The term "gene" refers to a segment of DNA involved in producing a protein. It includes the regions preceding and following the coding region (leader and trailer), as well as the intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements required during transcription and translation of a gene. Furthermore, a "protein gene product" is the protein expressed from a particular gene.

[0041] The term "expression" or "expressed" as used herein with respect to a gene refers to the transcription and / or translation product of that gene. The expression level of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell. The expression level of a non-coding nucleic acid molecule can be detected by standard PCR or Northern blot techniques well known in the art. See Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0042] Expression of the transfected gene may occur transiently or stably in the cell. During "transient expression", the transfected gene is not transmitted to daughter cells during cell division. Since the expression of the gene is limited to the transfected cell, the expression of the gene is lost over time. In contrast, stable expression of the transfected gene can be achieved by co-transfecting the transfected gene with another gene that confers an advantage during selection to the transfected cell. Such a selection advantage may be resistance to a particular toxin presented to the cell.

[0043] The terms "transfection", "transduction", "transfecting" or "transducing" may be used interchangeably throughout and are defined as the process of introducing a nucleic acid molecule and / or a protein into a cell. Non-viral or virus-based methods can be used to introduce a nucleic acid into a cell. The nucleic acid molecule may be a genetic sequence encoding a complete protein or a functional portion thereof. Non-viral methods of transfection include any suitable transfection method that does not use viral DNA or viral particles as a delivery system to introduce a nucleic acid molecule into a cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection via heat shock, magnetofection and electroporation. In some embodiments, the nucleic acid molecule is introduced into a cell using electroporation according to standard procedures well known in the art. In the case of viral-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some embodiments, the nucleic acid molecule is introduced into the cell using a retroviral vector, according to standard procedures well known in the art. The term "transfection" or "transduction" also refers to the introduction of a protein into a cell from the external environment. Typically, protein transduction or transfection relies on the attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, for example, Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0044] The term "plasmid" or "expression vector" refers to a nucleic acid molecule that encodes a gene and / or regulatory elements necessary for expression of the gene. Expression of a gene from a plasmid can occur in cis or trans. When a gene is expressed in cis, the gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to when the gene and regulatory elements are encoded by separate plasmids.

[0045] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0046] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0047] When used in the context of numbering of a given amino acid or polynucleotide sequence, the term "numbered with reference to" or "corresponding to" refers to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue of a protein "corresponds" to a given residue if it occupies the same essential structural position in the protein as the given residue. A person skilled in the art will immediately recognize the identity and position of a residue that corresponds to a particular position of a protein (e.g., ROR-1) in other proteins using different numbering systems. For example, by performing a simple sequence alignment with a protein (e.g., ROR-1), the identity and position of a residue that corresponds to a particular position of a protein is identified in other protein sequences that align to this protein. For example, a selected residue of a selected protein corresponds to a glutamic acid at position 138 if the selected residue occupies the same essential spatial or other structural relationship as the glutamic acid at position 138. In some embodiments, when the selected proteins are aligned for maximum homology in the proteins, positions in the aligned selected proteins that align with glutamic acid 138 are said to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three-dimensional structural alignment can also be used, for example, the structures of the selected proteins are aligned for maximum correspondence with glutamic acid at position 138 and the overall structures are compared. In this case, the amino acid occupying the same essential position in the structural model as glutamic acid 138 is the glutamic acid 138 residue.

[0048] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which may optionally be linked to moieties not composed of amino acids. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0049] The term "recombinant," when used with reference to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by, or is the result of, a laboratory method. Thus, for example, recombinant protein includes proteins produced by laboratory methods. Recombinant proteins may contain amino acid residues that are not found in the naturally occurring (non-recombinant) form of the protein or may contain modified (e.g., labeled) amino acid residues.

[0050] The term "isolated," as applied to a nucleic acid or protein, means that the nucleic acid or protein is essentially free of other cellular components with which it is naturally associated. It can be, for example, in a homogeneous state, either dry or in an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is present as the predominant species in a preparation is substantially purified.

[0051] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a certain percentage (i.e., about 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a particular region, e.g., an entire polypeptide sequence of the invention or an individual domain of a polypeptide of the invention) of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are thus "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, and more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0052] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). This percentage is calculated by determining the number of positions where an identical nucleic acid base or amino acid residue is present in both sequences to generate a number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate a percentage of sequence identity.

[0053] In sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence compared to reference sequence based on program parameters.

[0054] A "comparison window" as used herein includes reference to, for example, a full length sequence or any segment of a number of contiguous positions selected from the group consisting of 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, in which the same number of contiguous positions of a sequence can be compared with a reference sequence after optimally aligning the two sequences. Any method of alignment of sequences for comparison known in the art is contemplated. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similar methods of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0055] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is halted when the cumulative alignment score falls off its maximum achieved value by an amount X, when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length of 3 and an expectation (E) of 10, with the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) using as defaults an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0056] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0057] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with an antibody against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically or substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0058] Antibodies are large, complex molecules (molecular weight about 150,000, or about 1320 amino acids) with a complex internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light and one heavy chain. The light and heavy chains each consist of two regions: a variable ("V") region, which is responsible for binding the target antigen, and a constant ("C") region, which interacts with other components of the immune system. The variable regions of the light and heavy chains come together in three-dimensional space to form the variable region that binds to the antigen (e.g., a receptor on the surface of a cell). Within each variable region of the light or heavy chain, there are three short segments (average 10 amino acids long) called complementarity determining regions ("CDRs"). The six CDRs of an antibody variable domain (three from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody binding site, which docks to the target antigen. The positions and lengths of the CDRs are precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1983, 1987. The parts of the variable regions not included in the CDRs are called the framework ("FR") and form the surroundings of the CDRs.

[0059] An "antibody variant" as provided herein refers to a polypeptide capable of binding to an antigen and comprising one or more structural domains of an antibody or fragment thereof (e.g., a light chain variable domain, a heavy chain variable domain). Non-limiting examples of antibody variants include single domain antibodies or nanobodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A "peptibody" as provided herein refers to a peptide moiety attached (via a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies and their variable regions from camelids, and methods of their production, isolation, and use can be found in references WO 97 / 49805 and WO 97 / 49805, which are incorporated herein by reference in their entirety for all purposes. Similarly, antibodies and their variable regions from cartilaginous fish, and methods of their production, isolation, and use can be found in WO 2005 / 118629, which are incorporated herein by reference in their entirety for all purposes.

[0060] The terms "CDR L1", "CDR L2" and "CDR L3" provided herein refer to complementarity determining regions (CDRs) 1, 2 and 3 of the variable light chain (L) of an antibody. In some embodiments, the variable light chain provided herein comprises, from N-terminus to C-terminus, CDR L1, CDR L2 and CDR L3. Similarly, the terms "CDR H1", "CDR H2" and "CDR H3" provided herein refer to complementarity determining regions (CDRs) 1, 2 and 3 of the variable heavy chain (H) of an antibody. In some embodiments, the variable light chain provided herein comprises, from N-terminus to C-terminus, CDR L1, CDR L2 and CDR L3.

[0061] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bonds in the hinge region to produce dimers of Fab, F(ab)'2, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments have been defined in terms of the digestion of intact antibodies, one of skill in the art will understand that such fragments can be synthesized chemically or de novo using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodology (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0062] The term "antigen" as provided herein refers to a molecule that can bind to an antibody binding domain as provided herein. An "antigen binding domain" as provided herein is a region of an antibody that binds to an antigen (epitope). As described above, an antigen binding domain is generally composed of one constant domain and one variable domain of each of the heavy and light chains (VL, VH, CL and CH1, respectively). The paratope or antigen binding site is formed at the N-terminus of the antigen binding domain. The two variable domains of the antigen binding domain usually bind to an epitope on the antigen.

[0063] Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bonds in the hinge region to produce dimers of Fab, F(ab)'2, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions to break the disulfide bonds in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially the antigen-binding portion with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments have been defined in terms of the digestion of intact antibodies, one of skill in the art will understand that such fragments can be synthesized chemically or de novo using recombinant DNA methodology. Thus, the term antibody as used herein also includes antibody fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodology (e.g., single chain Fvs) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0064] Single chain variable fragments (scFv) are typically fusion proteins of the variable regions of immunoglobulin heavy chains (VH) and light chains (VL), connected by a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, or rich in serine or threonine for solubility. The linker may connect the N-terminus of VH to the C-terminus of VL, or vice versa.

[0065] The term "polypeptide linker", otherwise referred to as "polypeptide sequence", refers to a polypeptide segment that covalently links two adjacent domains in a protein. As used herein, a "polypeptide linker" or "polypeptide sequence" covalently connects a light chain variable domain to a heavy chain variable domain. A linker is a VH N-terminus of V L or vice versa.

[0066] Polypeptide linkers may range from about 20 to about 60 amino acids in length. Short linker peptides may contain about 10 to about 25 amino acids. In embodiments, polypeptide linkers contain 10-50 amino acids. In embodiments, polypeptide linkers contain 10-40 amino acids. In embodiments, polypeptide linkers contain 10-30 amino acids. In embodiments, polypeptide linkers contain 10-20 amino acids. In embodiments, polypeptide linkers contain 10-15 amino acids. The linker is typically rich in glycine for flexibility and may be rich in serine or threonine for solubility.

[0067] The epitope of an antibody is the region of its antigen to which the antibody binds. Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 30%, preferably 50%, 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

[0068] The term "ROR-1" or "ROR1" as used herein refers to either recombinant or naturally occurring forms of tyrosine kinase-like orphan receptor 1 (ROR-1) or variants or homologs thereof that maintain ROR-1 activity (e.g., in the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to ROR-1). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring ROR-1 protein. In embodiments, the ROR-1 protein is substantially identical to a protein identified by Accession Number NP_005003.1, or a variant or homolog having substantial identity thereto. In an embodiment, the ROR-1 protein comprises the amino acid sequence of SEQ ID NO: 55. In an embodiment, the ROR-1 protein is the amino acid sequence of SEQ ID NO: 55. In an embodiment, the ROR-1 protein comprises the amino acid sequence of SEQ ID NO: 56. In an embodiment, the ROR-1 protein comprises the amino acid sequence of SEQ ID NO: 57.

[0069] The terms "cirmutuzumab," "UC-961," and "99961.1" refer to a humanized monoclonal antibody capable of binding to the extracellular domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR-1). In embodiments, cirmutuzumab is any one of the antibodies or fragments thereof disclosed in U.S. Patent Application No. 14 / 422,519, which is incorporated herein by reference in its entirety for all purposes.

[0070] As used herein, the term "CD28 transmembrane domain" refers to either a recombinant or naturally occurring form of the transmembrane domain of CD28, or a variant or homolog thereof, that maintains CD28 transmembrane activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD28 transmembrane domain). In some embodiments, a variant or homolog will retain the entire sequence or a portion of the sequence (e.g., In embodiments, the CD28 transmembrane domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over a portion of at least one contiguous amino acid (e.g., 50, 100, 150, or 200 contiguous amino acids). In embodiments, the CD28 transmembrane domain is a human CD28 transmembrane domain protein. In embodiments, the variant or mutant of the CD28 transmembrane domain protein comprises no more than 5, 4, 3, 2, or 1 deletion compared to a naturally occurring ... In an embodiment, the CD28 transmembrane domain protein variant or mutant comprises no more than 5, 4, 3, 2, or 1 insertion compared to a naturally occurring CD28 transmembrane domain protein. In an embodiment, the CD28 transmembrane domain protein variant or mutant comprises no more than a deletion compared to a naturally occurring CD28 transmembrane domain protein. In an embodiment, the CD28 transmembrane domain protein variant or mutant comprises no more than an insertion compared to a naturally occurring CD28 transmembrane domain protein. In an embodiment, the CD28 transmembrane domain protein variant or mutant comprises no more than an insertion compared to a naturally occurring CD28 transmembrane domain protein. In an embodiment, the CD28 transmembrane domain protein variant or mutant comprises a substitution that is a conservative substitution compared to a naturally occurring CD28 transmembrane domain protein. In an embodiment, the CD28 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001230006.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD28 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001230007.1, or an isoform or naturally occurring mutant or variant thereof.In embodiments, the CD28 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_006130.1, or an isoform or naturally occurring mutant or variant thereof.

[0071] The term "CD4 transmembrane domain" as used herein refers to either recombinant or naturally occurring forms of the transmembrane domain of CD4 or variants or homologues thereof that maintain CD4 transmembrane domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD4 transmembrane domain). In some aspects, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring CD4 transmembrane domain polypeptide. In embodiments, the CD4 transmembrane domain is a human CD4 transmembrane domain protein. In embodiments, the variant or mutant of the CD4 transmembrane domain protein comprises no more than 5, 4, 3, 2, or 1 deletion compared to a naturally occurring CD4 transmembrane domain protein. In an embodiment, the variant or mutant of the CD4 transmembrane domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to a naturally occurring CD4 transmembrane domain protein. In an embodiment, the variant or mutant of the CD4 transmembrane domain protein comprises no deletions compared to a naturally occurring CD4 transmembrane domain protein. In an embodiment, the variant or mutant of the CD4 transmembrane domain protein comprises no insertions compared to a naturally occurring CD4 transmembrane domain protein. In an embodiment, the variant or mutant of the CD4 transmembrane domain protein comprises substitutions that are conservative substitutions compared to a naturally occurring CD4 transmembrane domain protein. In an embodiment, the CD4 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_000607.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD4 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001181943.1, or an isoform or naturally occurring mutant or variant thereof.In embodiments, the CD4 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001181944.1, or an isoform or naturally occurring mutant or variant thereof. In embodiments, the CD4 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001181945.1, or an isoform or naturally occurring mutant or variant thereof. In embodiments, the CD4 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001181946.1, or an isoform or naturally occurring mutant or variant thereof.

[0072] The term "CD8 transmembrane domain" as used herein refers to either a recombinant or naturally occurring form of the transmembrane domain of CD8 or a variant or homolog thereof that maintains CD8 transmembrane domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD8 transmembrane domain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring CD8 transmembrane domain polypeptide. In embodiments, the CD8 transmembrane domain is a CD8A transmembrane domain. In embodiments, the CD8 transmembrane domain is a CD8B transmembrane domain. In embodiments, the CD8 transmembrane domain is a human CD8 transmembrane domain protein. In an embodiment, the variant or mutant of the CD8 transmembrane domain protein comprises no more than 5, 4, 3, 2, or 1 deletion compared to a naturally occurring CD8 transmembrane domain protein. In an embodiment, the variant or mutant of the CD8 transmembrane domain protein comprises no more than 5, 4, 3, 2, or 1 insertion ... a deletion compared to a naturally occurring CD8 transmembrane domain protein. In an embodiment, the variant or mutant of the CD8 transmembrane domain protein comprises no more than an insertion compared to a naturally occurring CD8 transmembrane domain protein. In an embodiment, the variant or mutant of the CD8 transmembrane domain protein comprises substitutions that are conservative substitutions compared to a naturally occurring CD8 transmembrane domain protein. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001139345.1, or an isoform or naturally occurring mutant or variant thereof.In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_001181943.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_001181944.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_001181945.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_741969.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_001759.3, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference XP_011531466.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_001171571.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_757362.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_742100.1, or an isoform or naturally occurring mutant or variant thereof. In an embodiment, the CD8 transmembrane domain comprises all or a portion of a protein specified by NCBI sequence reference NP_742099.1, or an isoform or naturally occurring mutant or variant thereof.In embodiments, the CD8 transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_004922.1, or an isoform or naturally occurring mutant or variant thereof.

[0073] As used herein, the term "CD3-zeta transmembrane domain" refers to either a recombinant or naturally occurring form of a transmembrane domain of CD3-zeta, or a variant or homolog thereof, that maintains CD3-zeta transmembrane domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to a CD3-zeta transmembrane domain). In some embodiments, the variant or homolog is a naturally occurring CD3-zeta transmembrane domain. Compared to the main peptide, the CD3-zeta transmembrane domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion). In embodiments, the CD3-zeta transmembrane domain is a human CD3-zeta transmembrane domain protein. In embodiments, the variant or mutant of the CD3-zeta transmembrane domain protein has a similar amino acid sequence identity to a naturally occurring CD3-zeta transmembrane domain protein. In embodiments, the CD3-zeta transmembrane domain protein variant or mutant comprises no more than 5, 4, 3, 2, or 1 deletion compared to a naturally occurring CD3-zeta transmembrane domain protein. In embodiments, the CD3-zeta transmembrane domain protein variant or mutant comprises no more than 5, 4, 3, 2, or 1 insertion ... deletion compared to a naturally occurring CD3-zeta transmembrane domain protein. In embodiments, the CD3-zeta transmembrane domain comprises all or a portion of a protein identified by NCBI sequence reference NP_000725.1, or an isoform or naturally occurring mutant or variant thereof.In embodiments, the CD3-zeta transmembrane domain comprises all or a portion of the protein identified by NCBI sequence reference NP_932170.1, or an isoform or naturally occurring mutant or variant thereof.

[0074] As used herein, the term "CD28 costimulatory domain" refers to either a recombinant or naturally occurring form of a costimulatory domain of CD28, or a variant or homolog thereof, that maintains CD28 costimulatory domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to a CD28 costimulatory domain). In some embodiments, a variant or homolog is an entire sequence or a portion of a sequence (e.g., a nucleotide sequence similar to that of a naturally occurring CD28 costimulatory domain polypeptide). In embodiments, the CD28 costimulatory domain has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over a portion of the CD28 costimulatory domain (e.g., a 50, 100, 150, or 200 contiguous amino acid portion). In embodiments, the CD28 costimulatory domain is a human CD28 costimulatory domain protein. In embodiments, the variant or mutant of the CD28 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 deletion compared to a naturally occurring CD28 costimulatory domain protein. In embodiments, the barrier of the CD28 costimulatory domain protein is In embodiments, the variant or mutant of the CD28 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to the naturally occurring CD28 costimulatory domain protein. In embodiments, the variant or mutant of the CD28 costimulatory domain protein comprises no deletions compared to the naturally occurring CD28 costimulatory domain protein. In embodiments, the variant or mutant of the CD28 costimulatory domain protein comprises no insertions compared to the naturally occurring CD28 costimulatory domain protein. In embodiments, the variant or mutant of the CD28 costimulatory domain protein comprises substitutions that are conservative substitutions compared to the naturally occurring CD28 costimulatory domain protein. In embodiments, the CD28 costimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001230006.1, or an isoform or naturally occurring mutant or variant thereof. In embodiments, the CD28 costimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_001230007.1, or an isoform or naturally occurring mutant or variant thereof.In embodiments, the CD28 costimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_006130.1, or an isoform or naturally occurring mutant or variant thereof.

[0075] As used herein, the term "4-1BB costimulatory domain" refers to either a recombinant or naturally occurring form of the costimulatory domain of 4-1BB, or a variant or homolog thereof, that maintains 4-1BB costimulatory domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the 4-1BB costimulatory domain). In some embodiments, the variant or homolog is a naturally occurring 4-1BB costimulatory domain polypeptide. In embodiments, the 4-1BB costimulatory domain is a human 4-1BB costimulatory domain protein. In embodiments, the variant or mutant of a 4-1BB costimulatory domain protein has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring 4-1BB costimulatory domain protein. In embodiments, the variant or mutant of a 4-1BB costimulatory domain protein has 5, 4, 3, 2, or 3 amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring 4-1BB costimulatory domain protein. In an embodiment, the variant or mutant of the 4-1BB costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to the naturally occurring 4-1BB costimulatory domain protein. In an embodiment, the variant or mutant of the 4-1BB costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to the naturally occurring 4-1BB costimulatory domain protein. In an embodiment, the variant or mutant of the 4-1BB costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to the naturally occurring 4-1BB costimulatory domain protein. In an embodiment, the variant or mutant of the 4-1BB costimulatory domain protein comprises substitutions that are conservative substitutions compared to the naturally occurring 4-1BB costimulatory domain protein. In an embodiment, the amino acid sequence of the 4-1BB costimulatory domain protein has at least or about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14.In embodiments, the 4-1BB costimulatory domain comprises all or a portion of the protein identified by NCBI sequence reference NP_001552.2, or an isoform or naturally occurring mutant or variant thereof.

[0076] As used herein, the term "ICOS costimulatory domain" refers to either a recombinant or naturally occurring form of the costimulatory domain of ICOS or a variant or homolog thereof that maintains ICOS costimulatory domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the ICOS costimulatory domain). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring ICOS costimulatory domain polypeptide. In embodiments, the ICOS costimulatory domain is a human "ICOS costimulatory domain protein. In embodiments, the variant or mutant of the ICOS costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 deletions compared to a naturally occurring "ICOS costimulatory domain protein. In embodiments, the variant or mutant of the "ICOS costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to a naturally occurring "ICOS costimulatory domain protein. In embodiments, the variant or mutant of the ICOS costimulatory domain protein does not comprise a deletion compared to a naturally occurring ICOS costimulatory domain protein. In embodiments, the variant or mutant of the ICOS costimulatory domain protein does not comprise an insertion compared to a naturally occurring ICOS costimulatory domain protein. In embodiments, the variant or mutant of the ICOS costimulatory domain protein comprises a substitution that is a conservative substitution compared to a naturally occurring ICOS costimulatory domain protein. In embodiments, the ICOS costimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_036224.1, or an isoform or naturally occurring mutant or variant thereof.

[0077] As used herein, the term "OX-40 costimulatory domain" refers to either a recombinant or naturally occurring form of the costimulatory domain of OX-40, or a variant or homolog thereof, that maintains OX-40 costimulatory domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the OX-40 costimulatory domain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring OX-40 costimulatory domain polypeptide. In embodiments, the OX-40 costimulatory domain is a human OX-40 costimulatory domain protein. In embodiments, the variant or mutant of the OX-40 costimulatory domain protein is a human OX-40 costimulatory domain protein that ... In embodiments, the variant or mutant of the OX-40 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 deletions compared to the naturally occurring OX-40 costimulatory domain protein. In embodiments, the variant or mutant of the OX-40 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to the naturally occurring OX-40 costimulatory domain protein. In embodiments, the variant or mutant of the OX-40 costimulatory domain protein comprises no more than a deletion compared to the naturally occurring OX-40 costimulatory domain protein. In embodiments, the variant or mutant of the OX-40 costimulatory domain protein comprises no more than an insertion compared to the naturally occurring OX-40 costimulatory domain protein. In embodiments, the variant or mutant of the OX-40 costimulatory domain protein comprises a substitution that is a conservative substitution compared to the naturally occurring OX-40 costimulatory domain protein. In embodiments, the OX-40 costimulatory domain comprises all or a portion of a protein identified by NCBI sequence reference NP_003318.1, or an isoform or naturally occurring mutant or variant thereof.

[0078] As used herein, the term "CTLA-4 costimulatory domain" refers to either a recombinant or naturally occurring form of the costimulatory domain of CTLA-4, or a variant or homolog thereof, that maintains CTLA-4 costimulatory domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to a CTLA-4 costimulatory domain). In some embodiments, a variant or homolog is a partial or partial CTLA-4 costimulatory domain that is a ... have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion). In embodiments, the CTLA-4 costimulatory domain protein is a human CTLA-4 costimulatory domain protein. In embodiments, the CTLA-4 costimulatory domain comprises no more than 5, 4, 3, 2, or 1 deletions. In embodiments, the CTLA-4 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions. In embodiments, In embodiments, the CTLA-4 costimulatory domain protein does not comprise a deletion. In embodiments, the CTLA-4 costimulatory domain protein does not comprise an insertion. In embodiments, the CTLA-4 costimulatory domain protein comprises a substitution that is a conservative substitution. In embodiments, the CTLA-4 costimulatory domain is a human CTLA-4 costimulatory domain protein. In embodiments, the variant or mutant of the CTLA-4 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 deletions compared to a naturally occurring CTLA-4 costimulatory domain protein. In embodiments, the variant or mutant of the CTLA-4 costimulatory domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to a naturally occurring CTLA-4 costimulatory domain protein. In embodiments, the variant or mutant of the CTLA-4 costimulatory domain protein does not comprise a deletion compared to a naturally occurring CTLA-4 costimulatory domain protein. In embodiments, the variant or mutant of the CTLA-4 costimulatory domain protein does not comprise an insertion compared to a naturally occurring CTLA-4 costimulatory domain protein.In embodiments, the variants or mutants of the CTLA-4 costimulatory domain proteins contain substitutions that are conservative substitutions compared to naturally occurring CTLA-4 costimulatory domain proteins.

[0079] The term "CD3ζ intracellular T cell signaling domain" as used herein refers to either a recombinant or naturally occurring form of the CD3ζ intracellular T cell signaling domain or a variant or homolog thereof that maintains CD3ζ intracellular T cell signaling domain activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD3ζ intracellular T cell signaling domain). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring CD3ζ intracellular T cell signaling domain polypeptide. In embodiments, the CD3ζ intracellular T cell signaling domain is a human CD3ζ intracellular T cell signaling domain protein. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain protein comprises no more than 5, 4, 3, 2, or 1 deletions compared to a naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain protein comprises no more than 5, 4, 3, 2, or 1 insertions compared to a naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain protein does not comprise a deletion compared to a naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain protein does not comprise an insertion compared to a naturally occurring CD3ζ intracellular T cell signaling domain protein. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain protein comprises substitutions that are conservative substitutions compared to a naturally occurring CD3ζ intracellular T cell signaling domain protein.In embodiments, the CD3 zeta intracellular T cell signaling domain comprises all or a portion of a protein identified by NCBI sequence reference NP_000725.1, or an isoform or naturally occurring mutant or variant thereof. In embodiments, the CD3 zeta intracellular T cell signaling domain comprises all or a portion of a protein identified by NCBI sequence reference NP_932170.1, or an isoform or naturally occurring mutant or variant thereof. Non-limiting examples of human CD3-zeta amino acid sequences available under NCBI sequence references are identified above.

[0080] In embodiments, the CD3ζ intracellular T cell signaling domain is encoded by all or a portion of the nucleic acid sequence identified by NCBI sequence reference NM_000734.3, or an isoform or naturally occurring mutant or variant thereof. In embodiments, the CD3ζ intracellular T cell signaling domain is encoded by all or a portion of the nucleic acid sequence identified by NCBI sequence reference NM_198053.2, or an isoform or naturally occurring mutant or variant thereof. In embodiments, the variant or variant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence comprises no more than 5, 4, 3, 2, or 1 deletion compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In embodiments, the variant or variant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence comprises no more than 5, 4, 3, 2, or 1 insertion compared to the naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence does not comprise a deletion compared to a naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence does not comprise an insertion compared to a naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence. In embodiments, the variant or mutant of the CD3ζ intracellular T cell signaling domain nucleic acid sequence comprises substitutions that are conservative substitutions compared to a naturally occurring CD3ζ intracellular T cell signaling domain nucleic acid sequence.

[0081] As used herein, "T cells" or "T lymphocytes" are a type of lymphocyte (a subtype of white blood cells) that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of T cell receptors on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTL), regulatory T (Treg) cells, and T helper cells. The various types of T cells can be distinguished using T cell detection agents.

[0082] A "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison with a test sample. For example, a test sample can be taken from a test condition, for example, in the presence of a test compound, and compared to a sample from a known condition, for example, in the absence of a test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value collected from several tests or results. Those skilled in the art will recognize that a control can be designed for the evaluation of any number of parameters. For example, a control can be devised to compare the therapeutic effect based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). A control is also useful for determining the significance of data. For example, if the value of a given parameter varies widely in the control, the variation of the test sample is not considered significant.

[0083] As used herein, the term "cancer" or "tumor" refers to all types of cancer, neoplasm, or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that may be treated using the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that may be treated with the compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, and uterine cancer. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma of the skin, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, invasive carcinoma of the breast, lung adenocarcinoma, squamous cell carcinoma of the lung, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, endocrine or exocrine pancreatic neoplasms, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0084] As used herein, the term "neuroendocrine cancer" or "neuroendocrine tumor" refers to a cancer or tumor formed from cells that release hormones into the blood in response to signals from the nervous system. Neuroendocrine tumors may produce higher than normal amounts of hormones, which can cause many different symptoms. Neuroendocrine tumors can be benign (not cancer) or malignant (cancer). Neuroendocrine cancers and tumors include, but are not limited to, carcinoid tumors, pancreatic islet cell tumors, medullary thyroid carcinoma, pheochromocytoma, neuroendocrine carcinoma of the skin (Merkel cell carcinoma), small cell lung cancer, large cell neuroendocrine carcinoma, neuroendocrine prostate cancer (NEPC), and metastatic castration-resistant prostate cancer (CRPC).

[0085] As used herein, the terms "metastasis", "metastatic" and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder (e.g., cancer) from one organ or another non-adjacent organ or part of the body. "Metastatic cancer" is also referred to as "stage IV cancer". Cancer begins at a site of origin, e.g., the breast, referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or site of origin acquire the ability to penetrate and invade surrounding normal tissues in the local area and / or penetrate the walls of the lymphatic or vascular system and circulate through the system to other sites and tissues of the body. A clinically detectable second tumor formed from cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, when lung cancer metastasizes to the breast, the secondary tumor at the breast site is composed of abnormal lung cells and not abnormal breast cells. A secondary tumor in the breast is referred to as metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or has had a primary tumor, and has one or more secondary tumors.The phrase non-metastatic cancer or a subject with non-metastatic cancer refers to a disease in which a subject has a primary tumor but does not have one or more secondary tumors.For example, metastatic lung cancer refers to a disease in a subject who has or has had a history of a primary lung tumor, and has one or more secondary tumors, for example, in a second location in the breast or in multiple locations.

[0086] As used herein (and as is well understood in the art), "treating" or "treatment" broadly includes any approach to obtain a beneficial or desired outcome in a subject's condition, such as a clinical outcome. Beneficial or desired clinical outcomes may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether partial or total, and whether detectable or undetectable, reduction in the extent of disease, stabilization (i.e., not worsening) of a disease condition, prevention of disease infection or spread, delay or slowing of disease progression, improvement or relief of a disease condition, reduction in disease recurrence, and remission. In other words, as used herein, "treatment" includes any cure, amelioration, or prevention of disease. Treatment may prevent the onset of disease, inhibit the spread of disease, reduce symptoms of disease, completely or partially eliminate the underlying cause of disease, shorten the duration of disease, or a combination thereof.

[0087] As used herein, "treating" and "treatment" include prophylactic treatment. The treatment method includes administering to the subject a therapeutically effective amount of an active agent. The administration step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It will also be understood that the effective dosage of the agent used for treatment or prevention may increase or decrease during a particular treatment or prevention regimen. Modifications in dosage may be effected and made evident by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the composition is administered to the subject in an amount and for a duration sufficient to treat the patient. In an embodiment, the treating or treatment is not a prophylactic treatment.

[0088] "Patient" or "subject in need of treatment" refers to an organism suffering from or susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0089] An "effective amount" is an amount sufficient for the compound to achieve a stated purpose (e.g., achieve an effect in the subject to which it is administered, treat a disease, reduce an enzyme activity, increase an enzyme activity, reduce a signal transduction pathway, or alleviate one or more symptoms of a disease or condition) compared to the absence of the compound. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of a symptom of a disease, which may also be referred to as a "therapeutically effective amount." "Alleviation" of a symptom(s) (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of a symptom, or the elimination of a symptom. A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, will have an intended prophylactic effect, e.g., an amount of a drug that prevents or delays the onset (or recurrence) of an injury, disease, pathology, or condition, or reduces the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or of the onset (or recurrence) of an injury, disease, pathology, or condition, or of the symptoms thereof. A complete prophylactic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. As used herein, "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of antagonist. As used herein, "function-interfering amount" refers to the amount of antagonist required to interfere with the function of an enzyme or protein compared to the absence of antagonist. The exact amount depends on the purpose of the treatment and can be ascertained by the skilled artisan using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0090] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration will be the concentration of active compound that is capable of achieving the methods described herein, as measured using methods described herein or known in the art.

[0091] As is well known in the art, the therapeutically effective amount for use in humans can also be determined from animal models.For example, human doses can be formulated to achieve concentrations that have been found to be effective in animals.Human dosages can be adjusted by monitoring the effectiveness of the compound and adjusting the dosage upwards or downwards, as described above.Based on the above and other methods, it is well within the capabilities of those skilled in the art to adjust dosages to achieve maximum effectiveness in humans.

[0092] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent sufficient to ameliorate a disorder, as described above. For example, for a given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can be at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effective than a control.

[0093] Dosage may vary depending on the patient's requirements and the compound used. In light of the present disclosure, the dose administered to a patient should be sufficient to cause the patient to have a beneficial therapeutic response over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the capabilities of one of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimal dose of the compound. Thereafter, dosage is increased by small increments until the optimal effect is reached under the circumstances. Dosage amount and interval can be individually adjusted to provide a level of administered compound that is effective for the particular clinical indication being treated. This provides a treatment regimen that is appropriate to the severity of the individual's condition.

[0094] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administration does not include administration of any active agent other than the listed active agents.

[0095] By "co-administered" is meant that the compositions described herein are administered simultaneously, immediately before, or immediately after the administration of one or more additional therapeutic agents. The compounds provided herein can be administered alone or co-administered to a patient. Co-administration is intended to include simultaneous or sequential administration of the compounds, individually or in combination (two or more compounds). Thus, the preparations can also be combined with other active substances, if desired (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally by topical routes, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0096] aris), D-82318 (Zentaris), SC-12983 (NCI), resverastatin sodium phosphate, BPR-OY-007 (National Health Research Institute Institutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin releasing hormone agonists (GnRH), such as goserelin or leuprolide, corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethylstilbestrol, ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), immunostimulants (e.g., BacillusCalmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., 111In, 90Y , or anti-CD20 monoclonal antibodies conjugated to 131I), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epithelial cell proliferation inhibitors, EGFR targeted therapies or therapeutic agents (e.g., gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OSI-420 / desmethylerlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, and the like.

[0097] "Selective" or "selectivity" of a compound, and the like, refers to the ability of the compound to distinguish between molecular targets (eg, a compound having selectivity for ROR1).

[0098] "Specific," "specifically," "specificity," and the like, of a compound refer to the ability of the compound to cause a particular effect, such as inhibition, on a particular molecular target with minimal or no effect on other proteins in the cell.

[0099] "ROR1 inhibitor" refers to a compound (e.g., a compound described herein) that reduces the activity of ROR1 compared to a control, such as no compound or a compound with a known inactivity.

[0100] "Contacting" is used according to its plain and ordinary meaning to refer to a process that allows at least two different species (e.g., chemical compounds, including biological molecules or cells) to come into sufficient proximity to react, interact, or come into physical contact. However, it should be understood that the resulting reaction product may be produced directly from the reaction between the added reagents, or from an intermediate from one or more of the added reagents that may be produced in the reaction mixture.

[0101] As defined herein, the terms "inhibit", "inhibit", "inhibiting" and the like with respect to protein-inhibitor interactions refer to adversely affecting (e.g., reducing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition refers to negatively affecting (e.g., reducing) the protein concentration or level compared to the protein concentration or level in the absence of the inhibitor. In embodiments, inhibition refers to a reduction in a disease or disease symptom. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Thus, inhibition includes at least partially, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating a signaling or enzyme activity or amount of a protein. In embodiments, inhibition refers to a reduction in the activity of a target protein due to a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction in the activity of a target protein due to an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein). A "ROR1 inhibitor" is a compound that adversely affects (eg, decreases) the activity or function of ROR1 compared to the activity or function of ROR1 in the absence of the inhibitor.

[0102] The term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0103] In the context of a substance or activity or function of a substance associated with a disease (e.g., a protein-related disease, a cancer associated with ROR1 activity, a ROR1-related cancer, a ROR1-related disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease)), the term "associated" or "related to" means that the disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease) is caused (in whole or in part) or a symptom of the disease is caused (in whole or in part) by the substance or activity or function of the substance. As used herein, something described as being associated with a disease may be a target for the treatment of the disease if it is a causative agent. For example, if increased activity or function (e.g., signal transduction pathway activity) of ROR1 causes the disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease), the cancer or ROR1-related disease (e.g., cancer, an inflammatory disease, an autoimmune disease, or an infectious disease) associated with ROR1 activity or function may be treated with a ROR1 modulator or a ROR1 inhibitor. For example, inflammatory diseases associated with ROR1 activity or function or ROR1-associated inflammatory diseases can be treated with ROR1 modulators or ROR1 inhibitors where increased ROR1 activity or function (e.g., signal transduction pathway activity) causes the disease.

[0104] As used herein, the term "signal transduction pathway" refers to a series of interactions between cellular and optionally extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that transmit a change in one component to one or more other components, which in turn may transmit the change to additional components, and optionally be propagated to other signal transduction pathway components.

[0105] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0106] Chimeric Antigen Receptor Provided herein are recombinant proteins / chimeric antigen receptors (these terms are used interchangeably throughout), nucleic acids encoding the recombinant proteins, cells expressing the chimeric antigen receptors disclosed herein, compositions thereof, and methods of using the same, which are useful, inter alia, for treating neuroendocrine cancer and treating or preventing metastases resulting from neuroendocrine cancer. Applicants have discovered that chimeric antigen receptors (CARs) against ROR-1 provide highly active and efficient immunotherapeutic compositions for treating neuroendocrine cancer and treating or preventing metastases in subjects with neuroendocrine cancer. Without being bound to a particular theory, antibodies known to inhibit the receptors they bind and downregulate their surface expression are generally not considered good clinical candidates for CARs. It was therefore highly surprising that CARs comprising the CDRs of the ROR-1 antibodies described herein, including embodiments thereof, exhibit effective neuroendocrine cancer-specific cytotoxicity when expressed by T cells.

[0107] In one aspect, the chimeric antigen receptor comprises an antibody binding region that specifically binds to ROR-1, a transmembrane domain, a spacer domain that connects the antigen binding region and the transmembrane domain, and an intracellular domain. The terms "antibody region," "antigen binding region," or "antigen binding domain" as provided herein are used interchangeably throughout and refer to a monovalent or multivalent protein moiety that forms part of the protein (i.e., recombinant protein, chimeric antigen receptor) provided herein, including embodiments thereof. Thus, one skilled in the art will immediately recognize that an antibody region or antigen binding region is a protein moiety that can bind to an antigen (epitope). The antibody region provided herein may comprise a domain of an antibody or a fragment thereof (e.g., Fab). Thus, the antibody region may comprise a light chain variable domain (VL) and / or a heavy chain variable domain (VH).

[0108] In an embodiment, the ROR-1 binding domain comprises an antibody region comprising a light chain variable region (VL) and a heavy chain variable region (VH). In an embodiment, the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48. It should be noted that these CDRs are of or derived from cirumutuzumab (also known as UC-961 or 99961.1). The development and structure of cirumutuzumab are disclosed in U.S. Patent No. 9,758,591, which is incorporated herein by reference in its entirety for all purposes. In an embodiment, the ROR-1 binding domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54.

[0109] In one embodiment, the chimeric antigen receptor comprises: i. an antigen-binding region that specifically binds to ROR-1, the antigen-binding region comprising a light chain variable domain and a heavy chain variable domain, (a) the light chain variable domain comprises CDR L1 described in SEQ ID NO: 43, CDR L2 described in SEQ ID NO: 44, and CDR L3 described in SEQ ID NO: 45, the heavy chain variable domain comprises CDR H1 described in SEQ ID NO: 46, CDR H2 described in SEQ ID NO: 47, and CDR H3 described in SEQ ID NO: 48, or (b) the light chain variable domain comprises CDR L1 described in SEQ ID NO: 49, CDR L2 described in SEQ ID NO: 50, and CDR L3 described in SEQ ID NO: 51, the heavy chain variable domain comprises CDR H1 described in SEQ ID NO: 52, CDR H2 described in SEQ ID NO: 53, and CDR H3 described in SEQ ID NO: 54; ii. a spacer domain having a length of 14 to 120 amino acids; iii. a transmembrane domain; and iv. an intracellular domain.

[0110] In one aspect, the chimeric antigen receptor is an antibody binding region that specifically binds to i.ROR-1, comprising a light chain variable domain and a heavy chain variable domain, wherein (a) the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48, or (b) the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54. an antigen-binding region comprising H3; ii. a spacer domain; iii. a transmembrane domain comprising a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof; and iv. an intracellular domain.

[0111] In one aspect, the chimeric antibody receptor is an antibody binding region that specifically binds to i.ROR-1, comprising a light chain variable domain and a heavy chain variable domain, wherein (a) the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48, or (b) the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54. H3; ii. a linker domain; iii. a transmembrane domain; and iv. an intracellular domain comprising an intracellular T cell signaling domain and an intracellular costimulatory domain selected from 4-1BB, ICOS, OX-40, and combinations thereof.

[0112] The light chain variable (VL) domain provided comprises the CDR sequences and framework region (FR) sequences of the light chain of an antibody, antibody variant or fragment thereof. In an embodiment, the antibody region or antigen-binding region comprises a variable light chain domain and a variable heavy chain domain. As provided herein, a "variable light chain domain" refers to a polypeptide contained in (forming part of) a light chain variable (VL) region. In an embodiment, the variable light chain region is a light chain variable (VL) domain. As provided herein, a "variable heavy chain domain" refers to a polypeptide contained in (forming part of) a heavy chain variable (VH) region. In an embodiment, the variable heavy chain region is a heavy chain variable (VH) domain. In an embodiment, the light chain variable (VL) domain comprises CDR L1 (SEQ ID NO: 43), CDR L2 (SEQ ID NO: 44), and CDR L3 (SEQ ID NO: 45). In an embodiment, the heavy chain variable (VH) domain comprises CDR H1 (SEQ ID NO: 46), CDR H2 (SEQ ID NO: 47), and CDR H3 (SEQ ID NO: 48).

[0113] In embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:21, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:21. In embodiments, the light chain variable domain has the amino acid sequence of SEQ ID NO:21, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:21. In embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:27, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:27. In embodiments, the heavy chain variable domain has the amino acid sequence of SEQ ID NO:27, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:27.

[0114] In embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 19. In embodiments, the light chain variable domain has the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 19. In embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 20. In embodiments, the light chain variable domain has the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 20. In embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 25. In embodiments, the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 25. In embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 26. In embodiments, the heavy chain variable domain has the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 26.

[0115] In an embodiment, the C-terminus of the light chain variable domain is linked to the N-terminus of the heavy chain variable domain. In an embodiment, the N-terminus of the light chain variable domain is linked to the C-terminus of the heavy chain variable domain. In an embodiment, the light chain variable domain is covalently linked to the heavy chain variable domain via a chemical linker. A "chemical linker" as provided herein is a covalent linker, a non-covalent linker, a peptide linker (a linker comprising a peptide moiety), a cleavable peptide linker, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, or any combination thereof. Thus, a chemical linker as provided herein may comprise multiple chemical moieties, where each of the multiple chemical moieties is chemically distinct. Alternatively, the chemical linker may be a non-covalent linker. Examples of non-covalent linkers include, but are not limited to, ionic bonds, hydrogen bonds, halogen bonds, van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), and hydrophobic interactions. In embodiments, the chemical linkers are formed using conjugation chemistries, including, but not limited to, nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and addition of carbon-carbon and carbon-heteroatom to multiple bonds (e.g., Michael reaction, Diels-Alder addition). In embodiments, the chemical linkers are peptide linkers of 5-100, 5-80, 5-70, 5-60, 5-50, 10-50, 10-40, or 10-30 amino acids in length. Any sequence that provides sufficient flexibility between the light chain variable domain and the heavy chain variable domain is contemplated for the recombinant proteins provided herein, including embodiments thereof. In embodiments, the peptide linker comprises or has the amino acid sequence of SEQ ID NO: 24. In embodiments, the peptide linker has an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:24.

[0116] The term "transmembrane domain" refers to a transmembrane protein domain capable of anchoring a protein to a membrane. Any transmembrane domain capable of anchoring a protein provided herein, including embodiments thereof, is contemplated. While any suitable transmembrane domain is contemplated herein, illustrative but non-limiting examples of transmembrane domains include the transmembrane domains of CD28, CD8, CD4, CD3ζ, or CD8α. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. As used herein, the term "CD28 transmembrane domain" refers to any recombinant or naturally occurring form of the transmembrane domain of CD28 or a variant or homolog thereof that maintains CD28 transmembrane domain activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the CD28 transmembrane domain). In some aspects, a variant or homolog has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity over the entire sequence or a portion of the sequence of a naturally occurring CD28 transmembrane domain polypeptide. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CD28 transmembrane domain is the amino acid sequence of SEQ ID NO: 32. In embodiments, the CD28 transmembrane domain in the ROR-1 CAR described herein comprises a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32.

[0117] In embodiments, the C-terminus of the heavy chain variable domain is linked to the N-terminus of the transmembrane domain. In embodiments, the C-terminus of the light chain variable domain is linked to the N-terminus of the transmembrane domain. In embodiments, the heavy chain variable domain or the light chain variable domain is covalently linked to the transmembrane domain through a spacer domain. In embodiments, the binding affinity of the antigen-binding region to the antigen is increased in a CAR construct having a spacer domain. In embodiments, the activity of a CAR having a spacer domain is higher than that of a CAR without a spacer domain. In embodiments, the flexibility of the antigen-binding region of the CAR is increased by the presence of a spacer domain.

[0118] The term "intracellular domain" refers to a portion of a receptor or protein that interacts with the interior of a cell or organelle via protein-protein interactions. In embodiments, the recombinant protein (e.g., intracellular domain) further comprises one or more intracellular costimulatory signaling domains. An "intracellular costimulatory signaling domain" provided herein comprises an amino acid sequence capable of providing costimulatory signaling in response to binding of an antigen to an antibody region provided herein, including embodiments thereof. In embodiments, signaling of the costimulatory signaling domain results in the production of a cytokine and proliferation of T cells expressing it. In embodiments, the intracellular costimulatory signaling domain comprises at least one or more of a CD28 intracellular costimulatory signaling domain, a 4-1BB (CD137) intracellular costimulatory signaling domain, an ICOS intracellular costimulatory signaling domain, an OX-40 intracellular costimulatory signaling domain, or any combination thereof. For example, a preferred intracellular costimulatory signaling domain comprises or consists of at least a portion of a CD28 intracellular costimulatory signaling domain, or at least a portion of a 4-1BB (CD137) intracellular costimulatory signaling domain. Another preferred intracellular costimulatory signaling domain comprises or consists of at least a portion of a CD28 intracellular costimulatory signaling domain combined with at least a portion of a 4-1BB (CD137) intracellular costimulatory signaling domain. In an embodiment, at least a portion of the CD28 intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32. In an embodiment, at least a portion of the CD28 intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32. In an embodiment, at least a portion of the 4-1BB intracellular costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 33.In embodiments, at least a portion of the 4-1BB intracellular costimulatory signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29. In embodiments, a chimeric antigen receptor or recombinant protein disclosed herein comprises the amino acid sequence of SEQ ID NO: 32 in combination with SEQ ID NO: 33 (either SEQ ID NO: 32 is present at the N'-terminus of SEQ ID NO: 33 or SEQ ID NO: 33 is present at the N'-terminus of SEQ ID NO: 32), or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to a combination of SEQ ID NO: 32 in combination with SEQ ID NO: 33 (either SEQ ID NO: 32 is present at the N'-terminus of SEQ ID NO: 33 or SEQ ID NO: 33 is present at the N'-terminus of SEQ ID NO: 32).

[0119] In embodiments, the recombinant protein further comprises an intracellular T cell signaling domain. An "intracellular T cell signaling domain" as provided herein comprises an amino acid sequence capable of providing primary signaling in response to binding of an antigen to an antibody region as provided herein, including embodiments thereof. In embodiments, signaling of the intracellular T cell signaling domain results in activation of a T cell expressing the chimeric antigen receptor. In embodiments, signaling of the intracellular T cell signaling domain results in proliferation (cell division) of the T cell expressing it. In embodiments, signaling of the intracellular T cell signaling domain results in expression of proteins by the T cell known in the art to be characteristic of activated T cells, e.g., CTLA-4, PD-1, CD28, C, and D69. In embodiments, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0120] In embodiments, the CD3 ζ intracellular T cell signaling domain comprises an amino acid sequence of SEQ ID NO: 34, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 34, generating a CD3 ζ intracellular T cell signaling domain with at least 80%, 85%, 90%, or 95% of the activity of the CD3 ζ intracellular T cell signaling domain having SEQ ID NO: 34. In embodiments, the CD3 ζ intracellular T cell signaling domain has an amino acid sequence of SEQ ID NO: 34, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 34, generating a peptide with at least 80%, 85%, 90%, or 95% of the activity of the CD3 ζ intracellular T cell signaling domain having SEQ ID NO: 34.

[0121] In embodiments, the ROR-1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27), or alternatively a ROR1 scFv (CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:20), ii) a spacer domain having a hinge and CH2 and CH3 domains, iii) a CD28 transmembrane domain, iv) a 4-1BB (CD137) costimulatory domain, and v) a CD3Z T cell activation domain.

[0122] Alternatively, in embodiments, the ROR1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:20); ii) a spacer domain having a hinge and a CH2 domain; iii) a CD28 transmembrane domain; iv) a 4-1BB (CD137) costimulatory domain; and v) a CD3Z T cell activation domain.

[0123] Alternatively, in embodiments, the ROR1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:20); ii) a spacer domain having a hinge and a CH3 domain; iii) a CD28 transmembrane domain; iv) a 4-1BB (CD137) costimulatory domain; and v) a CD3Z T cell activation domain.

[0124] Alternatively, in embodiments, the ROR1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:20); ii) a spacer domain comprising a hinge and a portion of the CH3 domain (e.g., half of the CH3 domain, the N-terminal 43 amino acids of the CH3 domain, etc.); iii) a CD28 transmembrane domain; iv) a 4-1BB (CD137) costimulatory domain; and v) a CD3Z T cell activation domain.

[0125] Alternatively, in embodiments, the ROR1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:20), ii) a spacer domain with a hinge domain, iii) a CD28 transmembrane domain, iv) a 4-1BB (CD137) costimulatory domain, and v) a CD3Z T cell activation domain.

[0126] Alternatively, in embodiments, the ROR1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:20); ii) a spacer domain having a hinge and CH2 and CH3 domains; iii) a CD28 transmembrane domain; iv) a CD28 costimulatory domain linked to a 4-1BB (CD137) costimulatory domain; and v) a CD3Z T cell activation domain.

[0127] Alternatively, in embodiments, the ROR-1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having the sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:19, and a VH domain having the sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:20), ii) a spacer domain having a hinge and a CH2 domain, iii) a CD28 transmembrane domain, iv) a CD28 costimulatory domain linked to a 4-1BB (CD137) costimulatory domain, and v) a CD3Z T cell activation domain.

[0128] Alternatively, in embodiments, the ROR-1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having the sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:19, and a VH domain having the sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90% or 95% identical to SEQ ID NO:20), ii) a spacer domain having a hinge and a CH3 domain, iii) a CD28 transmembrane domain, iv) a CD28 costimulatory domain linked to a 4-1BB (CD137) costimulatory domain, and v) a CD3Z T cell activation domain.

[0129] Alternatively, in embodiments, the ROR1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having a sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:19, and a VH domain having a sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:20); ii) a spacer domain comprising a hinge and a portion of a CH3 domain (e.g., half of the CH3 domain, the N-terminal 43 amino acids of the CH3 domain, etc.); iii) a CD28 transmembrane domain; iv) a CD28 costimulatory domain linked to a 4-1BB (CD137) costimulatory domain; and v) a CD3Z T cell activation domain.

[0130] Alternatively, in embodiments, the ROR-1 CAR disclosed herein comprises i) a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:21, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:21, with CDR L1 (SEQ ID NO:43), CDR L2 (SEQ ID NO:44), and CDR L3 (SEQ ID NO:45), CDR H1 (SEQ ID NO:46), CDR H2 (SEQ ID NO:47), and CDR H3 (SEQ ID NO:48), and a VH domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27) or, alternatively, a ROR1 scFv (a VL domain having a sequence of SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:27, with CDR L1 (SEQ ID NO:49), CDR L2 (SEQ ID NO:50), and CDR L3 (SEQ ID NO:51), CDR H1 (SEQ ID NO:52), CDR H2 (SEQ ID NO:53), and CDR H3 (SEQ ID NO:54), or a VL domain having the sequence of SEQ ID NO:19, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:19, and a VH domain having the sequence of SEQ ID NO:20, or a sequence at least 80%, 85%, 90%, or 95% identical to SEQ ID NO:20), ii) a spacer domain with a hinge domain, iii) a CD28 transmembrane domain, iv) a CD28 costimulatory domain linked to a 4-1BB (CD137) costimulatory domain, and v) a CD3Z T cell activation domain.

[0131] The chimeric antigen receptors described herein are contemplated to bind a peptide comprising a glutamic acid at a position corresponding to amino acids 130-160 of ROR-1 or a fragment thereof, preferably position 138 of the ROR-1 polypeptide. Alternatively and / or additionally, the chimeric antigen receptors described herein specifically bind to either the 3' or middle Ig-like region of the extracellular domain of the ROR-1 protein, preferably the 3' end of the Ig-like region of the extracellular domain of the ROR-1 protein at positions 1-147.

[0132] As a result, the ROR-1 CAR disclosed herein can bind to ROR-1 expressing cells and initiate or induce an immune response against ROR-1 expressing cells, such as neuroendocrine cancer cells, such as carcinoid tumors, pancreatic islet cell tumors, medullary thyroid carcinoma, pheochromocytoma, neuroendocrine carcinoma of the skin, small cell lung cancer, large cell neuroendocrine carcinoma, neuroendocrine prostate cancer (NEPC), or metastatic castration-resistant prostate cancer (CRPC).

[0133] In an embodiment, the recombinant protein provided herein, including the embodiments thereof, further comprises a detectable domain. A "detectable domain" provided herein is a peptide moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, a detectable domain provided herein can be a protein or other entity that can be made detectable, for example, by incorporating a radioactive label or by being specifically reactive with an antibody. Any suitable method known in the art for conjugating an antibody to a label (e.g., using the method described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego) can be used. In the present invention, a detectable domain is used to confirm transfection of T cells.

[0134] In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 500 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 550 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 600 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 650 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 700 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 750 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 800 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 850 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 900 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 950 pM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1.5 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2.5 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3.5 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4.5 nM to about 6 nM. In an embodiment, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5 nM to about 6 nM.In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5.5 nM to about 6 nM.

[0135] In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 500 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 500 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 550 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 550 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 600 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 600 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 650 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 650 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 700 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 700 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 750 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 750 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 800 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 800 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 850 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 850 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 900 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 900 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 950 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 950 pM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 1 nM.In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 1.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 1.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 2 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 2.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 2.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 3 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 3.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 3.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 4 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 4.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 4.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 5.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 5.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor has a binding affinity of 6 nM.

[0136] In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 40 nM (e.g., 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1 nM). In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 40 nM (e.g., 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1 nM). In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 35 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 35 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 30 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 30 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 25 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 25 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 20 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 20 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 15 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 15 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 10 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 10 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 9 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 9 nM.In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 8 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 8 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 7 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 7 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 6 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 5 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 5 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 4 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 4 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 3 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 3 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 2 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 2 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 1 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 1 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 0.5 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 0.5 nM.In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 0.25 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 0.25 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than about 0.1 nM. In embodiments, the recombinant protein or chimeric antigen receptor binds to the ROR-1 protein with a KD of less than 0.1 nM.

[0137] Further provided herein are recombinant nucleic acids encoding the recombinant proteins or chimeric antigen receptors provided herein, including embodiments thereof. In some embodiments, the chimeric antigen receptor is encoded by a single recombinant nucleic acid forming part of an expression vector. Any suitable expression vector capable of being transfected and expressed in immune cells (e.g., CD8+ T cells, CD4+ T cells, CD56+ immune cells, NK cells, or genetically modified or engineered NK cells) is contemplated. Exemplary and / or preferred expression vectors may include viral expression vectors (e.g., adenovirus, adeno-associated virus, alphavirus, herpesvirus, lentivirus, etc. expression vectors). In some embodiments, adenoviruses are replication-deficient and non-immunogenic viruses, which is typically achieved by targeted deletion of selected viral proteins (e.g., E1, E2b, E3 proteins). For example, the recombinant nucleic acid may be placed in a non-viral vector (e.g., a mammalian expression vector) and transfected into T cells using any commonly used transfection method. In another example, the recombinant nucleic acid is included in a viral vector, such that viral particles containing the recombinant nucleic acid infect T cells and deliver the recombinant nucleic acid to the T cells. In yet another example, when the recombinant nucleic acid is a self-replicating RNA-based vector, the self-replicating RNA-based vector can be formulated with a pharma- ceutically acceptable carrier (e.g., in a buffer or cell culture medium, preferably containing an RNAase inhibitor) so that the self-replicating RNA-based vector can be delivered to cells in a naked form by directly contacting the cell membrane. In some embodiments, the self-replicating RNA-based vector can be bound to a carrier molecule. Exemplary carrier molecules include protein A, protein G, protein Z, albumin, refolded albumin, nanoparticles (e.g., quantum dots, gold nanoparticles, magnetic nanoparticles, nanotubes, polymer nanoparticles, dendrimers, etc.), or beads (e.g., polystyrene beads, latex beads, dynabeads, etc.). Preferably, the nanoparticles and / or beads have a dimension of less than 1 μm, preferably less than 100 nm.In other embodiments, the self-replicating RNA-based vector can be coupled to a microparticle (e.g., PLG RG503 (50:50 lactide / glycolide molar ratio), where the self-replicating RNA-based vector can be absorbed into the microparticle for further delivery to cells. In yet other embodiments, the self-replicating RNA-based vector can be encapsulated within a liposome (e.g., PEG-based liposome, etc.) to protect the self-replicating RNA-based vector from RNAase digestion and deliver the RNA-based vector by fusing the liposome to the target cell membrane.

[0138] In embodiments, a recombinant nucleic acid encoding a recombinant protein or chimeric antigen receptor provided herein can be inserted into a vector carrying a cassette for gene editing (e.g., a CRISPR-CAS expression vector). Another exemplary expression vector can include a transposon-based expression system (e.g., the Sleeping Beauty system disclosed in Deninger et al., PLOS ONE, June 1, 2015).

[0139] Cells and pharmaceutical compositions thereof Provided herein is a cell expressing a recombinant protein described herein, comprising a chimeric antigen receptor (CAR) comprising a ROR-1-binding antibody or antibody fragment region, a linker domain, a transmembrane domain, and an intracellular domain. In embodiments, the ROR-1-binding domain is an antibody.

[0140] The chimeric antigen receptors described herein are expressed on the surface of T lymphocytes or T cells, such as CD8+ T cells and CD4+ T cells, isolated from an individual suffering from neuroendocrine cancer by genetically engineering the cells to express a heterologous nucleic acid sequence encoding the chimeric nucleic acid receptor. The CAR-T cells thus generated are then re-administered to the individual. Once administered, the CAR-T cells specifically bind to ROR-1 expressing cells, such as neuroendocrine cancer cells expressing ROR-1, and induce an immune response against the neuroendocrine cancer cells.

[0141] In embodiments, the chimeric antigen receptor described herein is expressed on the surface of natural killer cells isolated from an individual suffering from a neuroendocrine cancer, and then genetically modified to express the ROR-1 CAR. In certain embodiments, the NK cells are CD56 positive cells. CD56 positivity can be determined, for example, by flow cytometric analysis of a cell population, and is defined as at least 10-fold, 100-fold, or 1,000-fold more cells stained with an isotype control antibody compared to cells stained with an isotype control antibody. In certain embodiments, the NK cells are primary NK cells. In embodiments, the NK cells include NK cell lines. In certain embodiments, the NK cells are autologous to the individual being treated with the CAR expressing the NK cells of the present disclosure. In embodiments, the NK cells are xenogeneic to the individual being treated with the CAR expressing the NK cells of the present disclosure. In embodiments, the NK cells are allogeneic to the individual being treated with the CAR expressing the NK cells of the present disclosure. NK cells may be derived from any suitable source: bone marrow, induced pluripotent stem cells, peripheral blood mononuclear cells, fetal or placental cells.

[0142] In embodiments, the cells are administered as part of a pharmaceutical composition that includes, in addition to the cells expressing a CAR comprising a ROR-1-binding antibody region, a pharma- ceutically acceptable excipient, diluent, or carrier.

[0143] The chimeric antigen receptor and / or recombinant nucleic acid encoding the chimeric antigen receptor may optionally be formulated as a pharmaceutical composition together with any pharma- ceutically acceptable carrier (e.g., as a sterile injectable composition for immune cells expressing the chimeric antigen receptor, a pharma- ceutically acceptable salt for the recombinant nucleic acid encoding the chimeric antigen receptor, etc.). Although the dose or cell titer of the pharmaceutical composition may vary depending on the treatment protocol, treatment regimen, treatment condition, etc., an example of the dose or cell titer of the pharmaceutical composition may include a cell titer of at least 1×103 cells / mL, preferably at least 1×105 cells / mL, more preferably at least 1×106 cells / mL, and at least 1 mL, preferably at least 5 mL, more preferably and at least 20 mL per dosage unit.

[0144] In some embodiments, the pharmaceutical composition may comprise a homogenous cell or a plurality thereof (e.g., CD8+ T cells expressing a chimeric antigen receptor, CD4+ T cells expressing a chimeric antigen receptor, NK cells expressing a chimeric antigen receptor, etc.). In other embodiments, the composition may comprise a heterogeneous mixture of cells (e.g., a mixture of CD8+ T cells expressing a chimeric antigen receptor and NK cells expressing a chimeric antigen receptor in a ratio of 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1, etc.). The ratio of different types of cells may vary based on the type of neuroendocrine cancer, the age, sex, or health of the patient, the size of the tumor, and the cell count of the patient. The actual amount effective for a particular application will vary depending, among other things, on the condition being treated. When administered in a method for treating a disease, the recombinant proteins described herein contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule and / or reducing, eliminating, or delaying the progression of a disease symptom. Determination of a therapeutically effective amount of a compound of the present invention is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein.

[0145] The dosage and frequency (single or multiple doses) administered to a mammal may vary depending on a variety of factors, such as whether the mammal is suffering from another disease and its route of administration, the size, age, sex, health, weight, body mass index, and diet of the recipient, the nature and extent of symptoms of the disease being treated (e.g., cancer symptoms and the severity of such symptoms), the type of concomitant treatment, complications from the disease being treated or other health-related problems. Other treatment regimens or drugs may be used in conjunction with the methods and compounds of the present invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the capabilities of one of ordinary skill in the art.

[0146] For any composition (e.g., recombinant protein, nucleic acid) provided herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration will be the concentration of active compound that is capable of achieving the methods described herein, as measured using methods described herein or known in the art. As is well known in the art, effective amounts for use in humans can also be determined from animal models. For example, human doses can be formulated to achieve a concentration that has been found to be effective in animals. Dosages in humans can be adjusted by monitoring efficacy and adjusting dosage upwards or downwards, as described above. It is well within the capabilities of one of ordinary skill in the art to adjust doses to achieve maximum efficacy in humans based on the above and other methods.

[0147] Dosage may vary depending on the patient's requirements and the compound used. In the context of the present invention, the dose administered to a patient should be sufficient to induce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the capabilities of one of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimal dose of the compound. Dosage is then increased by small increments until the optimal effect under the circumstances is reached.

[0148] Dosage amount and interval may be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated, thereby providing a treatment regimen appropriate to the severity of the individual's disease condition.

[0149] Utilizing the teachings provided herein, one may design an effective prophylactic or therapeutic treatment regimen that does not cause substantial toxicity, and yet is effective in treating the clinical symptoms exhibited by a particular patient. This design should involve careful selection of an active compound by considering factors such as the potency of the compound, relative bioavailability, the patient's weight, and the presence and severity of adverse side effects.

[0150] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of active agents to and by a subject and can be included in the compositions of the present invention without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, saline (such as Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compounds of the present invention. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0151] The term "pharmaceutically acceptable salts" refers to salts derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and, where the molecule contains a basic functional group, refers to salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like.

[0152] The term "preparation" is intended to include the formulation of the active compound with an encapsulating material as a carrier to provide a capsule in which the active ingredient, with or without other carriers, is surrounded by the carrier and is therefore associated with the carrier. Also included are cachets and lozenges. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0153] The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate amounts of active ingredient. The unit dosage form can be a packaged preparation, the package containing discrete amounts of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, tablet, cachet, or lozenge itself, or the appropriate number of any of these in packaged form. The unit dosage form can be a frozen dispersion.

[0154] Treatment method The CAR-T cell compositions described herein are useful for treating neuroendocrine cancer in an individual in need thereof, and for preventing or treating metastasis from neuroendocrine cancer.

[0155] Thus, described herein are methods for treating neuroendocrine cancer, including the administration of cells expressing a chimeric antigen receptor that targets human ROR-1, and methods for preventing or treating metastases derived from neuroendocrine cancer. The chimeric antigen receptors described herein are expressed by T lymphocytes or natural killer cells that are isolated from and re-administered to an individual suffering from neuroendocrine cancer. The administration of T cells expressing the CAR described herein serves as an effective therapeutic treatment for neuroendocrine cancer expressing ROR-1 and its metastases.

[0156] The chimeric antigen receptor comprises: i) an antigen-binding region, the antigen-binding region specifically binds to ROR-1, the antigen-binding region comprising a light chain variable domain and a heavy chain variable domain; ii) a spacer domain; iii) a transmembrane domain; and iv) an intracellular domain. In some embodiments, the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48. Alternatively, the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54.

[0157] In some embodiments, the light chain variable domain is covalently linked to the heavy chain variable domain via a polypeptide linker. In some embodiments, the polypeptide linker comprises the amino acid sequence of SEQ ID NO:24.

[0158] In embodiments, the spacer domain comprises an antibody domain, hi some embodiments, the antibody domain comprises an immunoglobulin hinge domain, an immunoglobulin constant heavy 3 (CH3) domain, an immunoglobulin constant heavy 2 (CH2) domain, or a combination thereof.

[0159] In embodiments, the spacer domain comprises an amino acid sequence represented by SEQ ID NO:29, SEQ ID NO:41 or SEQ ID NO:42.

[0160] In embodiments, the light chain variable domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:21.

[0161] In embodiments, the heavy chain variable domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:27.

[0162] In embodiments, the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof. In some aspects, the transmembrane domain is a CD28 transmembrane domain.

[0163] In embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain, an intracellular T cell signaling domain, or a combination thereof. The intracellular costimulatory signaling domain is a 4-1BB intracellular costimulatory signaling domain, a CD28 intracellular costimulatory signaling domain, an ICOS intracellular costimulatory signaling domain, an OX-40 intracellular costimulatory signaling domain, or any combination thereof. In some embodiments, the intracellular costimulatory signaling domain comprises a 41-BB intracellular costimulatory signaling domain. In other embodiments, the intracellular costimulatory signaling domain comprises a CD28 intracellular costimulatory signaling domain and a 41-BB intracellular costimulatory signaling domain.

[0164] In embodiments, the intracellular costimulatory signaling domain further comprises an intracellular T cell signaling domain, hi some embodiments, the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0165] In embodiments, the chimeric antigen receptor binds to a cell expressing ROR-1. In some embodiments, the cell expressing ROR-1 is a neuroendocrine cancer cell.

[0166] In embodiments, the cell is a T lymphocyte. In some embodiments, the T lymphocyte is a CD4+ T lymphocyte or a CD8+ T lymphocyte.

[0167] In embodiments, the cells are natural killer cells. In some embodiments, the natural killer cells are autologous to the subject. In other embodiments, the natural killer cells are xenogeneic to the subject. In yet other embodiments, the natural killer cells are allogeneic to the subject.

[0168] Neuroendocrine cancers that may be treated by the methods provided herein include, but are not limited to, carcinoid tumors, pancreatic islet cell tumors, medullary thyroid carcinoma, pheochromocytoma, neuroendocrine carcinoma of the skin, small cell lung cancer, large cell neuroendocrine carcinoma, neuroendocrine prostate cancer (NEPC), and metastatic castration-resistant prostate cancer (CRPC). In some embodiments, the neuroendocrine cancer is metastatic castration-resistant prostate cancer (CRPC).

[0169] In embodiments, the neuroendocrine cancerous cells express ROR-1. Optionally, the subject has not responded to androgen deprivation therapy. Optionally, the neuroendocrine cancer has metastasized to bone.

[0170] In embodiments, the methods provided herein further comprise administering cirumutuzumab to the subject. In some embodiments, cirumutuzumab and the cells expressing a disclosed chimeric antigen receptor are administered separately. In other embodiments, cirumutuzumab and the cells are administered together.

[0171] In embodiments, the methods provided herein further include administering to the subject a platinum-based chemotherapy. Suitable platinum-based chemotherapy includes, but is not limited to, carboplatin, cisplatin, etoposide, taxanes, and any combination thereof.

[0172] Treatment refers to a method that seeks to improve or ameliorate the condition being treated. With respect to neuroendocrine cancer, treatment includes, but is not limited to, reducing tumor volume, reducing tumor volume growth, increasing progression-free survival, or increasing overall life expectancy. In certain embodiments, treatment will result in remission of the neuroendocrine cancer being treated. In certain embodiments, treatment includes use as a prophylactic or maintenance dose aimed at preventing recurrence or progression of a previously treated neuroendocrine cancer or tumor. Those skilled in the art understand that not all individuals will respond equally or fully to the treatment administered, yet these individuals are considered to be treated.

[0173] Anti-ROR-1 CAR T cells and anti-ROR-1 CAR-T cell compositions can be administered to a subject in need thereof by any route suitable for administration of a cell-containing pharmaceutical composition, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally.

[0174] Anti-ROR-1 CAR T cells and anti-ROR-1 CAR-T cell compositions can be administered according to a suitable dosing schedule. In certain embodiments, CAR T cells are administered once, with subsequent dosing depending on clinical criteria. If an individual does not respond or only partially responds, the patient can be administered a second, third, or fourth dose of anti-ROR-1 CAR-T cell composition until a desired clinical response is observed. A dose of CAR-T cells generally includes at least 1×106 cells, but may include up to 5×108 cells. Cells can be administered based on the total viable PBMCs of an individual transduced with the CAR construct. In certain embodiments, a single dose includes between 1 million transduced PBMCs and 100 million transduced PBMCs. In certain embodiments, a single dosage may be between 1 million transduced PBMCs and 2 million transduced PBMCs, between 1 million transduced PBMCs and 3 million transduced PBMCs, between 1 million transduced PBMCs and 4 million transduced PBMCs, between 1 million transduced PBMCs and 5 million transduced PBMCs, between 1 million transduced PBMCs and 6 million transduced PBMCs, between 1 million transduced PBMCs and 7 million transduced PBMCs, between 1 million transduced PBMCs and 8 million transduced PBMCs, between 1 million transduced PBMCs and 9 million transduced PBMCs, between 1 million transduced PBMCs and 1 million transduced PBMCs and 2 million transduced PBMCs. Transduced PBMCs - 10 million transduced PBMCs, 1 million transduced PBMCs - 50 million transduced PBMCs, 1 million transduced PBMCs - 100 million transduced PBMCs, 2 million transduced PBMCs - 3 million transduced PBMCs, 2 million transduced PBMCs - 4 million transduced PBMCs, 2 million transduced PBMCs - 5 million transduced PBMCs, 2 million transduced PBMCs - 6 million transduced PBMCs, 2 million transduced PBMCs - 7 million transduced PBMCs, 2 million transduced PBMCs - 8 million transduced PBMCs,2 million transduced PBMCs - 9 million transduced PBMCs, 2 million transduced PBMCs - 10 million transduced PBMCs, 2 million transduced PBMCs - 50 million transduced PBMCs, 2 million transduced PBMCs - 100 million transduced PBMCs, 3 million transduced PBMCs - 4 million transduced PBMCs, 3 million transduced PBMCs - 5 million transduced PBMCs, 3 million transduced PBMCs - 6 million transduced PBMCs transduced PBMC, 3 million transduced PBMC - 7 million transduced PBMC, 3 million transduced PBMC - 8 million transduced PBMC, 3 million transduced PBMC - 9 million transduced PBMC, 3 million transduced PBMC - 10 million transduced PBMC, 3 million transduced PBMC - 50 million transduced PBMC, 3 million transduced PBMC - 100 million transduced PBMC, 4 million transduced PBMC - 5 Million transduced PBMCs, 4 Million transduced PBMCs - 6 Million transduced PBMCs, 4 Million transduced PBMCs - 7 Million transduced PBMCs, 4 Million transduced PBMCs - 8 Million transduced PBMCs, 4 Million transduced PBMCs - 9 Million transduced PBMCs, 4 Million transduced PBMCs - 10 Million transduced PBMCs, 4 Million transduced PBMCs - 50 Million transduced PBMCs, 4 Million transduced PBMCs - Transduced PBMCs - 100 million transduced PBMCs, 5 million transduced PBMCs - 6 million transduced PBMCs, 5 million transduced PBMCs - 7 million transduced PBMCs, 5 million transduced PBMCs - 8 million transduced PBMCs, 5 million transduced PBMCs - 9 million transduced PBMCs, 5 million transduced PBMCs - 10 million transduced PBMCs, 5 million transduced PBMCs - 50 million transduced PBMCs,5 million transduced PBMCs - 100 million transduced PBMCs, 6 million transduced PBMCs - 7 million transduced PBMCs, 6 million transduced PBMCs - 8 million transduced PBMCs, 6 million transduced PBMCs - 9 million transduced PBMCs, 6 million transduced PBMCs - 10 million transduced PBMCs, 6 million transduced PBMCs - 500 million Million transduced PBMCs, 6 Million transduced PBMCs - 100 Million transduced PBMCs, 7 Million transduced PBMCs - 8 Million transduced PBMCs, 7 Million transduced PBMCs - 9 Million transduced PBMCs, 7 Million transduced PBMCs - 10 Million transduced PBMCs, 7 Million transduced PBMCs - 50 Million transduced PBMCs, 7 Million transduced PBMCs - 50 Million transduced PBMCs, 7 Million transduced PBMCs - 6 Million transduced PBMCs - 10 Million transduced PBMCs, 7 Million transduced PBMCs - 50 Million transduced PBMCs, 7 Million transduced PBMCs - 8 Million transduced PBMCs, 7 Million transduced PBMCs - 9 Million transduced PBMCs, 7 Million transduced PBMCs - 10 Million transduced PBMCs, 7 Million transduced PBMCs - 5 ... Million transduced PBMCs - 100 million transduced PBMCs, 8 million transduced PBMCs - 9 million transduced PBMCs, 8 million transduced PBMCs - 10 million transduced PBMCs, 8 million transduced PBMCs - 50 million transduced PBMCs, 8 million transduced PBMCs - 100 million transduced PBMCs, 9 million transduced PBMCs - 10 million transduced PBMCs, 9 million transduced PBMCs to 50 million transduced PBMCs, 9 million transduced PBMCs to 100 million transduced PBMCs, 10 million transduced PBMCs to 50 million transduced PBMCs, 10 million transduced PBMCs to 100 million transduced PBMCs, or 50 million transduced PBMCs to 100 million transduced PBMCs. In certain embodiments, a single dose comprises 1 million transduced PBMCs, 2 million transduced PBMCs, 3 million transduced PBMCs, 4 million transduced PBMCs, 5 million transduced PBMCs, 6 million transduced PBMCs, 7 million transduced PBMCs, 8 million transduced PBMCs, 9 million transduced PBMCs, 10 million transduced PBMCs,50 million transduced PBMCs or 100 million transduced PBMCs. In certain embodiments, a single dose comprises at least 1 million transduced PBMCs, 2 million transduced PBMCs, 3 million transduced PBMCs, 4 million transduced PBMCs, 5 million transduced PBMCs, 6 million transduced PBMCs, 7 million transduced PBMCs, 8 million transduced PBMCs, 9 million transduced PBMCs, 10 million transduced PBMCs, or 50 million transduced PBMCs. In certain embodiments, a single dose comprises up to 2 million transduced PBMCs, 3 million transduced PBMCs, 4 million transduced PBMCs, 5 million transduced PBMCs, 6 million transduced PBMCs, 7 million transduced PBMCs, 8 million transduced PBMCs, 9 million transduced PBMCs, 10 million transduced PBMCs, 50 million transduced PBMCs, or 100 million transduced PBMCs.

[0175] On a case-by-case basis, more or less cells can be used depending on the transduction efficiency of the individual's T cells. In certain embodiments, a single dose comprises 1 million CAR-T cells to 100 million CAR-T cells. In certain embodiments, a single dose comprises 1 million CAR-T cells to 2 million CAR-T cells, 1 million CAR-T cells to 3 million CAR-T cells, 1 million CAR-T cells to 4 million CAR-T cells, 1 million CAR-T cells to 5 million CAR-T cells, 1 million CAR-T cells to 6 million CAR-T cells, 1 million CAR-T cells to 7 million CAR-T cells, 1 million CAR-T cells to 8 million CAR-T cells, 1 million CAR-T cells to 9 million CAR-T cells, 1 million CAR-T cells to 20 million CAR-T cells, 1 million CAR-T cells to 30 million CAR-T cells, 1 million CAR-T cells to 40 million CAR-T cells, 1 million CAR-T cells to 50 million CAR-T cells, 1 million CAR-T cells to 60 million CAR-T cells, 1 million CAR-T cells to 70 million CAR-T cells, 1 million CAR-T cells to 80 million CAR-T cells, 1 million CAR-T cells to 90 million CAR-T cells, 1 million CAR-T cells to 100 million CAR-T cells, 1 million CAR-T cells to 100 million CAR-T cells, 1 million CAR-T cells to 20 million CAR-T cells, 1 million CAR-T cells to 30 million CAR-T cells, 1 million CAR-T cells to 40 million CAR-T cells, 1 million CAR-T cells to 50 million CAR-T cells, 1 million CAR-T cells to 60 million CAR-T cells, 1 million CAR-T cells to 100 CAR-T cells - 9 million CAR-T cells, 1 million CAR-T cells - 10 million CAR-T cells, 1 million CAR-T cells - 50 million CAR-T cells, 1 million CAR-T cells - 100 million CAR-T cells, 2 million CAR-T cells - 3 million CAR-T cells, 2 million CAR-T cells - 4 million CAR-T cells, 2 million CAR-T cells - 5 million CAR-T cells, 2 million CAR-T cells - 6 million CAR-T cells, 2 million CAR-T cells~7 million CAR-T cells, 2 million CAR-T cells~8 million CAR-T cells, 2 million CAR-T cells~9 million CAR-T cells, 2 million CAR-T cells~10 million CAR-T cells, 2 million CAR-T cells~50 million CAR-T cells, 2 million CAR-T cells~100 million CAR-T cells, 3 million CAR-T cells~4 million CAR-T cells, 3 million CAR-T cells~5 million CAR-T cells, 30 0M CAR-T cells~6M CAR-T cells, 3M CAR-T cells~7M CAR-T cells, 3M CAR-T cells~8M CAR-T cells, 3M CAR-T cells~9M CAR-T cells, 3M CAR-T cells~10M CAR-T cells, 3M CAR-T cells~50M CAR-T cells, 3M CAR-T cells~100M CAR-T cells, 4M CAR-T cells~5M CAR-T cells,4 million CAR-T cells - 6 million CAR-T cells, 4 million CAR-T cells - 7 million CAR-T cells, 4 million CAR-T cells - 8 million CAR-T cells, 4 million CAR-T cells - 9 million CAR-T cells, 4 million CAR-T cells - 10 million CAR-T cells, 4 million CAR-T cells - 50 million CAR-T cells, 4 million CAR-T cells - 100 million CAR-T cells, 5 million CAR-T cells - 6 million CAR-T cells, 5 million CAR-T cells - 7 million CAR CAR-T cells, 5 million CAR-T cells - 8 million CAR-T cells, 5 million CAR-T cells - 9 million CAR-T cells, 5 million CAR-T cells - 10 million CAR-T cells, 5 million CAR-T cells - 50 million CAR-T cells, 5 million CAR-T cells - 100 million CAR-T cells, 6 million CAR-T cells - 7 million CAR-T cells, 6 million CAR-T cells - 8 million CAR-T cells, 6 million CAR-T cells - 9 million CAR-T cells, 6 million CAR-T cells - 10 million CAR-T cells, 6 million CAR-T cells - 50 million CAR-T cells, 6 million CAR-T cells - 100 million CAR-T cells, 7 million CAR-T cells - 8 million CAR-T cells, 7 million CAR-T cells - 9 million CAR-T cells, 7 million CAR-T cells - 10 million CAR-T cells, 7 million CAR-T cells - 50 million CAR-T cells, 7 million CAR-T cells - 100 million CAR-T cells, 8 million CAR-T cells - 9 million CAR-T cells, 8 million CAR- T cells to 10 million CAR-T cells, 8 million CAR-T cells to 50 million CAR-T cells, 8 million CAR-T cells to 100 million CAR-T cells, 9 million CAR-T cells to 10 million CAR-T cells, 9 million CAR-T cells to 50 million CAR-T cells, 9 million CAR-T cells to 100 million CAR-T cells, 10 million CAR-T cells to 50 million CAR-T cells, 10 million CAR-T cells to 100 million CAR-T cells, or 50 million CAR-T cells to 100 million CAR-T cells. In certain embodiments,A single dose includes 1 million CAR-T cells, 2 million CAR-T cells, 3 million CAR-T cells, 4 million CAR-T cells, 5 million CAR-T cells, 6 million CAR-T cells, 7 million CAR-T cells, 8 million CAR-T cells, 9 million CAR-T cells, 10 million CAR-T cells, 50 million CAR-T cells, or 100 million CAR-T cells. In certain embodiments, a single dose comprises at least 1 million CAR-T cells, 2 million CAR-T cells, 3 million CAR-T cells, 4 million CAR-T cells, 5 million CAR-T cells, 6 million CAR-T cells, 7 million CAR-T cells, 8 million CAR-T cells, 9 million CAR-T cells, 10 million CAR-T cells, or 50 million CAR-T cells. In certain embodiments, a single dose comprises up to 2 million CAR-T cells, 3 million CAR-T cells, 4 million CAR-T cells, 5 million CAR-T cells, 6 million CAR-T cells, 7 million CAR-T cells, 8 million CAR-T cells, 9 million CAR-T cells, 10 million CAR-T cells, 50 million CAR-T cells, or 100 million CAR-T cells.

[0176] In certain embodiments, the population of anti-ROR-1 CAR-T cells of the present disclosure is included in a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, carriers, and diluents. In certain embodiments, the CAR-T cells of the present disclosure are administered suspended in a sterile, isotonic solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffering agent, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine; or a chelating agent, such as EDTA or EGTA. The CAR-T cells, once formulated, may be buffered at a particular pH, generally about 7.0 to about 8.0. In certain embodiments, the cells are buffered at a physiological pH of about 7.4 (average of about 7.35 to 7.45).

[0177] Also described herein are methods of treating an individual who has experienced one or more serious adverse events associated with CAR T cell therapy, comprising administering a bolus injection of cirumutuzumab, which can prevent CAR T cells from interacting with their targets and attenuate their activity.

[0178] Working Example The following illustrative examples are representative of embodiments of the compositions and methods described herein, and are not meant to be limiting in any way.

[0179] Example 1: Chimeric antigen receptor modified T cells (CAR-T) targeting neuroendocrine cancer cells expressing ROR-1 Preclinical studies have previously reported on the use of anti-human ROR1 T cell CARS against a wide variety of solid and liquid tumor cancers. However, when used clinically (NCT02706392 and NCT02194374), these same ROR1 CARS did not demonstrate any clinical benefit in patients with ROR1 陽性 had minimal activity in the treatment of hematological and solid tumor cancers. The lack of therapeutic efficacy in these studies is due to the use of T cell CARS containing rabbit / human chimeric ROR1 targeting domains. These domains present the potential for off-target binding and are recognized by the immune system as foreign antigens and therefore rapidly inactivated.

[0180] The anti-ROR1 T cell CAR constructed and tested in these examples was generated from fully humanized cirumutuzumab (Figure 2) and contains the complementarity determining regions (CDRs) and variable regions (V H / V L The examples presented herein show that a T cell CAR using cirumutuzumab as an antigen-binding component targets ROR1, which is resistant to other therapies. 陽性 Shown to be effective in treating neuroendocrine cancer.

[0181] Treatment failure The use of effective therapies aimed at inhibiting key oncogenic pathways active in epithelial cancers has resulted in multiple resistance mechanisms, including the development of highly aggressive neuroendocrine cancers such as small cell neuroendocrine carcinoma (SCNC). SCNC can arise from almost any epithelial organ, including the prostate and lung. Metastatic castration-resistant prostate cancer (CRPC) is one of several deadly neuroendocrine cancers. Small cell prostate cancer (SCPC) is a type of neuroendocrine prostate cancer (NEPC) and a particularly aggressive form of CRPC.

[0182] One in six men will be diagnosed with prostate cancer (PCa), and up to a quarter of PCa patients will develop advanced prostate cancer with poor prognosis and 5-year survival rates. The primary treatment for these types of prostate cancer is androgen deprivation therapy (ADT), which targets androgen receptor (AR) signaling. Inevitably, however, the cancer progresses to become resistant to ADT, and patients develop lethal castration-resistant prostate cancer (CRPC) and metastases. NEPC is emerging with increasing frequency in patients treated with ADT.

[0183] Most PCa metastasizes to bone, where it typically develops resistance to therapy and is incurable. ROR1 is an attractive therapeutic target because it is expressed on many highly malignant hematological and solid tumor cancer cells, such as CRPC and NEPC cells. However, despite the recent development of several novel therapies that have improved patient survival, almost all individuals with CRPC develop resistance to therapy and progress with the disease.

[0184] Innovation Since mCRPC and NEPC remain urgent unmet medical needs, the inventors have generated a series of second generation T cell CAR constructs that, when transduced into human T cells, show highly potent and specific anti-neuroendocrine tumor activity and specificity in in vitro and in vivo test systems. As shown in FIG. 2, the CARs of the present disclosure contain the light chain CDRs of anti-human ROR-1 mAb 4A5 or UC-961 linked to the heavy chain CDRs of the same mAb by a specific linker to generate high affinity single chain (scFv) molecules that specifically bind human ROR-1 with sufficient affinity to activate intracellular signaling and cytotoxicity in transduced T lymphocytes. To link the scFv to the transmembrane domain, a series of protein spacers made from IgG4 were generated. These spacers allow the anti-ROR-1 scFv binding domain sufficient flexibility to optimally bind to the target ROR-1 antigen.

[0185] The extracellular antigen-binding domain from the CAR is linked to the CD28 transmembrane domain, which is then linked to intracellular activation domains derived from CD28 and / or CD137, used alone (second generation CARs) or in combination (third generation CARs). These activation domains are then linked to the T cell receptor activation domain contained within the CD3 zeta chain (CD3ζ chain). Thus, the anti-ROR-1 CAR constructed and tested in these examples is expressed as a single polypeptide that includes, from the N-terminus: a leader that directs the construct to the cell surface, the light chain CDRs of either 4A5 or UC-961 mAb, a linker that joins the light and heavy chains, the heavy chain CDRs of 4A5 or UC-961 mAb complemented by the corresponding light chain molecule, a spacer of defined length made from IgG4 such as the IgG4 hinge region, the CD28 transmembrane domain, and the intracellular activation domains of CD28 and 41-BB (CD137) bound individually or in tandem to the T cell zeta chain extending to the carboxy terminus of the molecule. Using clinical grade procedures and processes, we have currently produced anti-ROR1 T cell CAR products from over 20 normal human donors and chronic lymphocytic leukemia (CLL) patient products from subjects ranging in age from 31 to 83 years old.

[0186] Figure 3 shows graphs depicting the in vitro cell killing activity of ROR1 CAR T cells derived from T cells of two healthy donors transduced with ROR1 in a 4-hour chromium release assay (left panel) and a 120-hour ACEA impedance assay (right panel), where T cells were transduced against leukemic Mec1 ROR1-positive cells at the indicated effect-to-target ratios (E:T) in the left panel, and against MB 231 ROR1-positive breast cancer cells in the ACEA impedance assay in the right panel. Anti-ROR1 CAR T cells showed high and specific cytotoxicity without significant killing of ROR1-negative target cells. In in vitro studies, both normal and patient-derived T cell CAR products showed dose-dependent activity against a wide variety of ROR1-positive solid and liquid tumor cell lines with little off-target activity (Figure 3).

[0187] To confirm the activity of these products, we also analyzed human ROR1 陽性 We tested our anti-ROR1 T cell CAR in a mouse leukemia model using CLL-derived cells as targets (Figures 4 and 5).

[0188] Figure 4 shows bioluminescence imaging of mice inoculated with MEC1-ROR1 cells and ROR1 CAR T cells. Animals treated with CAR-T cells had reduced disease burden compared to controls. 6 The ROR1-CAR T-cell) cohort showed a reduction in leukemic burden relative to background levels by day 30 and had minimal disease over the course of the study. Animals in the control group (untreated, mock transduced) had to be sacrificed on day 20. The right panel shows the total bioluminescence product harvested from the mice. The blue squares and circles are controls and the green triangles are ROR1-CAR T treated mice.

[0189] Figure 5 shows ROR-1 CAR T cell expression in mouse tissues at various time points. After ROR-1 CAR-T administration, animals were sacrificed on day 11 (upper panel) and day 25 (lower panel). Blood and organs were collected and subjected to flow analysis for CAR expression and confirmed ROR1 binding activity. ROR-1 CAR-T cell numbers were substantially higher in mice carrying MEC-1 ROR1 cells (CAR+MEC1-ROR1) versus control (CAR only), indicating elevated proliferation of ROR-1 CAR-T cells in tumor-burdened animals. Bars represent the mean of five mice in each group, and error bars represent the SE of the mean.

[0190] These results demonstrated that sirumtuzumab-based anti-ROR1 T cell CARS was highly effective and specific against tumor cells.

[0191] The CARs described herein can be contained in transposon-based or lentiviral vectors and can be introduced into target lymphocytes or natural killer cells using standard transfection or transduction techniques. To maximize stable transduction and long-term expression of the CAR product, retroviral delivery systems such as mouse gamma and human lentivirus are used. Currently, a third generation lentiviral four-plasmid system (Addgene, Inc.) is used to transgenic T lymphocytes for ROR-1 expressing the CAR. This third generation system is based on the lentiviral vector system described in Naldini et al., "Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector" Proc Natl Acad Sci USA. 1996 Oct 15; 93(21): 11382-11388. This four-plasmid system includes plasmid 1-gag / pol, plasmid 2-rev, plasmid 3 VSV-G protein, and plasmid 4, where the transfer plasmid contains the nucleic acid sequence encoding the anti-ROR1 CAR inserted using the restriction sites in the polylinker.

[0192] Example 2: ROR-1 is expressed in primary human prostate cancer cells. We conducted a study aimed to elucidate the expression of ROR1 in human neuroendocrine cancers, including prostate cancer.

[0193] Tissue microarray analysis Tissue microarray evaluation revealed that 90 percent (n=19 / 21) of prostate cancers had moderate to strong staining for ROR1 on the plasma membrane (Table 1). Furthermore, expression was associated with AKT activation and silencing of ROR1 expression, which impaired cell proliferation in vitro. Figure 6 shows results from analysis of tumor RNA sequences obtained from 66 prostate cancer samples, expressed as single sample gene set enrichment analysis profiles of non-canonical WNT and stem cell gene sets in association with expression of ROR1 (mRNA). The analysis shows significant enrichment of WNT non-canonical and stem cell gene sets for ROR1 mRNA expression.

[0194] Prostate cancer patient-derived xenograft models Despite the challenges posed by PCa bone metastases, which are not typically surgically removed or biopsied, the inventors established a biobank and novel patient-derived xenograft (PDX) and organoid models and used these as preclinical models to perform drug testing that accurately represent patient disease and predict patient response and drug efficacy.

[0195] Figure 4 shows the results obtained from whole-human genome HTA2.0 Affymetrix microarray and RNASeq gene expression profiling. These results show that Wnt5A was highly expressed in the patient prostate adenocarcinoma bone metastasis specimen and its corresponding PDX, PCSD1. Figure 7A shows that RNASeq analysis of another patient PCa bone metastasis-derived PDX, PCSD13 (small cell prostate cancer), showed significant ROR1 expression (Figure 4). These data indicate an interaction between Wnt5A, ROR-1, ROR-2, and bone metastatic prostate cancer, as progressive CRPC is associated with Wnt5A and ROR1 expression.

[0196] FACS analysis revealed that ROR1 protein was highly expressed in PCSD13 cells as well as PC3 and DU145 PCa cell lines (Figure 7B). We noted the reciprocal expression of Wnt5a and ROR1 in bone metastases from prostate cancer PCSD1 compared to small cell prostate cancer PCSD13. PCSD1 is PSMA+WNT5A+ROR1 低 ROR2 低 However, PCSD13 was - WNT5A - ROR1+ROR2+. This may reflect fundamental differences in prostate adenocarcinoma and small cell carcinoma of the prostate, i.e., NEPC. Our findings highlight the importance of characterizing ROR1 expression at various stages of prostate cancer to better understand the role of ROR1 in prostate cancer disease progression and the emergence of neuroendocrine prostate cancer (NEPC) and to identify patient populations most suitable for ROR1 targeting with cirumtuzumab-CART cells.

[0197] PDX-derived organoid cultures Three-dimensional (3D) organoid cell cultures from patient-derived xenografts (PDXs) and primary patient tumor cells were established. Figure 8 shows PCSD1 PDX tumor cells from 3D organoids replicating the tissue morphology of xenografts growing in femurs. The 3D cultures consisted of two major cell masses: spheroids and epithelial cysts that resembled the gland-like structures seen in sections from xenografts growing in patients and within the femurs replacing bone marrow. The spheroids were resistant to androgen deprivation-induced death. The spheroid masses contained cyst-like structures surrounded by tumor cells that resembled gland-like structures. Black arrows point to the cystic structures. The organoids are GFP+. The PDXs were obtained from prostate cancer bone metastases. PCSD1 expressing GFP-luciferase formed distinct morphologies under 3D culture conditions. Figure 8 also shows that enzalutamide treatment of PCSD1 reduced the luminal size and the number of epithelial cysts. Transcriptome analysis showed that AR-responsive genes such as PSA (KLK3) and TMPRSS2 were downregulated under ADT, while stem cell transcription factors, steroidogenic and neurogenic pathway genes were upregulated. Thus, although antiandrogens were still able to suppress canonical AR-responsive genes, tumor and organoid growth was resistant to ADT, including the antiandrogen enzalutamide.

[0198] Example 3: Anti-ROR-1 T cell CAR is highly effective in targeting and killing the neuroendocrine cancer cell line EXPRESSINg Ror-1 PCa is highly heterogeneous and shows high recurrence rates after standard treatment, therefore new therapeutic approaches are needed. We have demonstrated that treatment-resistant ROR1 陽性 To target cancer, we generated a cirumtuzumab-based T cell CAR. To test and demonstrate the activity of this CAR product, we created a series of second generation T cell CAR constructs that demonstrated highly potent and specific antitumor activity and specificity in in vitro and in vivo test systems of hematological and human solid tumor cancers when transduced into human T cells.

[0199] The objectives of these studies are to examine the efficacy of human ROR1 CAR-T cells on PCa cell lines, PDX models, and organoids of mCRPCNEPC in combination with androgen deprivation therapy and standard of care therapies such as androgen deprivation and docetaxel to determine efficacy and define the use of adjuvant therapy in conjunction with CAR T cells. Further objectives are to evaluate ROR1 expression and signaling in various clinical conditions of malignant prostate cancer and to define PCa cell types that express the ROR-1 oncoprotein. Further objectives are to determine the in vitro efficacy of ROR1 CAR cells on patient biopsy-derived organoids using ROR1 CAR engineered in TILs and PBMCs from the same patient.

[0200] As shown in Figure 9, the generated anti-ROR1 T cell CAR is highly effective in targeting and killing ROR1-expressing NEPC cell lines, even at low effector to target (E:F) ratios. Figure 10 shows that anti-ROR1 CAR-T cells can effectively target and kill ROR1-expressing NEPC cell lines in an effector:target (E:T) dose-dependent manner. 陽性 Thus, the anti-ROR1 T cell CAR is highly effective in targeting and killing ROR1-expressing NEPC cell lines, even at low effector-to-target (E:T) ratios.

[0201] Pilot Testing A pilot study was conducted to examine the effect of CAR T cells provided herein on subcutaneously implanted prostate cancer cell lines. Luciferase-transduced PC3 cells were implanted subcutaneously into the flanks of immunodeficient NSG mice. For the treatment group, we administered ROR1 T cell CARs both intravenously and by direct injection into the tumor. Control animals received mock-transduced activated T cells generated from the same donor leukapheresis product. As shown in Figure 11, PC3 cells rapidly proliferated on the backs of these animals, and by the fourth week, they had grown into large bulky tumors and the mice had to be sacrificed. In contrast, CAR-T-treated mice that received a single intravenous or intratumoral injection had reduced disease burden when compared to control animals, with minimal disease at the end of the study.

[0202] To investigate the efficacy of ROR 1 CAR T cells in PCa cell lines and PDX models of mCRPC and NEPC in vivo alone and in combination with docetaxel, the effect of anti-ROR-1 CAR T cells on tumor growth inhibition is examined in PCa cell lines and PDX PCSD 1 and PCSD 13 organoids.

[0203] Anti-ROR-1 CAR T cells will be tested in vitro in PCa cell lines and PCa organoids, and in vivo in PCa cell line xenografts and PDX injected subcutaneously, intrafemorally and intracardially.

[0204] An overview of these experiments in preclinical models of metastatic prostate cancer is provided below.

[0205] Cells: Prostate cancer cell line: AR+ROR1 低 :LNCaP;ARV7+:VCaP;AR-ROR1 高 ;PC3;AR-ROR1 高 :DU145, PCa cell lines all stably express RFP-luciferase and cytotoxicity assays are performed in Incucyte. Prostate cancer bone metastasis PDX organoids AR+ROR1 低:PCSD1 and AR+ROR1 高 PCSD13.

[0206] Therapy: Sirmutuzumab-based anti-ROR1 CART cells.

[0207] In Vitro Experiment 1: Perform a titration of effector:target (E:T) ratios of CART cells experiments on four PCa cell lines alone or in combination with docetaxel. Four effector to target (E:T) ratios, i.e. 30:1, 10:1, 3:1, 1:1 of ROR1 CART or control T cells alone + / - antiandrogen + / - docetaxel are tested in triplicates per 96 well plate x 2 plates per cell line x 4 prostate cancer cell lines. Cell viability is measured using Incucyte on RFP labeled cells in a 96 well plate assay. Cytotoxic killing is measured by LDH release assay.

[0208] In Vitro Experiment 2 Perform an E:T titration experiment for PCSD 1-GFP-luciferase, PCSD 13-GFP-luciferase against CART as described above in the Incucyte assay. Test 3D cultures of two PDOs (PCSD1 and PCSD13) in the following groups: vehicle control, CART or control T cells + / - enzalutamide + / - docetaxel in E:T ratios of 30:1, 10:1, 3:1, 1:1 in triplicate 24-well plates (see Figure 1) = 2 PDOs x 1 24-well plate x 2 experiments (in duplicate) = 4 plates. Weekly digital microscopic imaging and analysis of organoid cell number (viability) and size (proliferation). Immunohistochemistry (IHC) for AR and immunofluorescence (IFC) analysis of prostate markers, i.e., cytokeratin (CK) 5, CK8, p63, are used to examine AR protein levels and organoid differentiation, Ki67 to examine proliferation, and cleaved caspase 3 to examine apoptosis. qRT-PCR and RNAseq are used to measure effects on AR expression and AR-dependent gene expression. Experiments are performed in triplicate plates. Dose-escalating treatments begin 1 week after doming of organoids. Images pre-treatment vs. 3 hours (hrs), 6 hours, 12 hours, 24 hours post-treatment. Cells are harvested for RNA, lysates are harvested after 24 hours for AR degradation analysis by IHC, and FFPE sections are harvested for IHC and IFC.

[0209] In vivo PCa cell line xenograft and patient-derived xenograft (PDX) experiments: Mice: NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Labs stock number 005557), 6-8 weeks old, male

[0210] Cells: LNCaP (AR+, enzalutamide sensitive) a.) subcutaneous (sc), b.) intracardiac (ic) injection, 2. PC3 AR negative, enzalutamide resistant) a.) subcutaneous (sc), b.) intracardiac (ic) injection, 3. DU145 AR negative, enzalutamide resistant) a.) subcutaneous (sc), b.) intracardiac (ic) injection, 4. VCaP (AR+, enzalutamide resistant) a.) subcutaneous (sc), b.) intracardiac (ic) injection, 5. PCSD1 PDX cells from prostate cancer bone metastasis (AR+, enzalutamide resistant in femoral environment) a.) subcutaneous (sc), b.) intrafemoral (if) injection, 6. PCSD13 PDX cells from prostate small cell carcinoma bone metastasis (AR+, enzalutamide resistant in femoral environment) a.) subcutaneous (sc), b.) intrafemoral (if) injection

[0211] Therapy: Sirmutuzumab-based anti-ROR1 CART cell tumors at least 100 mm 3 For CART treatment groups, n=10 mice (depending on tumor size at treatment initiation): 1. Control T cells (no CAR), vehicle (n=10), 2. CART cells, vehicle (n=10), 3. Control T cells, docetaxel (n=10), 4. CART cells, docetaxel (n=10)

[0212] Total n = 40 mice x 6 xenograft (LNCaP, PC3, DU145, VCaP, PCSD1, PCSD13) models = 240 mice

[0213] For CART compared to control T cells, n=8-10 mice per treatment group (depending on tumor size at start of treatment): 1. Control T cells, vehicle (n=8-10), 2. CART cells, vehicle (n=8-10), 3. Control T cells, enzalutamide, (n=8-10), 4. CART cells, enzalutamide, (n=8-10) Total n=40 mice x 4 (LNCaP, VCaP, PCSD1, PCSD13) xenograft mouse models=240 mice

[0214] Clinical signs are monitored daily, caliper measurements of tumor volume, body weight and health report are monitored twice weekly. After 4 weeks of treatment, the experiment is terminated and a.) peripheral blood: plasma for drug concentration (PK / PD), serum (PSA), b. tumors from all mice are harvested and (1 / 4 of tumor) fixed in 4% PFA, (1 / 4) snap frozen in liquid nitrogen for DNA, RNA, protein analysis, half of the tumor dissociated into single cells for organoids, cryopreserved in 10% DMSO, 90% FBS for FACS on tumor cells, and reimplanted in the future to measure tumor-initiating cancer stem cells. Injected femurs with surrounding tissue and contralateral non-injected femurs are formalin fixed and decalcified for IHC and IFC.

[0215] Characterization and improved efficacy of ROR-1 CART cells in PCa cell line xenograft and PDX models of mCRPC and NEPC alone or followed by in vivo treatment with docetaxel

[0216] The ability of ROR-1 CAR T cells to eradicate various different types of already established PCa tumors is tested in vivo. Human PCa cell lines are implanted subcutaneously (sc) in immunodeficient mice (NSG). Human patient-derived PCa bone metastasis xenografts (PDXs) are either injected intrafemorally (if) directly into the intraosseous medullary space of the right femur to test the efficacy of CART against bone metastases, or subcutaneously (sc) for comparison with PCa cell lines. Tumor growth inhibition by CART cells alone is compared to SOC therapy, i.e., docetaxel and ADT, enzalutamide alone and in combination.

[0217] Overview of in vivo PCa cell line xenograft and patient-derived xenograft (PDX) experiments Mice: NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Labs stock number 005557), 6-8 weeks old, male

[0218] Cell: 1.PC3(AR 陰性 , ROR1 陽性enzalutamide resistance) subcutaneous (sc), 2.DU145 (AR 陰性 , ROR1 陽性 , Enzalutamide resistance) subcutaneous (sc), 3. PCSD13 prostate cancer PDX cell metastasis to bone (AR 陽性 , ROR1 陽性 Cone resistance in femoral environment a.) Subcutaneous (sc), b.) Intrafemoral (if)

[0219] Therapy: Sirmutuzumab anti-ROR1 CART cells Tumors at least 100 mm 3 Treatment begins when the condition becomes 陽性 , ROR1 低 Enzalutamide sensitive), subcutaneous (sc

[0220] In vivo experiment 1: CART + docetaxel: For CART treatment groups, n=10 mice (depending on tumor size at the start of treatment): 1. Control T cells (no CAR), vehicle (n=10), 2. CART cells, vehicle (n=10), 3. Control T cells, docetaxel (n=10), 4. CART cells, docetaxel (n=10)

[0221] Tested ROR1+ cells: total n=40 mice x 4 xenografts PC3 sc, DU145 sc, PCSD13 sc, if model=160 mice

[0222] In vivo Example 2: CART + Enzalutamide: n=10 mice per treatment group (depending on tumor size at start of treatment) for CART compared to control T cells: 1. Control T cells, vehicle (n=10), 2. CART cells, vehicle (n=10), 3. Control T cells, enzalutamide, (n=10), 4. CART cells, enzalutamide, (n=10)

[0223] Test AR+, ROR1+ cells: 1. LNCaP (sc), 2. PCSD13 (sc) and 3. PCSD13 (if)

[0224] Total n = 40 mice × 3 (LNCaP AR 陽性, ROR1 低 sc, PCSD13 sc, PCSD13 if) xenograft mouse model = 120 mice

[0225] Primary Endpoint: Tumor growth inhibition: Caliper measurements of tumor volume, body weight and health reports twice weekly + in vivo bioluminescence (IVIS) once weekly

[0226] Weekly serum collection: PSA assay and ALP assay

[0227] End point: After 4 weeks of treatment, peripheral blood, spleen and bone marrow are harvested and analyzed for CART presence using FACS, plasma drug concentrations (PK / PD), serum (PSA). Tumors from all mice: (Tumor 1 / 4 ) was fixed in 4% PFA, and ( 1 / 4 ) are snap frozen in liquid nitrogen for DNA, RNA, protein analysis, and half of the tumor is dissociated into single cells for organoids, FACS on tumor cells, and tumor-infiltrating CART cells, and cryopreserved in 10% DMSO, 90% FBS for future reimplantation to measure tumor-initiating cancer stem cells. The injected femur with surrounding tissue and the contralateral non-injected femur are formalin fixed and decalcified for IHC and IFC. Snap frozen samples are used to extract DNA, RNA for RNASeq, qPCR of PSA, AR, and proteins for immunoblotting analysis of signaling pathways (WNT5A, ROR1, ROR2, phosphotyrosine, AKT, and ERK). Femur + periosteal tumor tissue: formalin fixed for MicroCT, then paraffin embedded for FFPE sections, immunohistochemistry (PSA, ROR1 and ROR2, other Frizzled proteins) and immunofluorescence (AR, CK5, CK8, Ki67, cleaved caspase 3).

[0228] In vivo testing of anti-ROR1-CAR T cells in de novo bone metastasis models using intracardiac or intrailiac artery infusion models of bone metastasis alone or in combination with ADT (enzalutamide) and / or docetaxel

[0229] The efficacy of sirmutuzumab-based CAR T cells against de novo metastasis is tested in treating mice implanted with ROR1-expressing cell lines, PC3, DU145 and PDX PCSD13 by intracardiac and intrailiac xenograft arterial injection, alone or after treatment with docetaxel. Mice are first treated with docetaxel or enzalutamide for at least 28 days to debulk tumors. After chemotherapy treatment and washout, PCa are treated with CAR T cells.

[0230] Primary Endpoint: Tumor growth inhibition: weekly IVIS body weight, weekly IVIS, weekly serum collection, and weekly PSA and ALP assays.

[0231] End Endpoint: Tumor tissue is harvested which is snap frozen for subsequent extraction of DNA, RNA for RNA-Seq, qPCR of PSA, AR and ROR1, and protein for immunoblot analysis of signaling pathways (WNT5A, ROR1, ROR2, phospho-tyrosine, AKT, ERK).

[0232] Results: Sirmutuzumab-based CAR T cells inhibit tumor cell proliferation in ROR 1+ metastatic prostate cancer lines and PDXs in vitro and in vivo, either alone or in combination with conventional SOC docetaxel or enzalutamide.

[0233] Development of a companion diagnostic for detecting ROR1-expressing CSCs in patients with CRPC

[0234] The goal of this study was to develop a CLIA-certified IHC assay that can be used to detect ROR-1 in neuroendocrine cancers such as CPRC.

[0235] The anti-ROR1 antibody used in the development of this assay is mAb (4A5). In this process, 4A5 is tested using tissue microarrays (TMA) of normal, inflammatory and neoplastic (benign and malignant) tissues provided by the Biorepository Tissue Technology Shared Resource (BTTSR) facility in San Diego, USA. Both frozen and paraffin-embedded TMAs from relevant pathologies are used, including patient-derived prostate tumor samples taken in various pathological conditions and corresponding normal tissue controls. For validation purposes, the antibody is tested using two standard IHC techniques, the standard and highly sensitive ABC technique (Vector, CA) as described, and the ImmPRESS™ HRP Polymer Detection Kit, as well as standard indirect immunofluorescence.

[0236] TMA Selection: Prostate tissue TMAs are constructed as described using a MicaArray GEN 3.0 manual arrayer (Micatu, NY). A cohort of de-identified paraffin blocks representing a range of non-malignant and malignant prostate tumors are selected from the BTTSR. Negative tissue controls are selected from ROR1 of ovary, colon, lung, skin, and pancreas. 陽性 Arrayed alongside known positive samples previously detected in cancer.

[0237] Analysis of protein expression: A study pathologist and a pathologist blinded to the results or technique evaluate all TMAs and individual tissues prior to IHC analysis using H&E stained slides for relevance and tissue preservation. The number of positive cells is visually assessed for each core and results are expressed as percentage of stained cells / total number of cells. Based on their immunoreactivity, TMA cores are divided and scored into five categories: 0, less than 10% of stained cells; 1, 10%-25% stained cells; 2, 25%-50% stained cells; 3, 50%-75% stained cells; 4, 75%-100% stained cells. Consider quantitative automated image analysis.

[0238] Statistical analysis: Use Pearson's correlation coefficient to quantify the agreement between measurements, as described by Anagnostou et al.. Cohen's weighted kappa is also used to measure the agreement between antibody pairs.

[0239] CLIA Assay Validation - Specificity, Sensitivity, Reproducibility, and Limitations. A study pathologist will analyze consecutive sets of samples from the CRPC TMA to examine day-to-day variability, reproducibility, and robustness of the assay performance. In a second set of studies, two independent pathologists will 陽性 and 陰性 Forty specimens were read from the sample. Interrater agreement was quantified by Pearson correlation and Cohen's weighted kappa with their 95% confidence limits. Based on the results of these studies, a CLIA-validated IHC companion assay was developed to evaluate the ROR1 antibody. 陽性 It will enable patients with CRPC to enroll in clinical trials using cirumutuzumab-based T-cell CARs.

[0240] Biosystem Culture The goal of this study was to determine the in vitro efficacy of ROR1 CART cells against patient biopsy-derived organoids using ROR1 CAR engineered into TILs and PBMCs from the same patient. Individualized mini-tumors in cell culture have been successfully generated from tumor tissue of prostate cancer (PCa) patients (Lee et al., 2020). Samples of each patient's tumor tissue were taken immediately after surgical removal and used to establish 3D-organoids or mini-tumors. In parallel, we expanded each patient's own immune cells present within the tumor tissue, known as tumor-infiltrating lymphocytes (TILs). We then combined each patient's mini-tumors with their own TILs to establish individualized 3D-biosystem cultures as shown in Figure 13. These individual patient-derived mini-tumors are being used to determine responses to immunotherapies in combination with current and novel tumor-targeted therapies.

[0241] Organoids are established and TILs expanded from n=5-10 patient biopsies from prostate cancer metastases. Sirmutuzumab-based CART cells are generated from TILs and PBMCs from the same patients as the organoids using our lentiviral constructs that we used in our CART products. Organoids and patient-derived cirumtuzumab-based CART are co-cultured. Cytotoxicity killing is measured by digital microscopic imaging daily on days 1-7. Single-cell RNA sequencing (UCSD IGM core, 10X Genomics) and immunofluorescence cytochemistry of the co-cultures with immune cells and prostate markers.

[0242] The results of this study will enable the development of strategies to tailor immunotherapy for individual patients through a better understanding of the mechanisms of response and resistance.

[0243] Evaluating ROR1 expression and signaling in various clinical states of malignant prostate cancer and defining ROR-1-expressing PCa cell types

[0244] ROR1 expression patterns and signaling will be examined in samples from patients with localized prostate cancer of different Gleason scores, metastatic hormone-sensitive prostate cancer, metastatic CRPC, and neuroendocrine prostate cancer to determine whether ROR1 expression is increased in high-grade and castration-resistant tumors.

[0245] FFPE tissues from standard of care radical prostatectomies, prostate biopsies, and metastatic biopsies stored in the UCSD Pathology Biorepository are analyzed. IHC staining is performed using a commercially available anti-ROR1 antibody of choice, e.g., 4A5 (BD) or rabbit polyclonal from Protein Tech, UCSD Tissue Technology Shared Resource histology core. IHC stained slides are inspected and areas of tumor are manually identified and marked. All slides are scanned at the Tissue Technology Shared Resource using an Aperio ScanScope XT system (Aperio®, Vista, CA). Spectrum Analysis algorithm package and ImageScope analysis software are applied to quantify IHC staining using color deconvolution algorithms as previously described (60, 61).

[0246] Clinical specimens are also examined to confirm Gleason score and the presence of neuroendocrine differentiation. Clinical parameters such as age, race, ethnicity, PSA, stage, and previous treatment status are incorporated and correlated with pathological and ROR1 expression data. ROR1 expression is assessed and graded as follows: 0: no positive cancer cells, 1: low to moderate staining in less than 50% of cancer cells, 2: strong staining in less than 50% of cancer cells or moderate staining in more than 50% of cancer cells, 3: strong staining in more than 50% of cancer cells. All staining is assessed and ROR1 levels are correlated with disease parameters such as measurable disease imaging response, PSA response (≥50% PSA decline from baseline), or stable disease for ≥6 months. Additionally, the effect of cirumutuzumab-based CART inhibition of ROR1 signaling is assessed in serial sections of biopsies using antibodies against phospho-SRC, phospho-AKT, phospho-CREB, YAP / TAZ, and BMI1.

[0247] Statistical Analysis: Table 1 details the number of available archived specimens. For the primary analysis, we examine the similarities in ROR1 expression and downstream signaling between castration-resistant disease (n=20) and hormone-sensitive disease (n=70). We define positive ROR1 expression as an IHC grading of 1-3. We calculate the proportion of ROR1 positive specimens for each clinical condition and their 95% CI. Fisher's exact test is used to compare the proportion of ROR1 positivity between castration-resistant and hormone-sensitive clinical conditions.

[0248] The results indicate that with our current sample size, the ROR1 expression rate is 20% in hormone-sensitive specimens and 60% in castration-resistant specimens with 90% power and a two-sided alpha of 0.05.

[0249] [Table 1]

[0250] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in the practice of the invention.

[0251] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the text incorporated by reference are excluded only to the extent that they conflict with definitions in this disclosure.

[0252] Embodiment Embodiment 1. A chimeric antigen receptor (CAR), comprising: i. an antigen-binding region that specifically binds to ROR-1 and comprises a light chain variable domain and a heavy chain variable domain; an antigen-binding region, wherein the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48; ii. a spacer domain; iii. a transmembrane domain; iv. A chimeric antigen receptor comprising:

[0253] Embodiment 2. A chimeric antigen receptor (CAR), comprising: v. an antigen-binding region that specifically binds to ROR-1 and comprises a light chain variable domain and a heavy chain variable domain; an antigen-binding region, wherein the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54; vi. a spacer domain; vii. a transmembrane domain; viii. A chimeric antigen receptor comprising an intracellular domain.

[0254] Embodiment 3. The chimeric antigen receptor according to any one of embodiments 1 to 2, wherein the spacer domain is 14 to 120 amino acids in length.

[0255] Embodiment 4. The chimeric antigen receptor of any one of embodiments 1 to 3, wherein the light chain variable domain is linked to the N-terminus or C-terminus of the heavy chain variable domain.

[0256] Embodiment 5. The chimeric antigen receptor of any one of embodiments 1 to 4, wherein the light chain variable domain is covalently linked to the heavy chain variable domain via a polypeptide linker.

[0257] Embodiment 6 The chimeric antigen receptor of embodiment 5, wherein the polypeptide linker comprises the amino acid sequence of SEQ ID NO:24.

[0258] Embodiment 7. The chimeric antigen receptor of any one of embodiments 1 to 6, wherein the spacer domain comprises an antibody domain.

[0259] Embodiment 8. The chimeric antigen receptor of embodiment 7, wherein the antibody domain comprises an immunoglobulin hinge domain, an immunoglobulin constant heavy 3 (CH3) domain, an immunoglobulin constant heavy 2 (CH2) domain, or a combination thereof.

[0260] Embodiment 9. The chimeric antigen receptor of any one of embodiments 1 to 8, wherein the spacer domain comprises the amino acid sequence of SEQ ID NO:29, SEQ ID NO:41, or SEQ ID NO:42.

[0261] Embodiment 10. The chimeric antigen receptor of any one of embodiments 1 to 9, wherein the light chain variable domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:21.

[0262] Embodiment 11. The chimeric antigen receptor of any one of embodiments 1 to 10, wherein the heavy chain variable domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:27.

[0263] Embodiment 12. The chimeric antigen receptor according to any one of embodiments 1 to 11, wherein the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination thereof.

[0264] Embodiment 13. The chimeric antigen receptor of embodiment 12, wherein the transmembrane domain is the CD28 transmembrane domain.

[0265] Embodiment 14. The chimeric antigen receptor of any one of embodiments 1 to 13, wherein the intracellular domain comprises an intracellular costimulatory signaling domain, an intracellular T cell signaling domain, or a combination thereof.

[0266] Embodiment 15. The chimeric antigen receptor of embodiment 14, wherein the intracellular costimulatory signaling domain is a 4-1BB intracellular costimulatory signaling domain, a CD28 intracellular costimulatory signaling domain, an ICOS intracellular costimulatory signaling domain, an OX-40 intracellular costimulatory signaling domain, or any combination thereof.

[0267] Embodiment 16 The chimeric antigen receptor of embodiment 15, wherein the intracellular costimulatory signaling domain comprises a 41-BB intracellular costimulatory signaling domain.

[0268] Embodiment 17. The chimeric antigen receptor of embodiment 16, wherein the intracellular costimulatory signaling domain comprises a CD28 intracellular costimulatory signaling domain and a 41-BB intracellular costimulatory signaling domain.

[0269] Embodiment 18. The chimeric antigen receptor of any one of embodiments 15 to 17, wherein the intracellular costimulatory signaling domain further comprises an intracellular T cell signaling domain.

[0270] Embodiment 19. The chimeric antigen receptor of embodiment 18, wherein the intracellular T cell signaling domain is a CD3ζ intracellular T cell signaling domain.

[0271] Embodiment 20. A chimeric antigen receptor according to any one of embodiments 1 to 19, which binds to a cell.

[0272] Embodiment 21. The chimeric antigen receptor of embodiment 20, wherein the cell is a neuroendocrine cancer cell.

[0273] Embodiment 22. The chimeric antigen receptor of embodiment 21, wherein the neuroendocrine cancerous cell is a carcinoid tumor cell, a pancreatic islet cell tumor cell, a medullary thyroid carcinoma cell, a pheochromocytoma cell, a neuroendocrine carcinoma of a skin cell, a small cell lung carcinoma cell, a large cell neuroendocrine carcinoma cell, a neuroendocrine prostate cancer (NEPC) cell, or a metastatic castration-resistant prostate cancer (CRPC) cell.

[0274] Embodiment 23. The chimeric antigen receptor of any one of embodiments 1 to 22, wherein the chimeric antigen receptor forms part of a cell.

[0275] Embodiment 24. A chimeric antigen receptor according to any one of embodiments 1 to 23, which forms part of a T cell.

[0276] Embodiment 25. An isolated nucleic acid encoding the chimeric antigen receptor of any one of embodiments 1 to 24.

[0277] Embodiment 26. A therapeutically effective amount of the chimeric antigen receptor of any one of embodiments 1 to 25 and a pharma- ceutically acceptable excipient.

[0278] Embodiment 27. A method for treating neuroendocrine cancer in a subject in need of such treatment, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 26.

[0279] Embodiment 28. The method of embodiment 27, wherein the chimeric antigen receptor (CAR) binds to the neuroendocrine cancerous cells.

[0280] Embodiment 29. The method of any one of embodiments 27 to 28, wherein the cell is a T lymphocyte.

[0281] Embodiment 30 The method of embodiment 29, wherein the T lymphocytes are CD4+ T lymphocytes or CD8+ T lymphocytes.

[0282] Embodiment 31. The method of any one of embodiments 27 to 30, wherein the cell is a natural killer cell.

[0283] Embodiment 32 The method of embodiment 31, wherein the natural killer cells are autologous to the subject.

[0284] Embodiment 33 The method of embodiment 31, wherein the natural killer cells are xenogeneic to the subject.

[0285] Embodiment 34 The method of embodiment 31, wherein the natural killer cells are allogeneic to the subject.

[0286] Embodiment 35. The method of any one of embodiments 27 to 34, wherein the neuroendocrine cancer is a carcinoid tumor, a pancreatic islet cell tumor, a medullary thyroid carcinoma, a pheochromocytoma, a neuroendocrine carcinoma of the skin, a small cell lung cancer, a large cell neuroendocrine carcinoma, a neuroendocrine prostate cancer (NEPC), or a metastatic castration-resistant prostate cancer (CRPC).

[0287] Embodiment 36. The method of any one of embodiments 27 to 35, wherein the neuroendocrine cancer is metastatic castration-resistant prostate cancer (CRPC).

[0288] Embodiment 37. The method of any one of embodiments 27 to 36, wherein the neuroendocrine cancerous cells express ROR-1.

[0289] Embodiment 38. The method of any one of embodiments 27-37, wherein the subject has not responded to androgen deprivation therapy.

[0290] Embodiment 39. The method of any one of embodiments 27 to 38, wherein the neuroendocrine cancer has metastasized to bone.

[0291] Embodiment 40. The method of any one of embodiments 27-39, further comprising administering cirumutuzumab to the subject.

[0292] Embodiment 41 The method of embodiment 40, wherein cirumutuzumab and the cells are administered separately.

[0293] Embodiment 42 The method of embodiment 40, wherein cirumutuzumab and the cells are administered together.

[0294] Embodiment 43. The method of any one of embodiments 27-42, further comprising administering to the subject platinum-based chemotherapy.

[0295] Embodiment 44. The method of embodiment 43, wherein the platinum-based chemotherapy comprises carboplatin, cisplatin, etoposide, a taxane, or any combination thereof.

[0296] Embodiment 45. A method for preventing or treating metastasis in a subject having a neuroendocrine cancer, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 26.

[0297] Embodiment 46. The method of embodiment 45, wherein the chimeric antigen receptor (CAR) binds to the neuroendocrine cancerous cells.

[0298] Embodiment 47. The method of any one of embodiments 45 to 46, wherein the cell is a T lymphocyte.

[0299] Embodiment 48 The method of embodiment 47, wherein the T lymphocytes are CD4+ T lymphocytes or CD8+ T lymphocytes.

[0300] Embodiment 49. The method of any one of embodiments 45 to 48, wherein the cell is a natural killer cell.

[0301] Embodiment 50 The method of embodiment 49, wherein the natural killer cells are autologous to the subject.

[0302] Embodiment 51 The method of embodiment 49, wherein the natural killer cells are xenogeneic to the subject.

[0303] Embodiment 52 The method of embodiment 49, wherein the natural killer cells are allogeneic to the subject.

[0304] Embodiment 53. The method of any one of embodiments 45 to 52, wherein the neuroendocrine cancer is a carcinoid tumor, a pancreatic islet cell tumor, a medullary thyroid carcinoma, a pheochromocytoma, a neuroendocrine carcinoma of the skin, a small cell lung cancer, a large cell neuroendocrine carcinoma, a neuroendocrine prostate cancer (NEPC), or a metastatic castration-resistant prostate cancer (CRPC).

[0305] Embodiment 54. The method of any one of embodiments 45 to 53, wherein the neuroendocrine cancer is metastatic castration-resistant prostate cancer (CRPC).

[0306] Embodiment 55. The method of any one of embodiments 45 to 54, wherein the neuroendocrine cancerous cells express ROR-1.

[0307] Embodiment 56. The method of any one of embodiments 45 to 55, wherein the subject has not responded to androgen deprivation therapy.

[0308] Embodiment 57. The method of any one of embodiments 45 to 56, wherein the neuroendocrine cancer has metastasized to bone.

[0309] Embodiment 58. The method of any one of embodiments 45 to 57, further comprising administering silmutuzumab to the subject.

[0310] Embodiment 59 The method of embodiment 58, wherein cirumutuzumab and the cells are administered separately.

[0311] Embodiment 60 The method of embodiment 58, wherein cirumutuzumab and the cells are administered together.

[0312] Embodiment 61. The method of any one of embodiments 45 to 60, further comprising administering to the subject platinum-based chemotherapy.

[0313] Embodiment 62. The method of embodiment 61, wherein the platinum-based chemotherapy comprises carboplatin, cisplatin, etoposide, a taxane, or any combination thereof.

[0314] Embodiment 63 The method of any one of embodiments 43 and 61, wherein the platinum-based chemotherapy comprises administering an antibody-drug conjugate.

[0315] References 1. Siegel R, Ma J, Zou Z, & Jemal A. Cancer statistics. CA Cancer J Clin 2014:64(1):9-29. 2. Oster G, Lamerato L, Glass AG, Richert-Boe KE, Lopez A, Chung K, Richhariya A, Dodge T, Wolff GG, Balakumaran A, Edelsberg J. Natural history of skeletal-related events in patients with breast, lung, or prostate cancer and metastases to bone:a 15-year study in two large US health systems.Support Care in Cancer.2013 Dec 21(12):3279-3286. 3.Logothetis C,Morris MJ,Den R,Coleman RE.Current perspectives on bone metastases in castrate-resistant prostate cancer.Cancer Metastasis Rev.2018 Mar;37(1):189-196.doi:10.1007 / s10555-017-9719-4.Review.PMID:29380085 4.Garnero P,Buchs N,Zekri J,Rizzoli R,Coleman RE,Delmas PD.Markers of bone turnover for the management of patients with bone metastases from prostate cancer.Br J Cancer.2000 82(4):858-864. 5.Shiao SL,Chu GC,Chung LW.Regulation of prostate cancer progression by the tumor microenvironment.Cancer Lett.2016 Jan 28.pii:S0304-3835(15)00773-9.doi:10.1016 / j.canlet.2015.12.022.[Epub ahead of print]. 6.Vargas HA,Wassberg C,Fox JJ,Wibmer A,Goldman DA,Kuk D,Gonen M,Larson SM,Morris MJ,Scher HI,Hricak H.Bone metastases in castration-resistant prostate cancer:associations between morphologic CT patterns,glycolytic activity,and androgen receptor expression on PET and overall survival.Radiology.2014 Apr;271(1):220-9.doi:10.1148 / radiol.13130625.Epub 2013 Nov 18.PMID:24475817 7.Amend SR1,Roy S2,Brown JS3,Pienta KJ2.Ecological paradigms to understand the dynamics of metastasis.Cancer Lett.2016 Sep 28;380(1):237-42.doi:10.1016 / j.canlet.2015.10.005.Epub 2015 Oct 10. 8.Shiozawa Y1,2,Berry JE1,Eber MR1,2,Jung Y1,Yumoto K1,Cackowski FC1,Yoon HJ3,Parsana P4,5,Mehra R6,Wang J1,McGee S1,Lee E1,Nagrath S3,Pienta KJ4,Taichman RS1.The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer.Oncotarget.2016 Jul 5;7(27):41217-41232.doi:10.18632 / oncotarget.9251. 9.Nguyen HM,Vessella RL,Morrissey C,Brown LG,Coleman IM,Higano CS,Mostaghel EA,Zhang X,True LD,Lam HM,Roudier M,Lange PH,Nelson PS,Corey E.LuCaP Prostate Cancer Patient-Derived Xenografts Reflect the Molecular Heterogeneity of Advanced Disease an--d Serve as Models for Evaluating Cancer Therapeutics.Prostate.2017 May;77(6):654-671.doi:10.1002 / pros.23313.Epub 2017 Feb 3.PMID:28156002 10.Kleb B,Estecio MR,Zhang J,Tzelepi V,Chung W,Jelinek J,Navone NM,Tahir S,Marquez VE,Issa JP,Maity S,Aparicio A.Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.Epigenetics.2016 Mar 3;11(3):184-93.doi:10.1080 / 15592294.2016.1146851.Epub 2016 Feb 18.PMID:26890396 11.Hao J,Ci X,Xue H,Wu R,Dong X,Choi SYC,He H,Wang Y,Zhang F,Qu S,Zhang F,Haegert AM,Gout PW,Zoubeidi A,Collins C,Gleave ME,Lin D,Wang Y.Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant ProstateCancer.Eur Urol.2018 Mar 12.pii:S0302-2838(18)30162-3.doi:10.1016 / j.eururo.2018.02.019.[Epub ahead of print]PMID:29544736 12.Robinson D,Van Allen EM,Wu YM,Schultz N,Lonigro RJ,Mosquera JM,Montgomery B,Taplin ME,Pritchard CC,Attard G,Beltran H,Abida W,Bradley RK,Vinson J,Cao X,Vats P,Kunjussa,MLPF,Humphrey,FinY,Y SA,Cooney KA,Smith DC,Brennan C,Siddiqui J,Mehra R,Chen Y,Rathkopf DE,Morris MJ,Solomon SB,Durack JC,Reuter VE,Gopalan A,Gao J,Loda M,Lis RT,Bowden M,Balk SP,Gaviola G,Soughelez,Mostag,MY,EY EA,Cheng HH,Mulcahy H,True LD,Plymate SR,Dvinge H,Ferraldeschi R,Flohr P,Miranda S,Zafeiriou Z,Tunariu N,Mateo J,Perez-Lopez R,Demichelis F,Robinson BD,Sboner A,Schiffman M,Tagara ST,Singas ADM,Singawa KW,Elemento O,Sboner A,Heath EI,Scher HI,Pienta KJ,Kantoff P,de Bono JS,Rubin MA,Nelson PS,Garraway LA,Sawyers CL,Chinnaiyan AM.Integrative Clinical Genomics of Advanced Prostate Cancer.Cell.2015 May. 21;161(5):1215-1228.doi:10.1016 / j.cell.2015.05.001.Erratum in:Cell.2015 Jul 16;162(2):454. 13.Beltran H,Wyatt AW,Chedgy EC,Donoghue A,Annala M,Warner EW,Beja K,Sigouros M,Mo F,Fazli L,Collins CC,Eastham J,Morris M,Taplin ME,Sboner A,Halabi S,Gleave ME.Impact of Therapy on Genomics and Transcriptomics in High-Risk Prostate Cancer Treated with Neoadjuvant Docetaxel and Androgen Deprivation Therapy.Clin Cancer Res.2017 Nov 15;23(22):6802-6811.doi:10.1158 / 1078-0432.CCR-17-1034.Epub 2017 Aug 25.PMID:28842510 14.Pritchard CC,Mateo J,Walsh MF,De Sarkar N,Abida W,Beltran H,Garofalo A,Gulati R,Carreira S,Eeles R,Elemento O,Rubin MA,Robinson D,Lonigro R,Hussain M,Chinnaiyan A,Vinson J,Filipenway J,Taplinko L,L ME,AlDubayan S,Han GC,Beightol M,Morrissey C,Nghiem B,Cheng HH,Montgomery B,Walsh T,Casadei S,Berger M,Zhang L,Zehir A,Vijai J,Scher HI,Sawyers C,Schultz N,Kantoff PW,Solit,Robson M,Offit EM,Offit K,de Bono J,Nelson PS.Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer.N Engl J Med.2016 Aug 4;375(5):443-53.doi:10.1056 / NEJMoa1603144.Epub 2016.PMID: Jul 2647426383. 15.Luo J,Attard G,Balk SP,Bevan C,Burnstein K,Cato L,Cherkasov A,De Bono JS,Dong Y,Gao AC,Gleave M,Heemers H,Kanayama M,Kittler R,Lang JM,Lee RJ,Logothetis CJ,Matusik R,Plymate S,Sawyers CL,Selth LA,Soule H,Tilley W,Weigel NL,Zoubeidi A,Dehm SM,Raj GV.Role of Androgen Receptor Variants in Prostate Cancer:Report from the 2017 Mission Androgen Receptor Variants Meeting.Eur Urol.2018 May;73(5):715-723.doi:10.1016 / j.eururo.2017.11.038.Epub 2017 Dec 16.PMID:29258679 16.Armenia J,Wankowicz SAM,Liu D,Gao J,Kundra R,Reznik E,Chatila WK,Chakravarty D,Han GC,Coleman I,Montgomery B,Pritchard C,Morrissey C,Barbieri CE,Beltran H,Sboner A,Zafeiriou Z,Miranda S,Bielski CM,Penson AV,Tolonen C,Huang FW,Robinson D,Wu YM,Lonigro R,Garraway LA,Demichelis F,Kantoff PW,Taplin ME,Abida W,Taylor BS,Scher HI,Nelson PS,de Bono JS,Rubin MA,Sawyers CL,Chinnaiyan AM;PCF / SU2C International Prostate Cancer Dream Team,Schultz N,Van Allen EM;PCF / SU2C International Prostate Cancer Dream Team.The long tail of oncogenic drivers in prostate cancer.Nat Genet.2018 Apr 2.doi:10.1038 / s41588-018-0078-z.[Epub ahead of print]PMID:29610475 17.Carlsson A,Kuhn P,Luttgen MS,Dizon KK,Troncoso P,Corn PG,Kolatkar A,Hicks JB,Logothetis CJ,Zurita AJ.Paired High-Content Analysis of Prostate Cancer Cells in Bone Marrow and Blood Characterizes Increased Androgen Receptor Expression in Tumor Cell Clusters.Clin Cancer Res.2017 Apr 1;23(7):1722-1732.doi:10.1158 / 1078-0432.CCR-16-1355.Epub 2016 Oct 4.PMID:27702818 18.Don-Doncow N1,Marginean F2,Coleman I3,Nelson PS3,Ehrnstrom R4,Krzyzanowska A1,Morrissey C5,Hellsten R1,Bjartell A6.Expression of STAT3 in Prostate Cancer Metastases.Eur Urol.2017 Mar;71(3):313-316.doi:10.1016 / j.eururo.2016.06.018.Epub 2016 Jun 22. 19.Wu Y,Schoenborn JR,Morrissey C,Xia J,Larson S,Brown LG,Qu X,Lange PH,Nelson PS,Vessella RL,Fang M.High-Resolution Genomic Profiling of Disseminated Tumor Cells in Prostate Cancer.J Mol Diagn.2016 Jan;18(1):131-43.doi:10.1016 / j.jmoldx.2015.08.004.Epub 2015 Nov 20.PMID:26607774 20.Haider M,Zhang X,Coleman I,Ericson N,True LD,Lam HM,Brown LG,Ketchanji M,Nghiem B,Lakely B,Coleman R,Montgomery B,Lange PH,Roudier M,Higano CS,Bielas JH,Nelson PS,Vessella RL,Morrissey C.Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases.Clin Exp Metastasis.2016 Mar;33(3):239-48.doi:10.1007 / s10585-015-9773-7.Epub 2015 Dec 14. 21.Bluemn EG,Coleman IM,Lucas JM,Coleman RT,Hernandez-Lopez S,Tharakan R,Bianchi-Frias D,Dumpit RF,Kaipainen A,Corella AN,Yang YC,Nyquist MD,Mostaghel E,Hsieh AC,Zhang X,Corey E,Brown LG,Nguyen HM,Pienta K,Ittmann M,Schweizer M,True LD,Wise D,Rennie PS,Vessella RL,Morrissey C,Nelson PS.Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling.Cancer Cell.2017 Oct 9;32(4):474-489.e6.doi:10.1016 / j.ccell.2017.09.003.PMID:29017058 22.Cho WJ,Oliveira DS,Najy AJ,Mainetti LE,Aoun HD,Cher ML,Heath E,Kim HR,Bonfil RD.Gene expression analysis of bone metastasis and circulating tumor cells from metastatic castrate-resistant prostate cancer patients.J Transl Med.2016 Mar 15;14:72.doi:10.1186 / s12967-016-0829-5.PMID:26975354. 23.Davies AH,Beltran H,Zoubeidi A.Cellular plasticity and the neuroendocrine phenotype in prostate cancer.Nat Rev Urol.2018 Feb 20.doi:10.1038 / nrurol.2018.22.[Epub ahead of print]Review.PMID:29460922 24.Bishop JL,Thaper D,Vahid S,Davies A,Ketola K,Kuruma H,Jama R,Nip KM,Angeles A,Johnson F,Wyatt AW,Fazli L,Gleave ME,Lin D,Rubin MA,Collins CC,Wang Y,Beltran H,Zoubeidi A.The Master Neural Transcription Factor BRN2 Is an Androgen Receptor-Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer.Cancer Discov.2017 Jan;7(1):54-71.doi:10.1158 / 2159-8290.CD-15-1263.Epub 2016 Oct 26.PMID:2778470 25.Dardenne E,Beltran H,Benelli M,Gayvert K,Berger A,Puca L,Cyrta J,Sboner A,Noorzad Z,MacDonald T,Cheung C,Yuen KS,Gao D,Chen Y,Eilers M,Mosquera JM,Robinson BD,Elemento O,Rubin MA,Demichelis F,Rickman DS.N-Myc Induces an EZH2-Mediated Transcriptional Program Driving Neuroendocrine Prostate Cancer.Cancer Cell.2016 Oct 10;30(4):563-577.doi:10.1016 / j.ccell.2016.09.005.PMID:27728805 26. Zhang W, Liu B, Wu W, Li L, Broom BM, Basourakos SP, Korentzelos D, Luan Y, Wang J, Yang G, Park S, Azad AK, Cao X, Kim J, Corn PG, Logothetis CJ, Aparicio AM, Chinnaiyan AM, Navone N, Troncoso P, Thompson TC.Targeting the MYCN-PARP-DNA Damage Response Pathway in Neuroendocrine Prostate Cancer.Clin Cancer Res.2018 Feb 1;24(3):696-707.doi:10.1158 / 1078-0432.CCR-17-1872.Epub 2017 Nov 14.PMID:2913834 27.Lee JK,Phillips JW,Smith BA,Park JW,Stoyanova T,McCaffrey EF,Baertsch R,Sokolov A,Meyerowitz JG,Mathis C,Cheng D,Stuart JM,Shokat KM,Gustafson WC,Huang J,Witte ON.N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells.Cancer Cell.2016 Apr 11;29(4):536-547.doi:10.1016 / j.ccell.2016.03.001.Epub 2016 Mar 31.PMID:27050099 28.Singhal U,Wang Y,Henderson J,Niknafs YS,Qiao Y,Gursky A,Zaslavsky A,Chung JS,Smith DC,Karnes RJ,Chang SL,Feng FY,Palapattu GS,Taichman RS,Chinnaiyan AM,Tomlins SA,Morgan TM.Multigene Profiling of CTCs in mCRPC Identifies a Clinically Relevant Prognostic Signature.Mol Cancer Res.2018 Apr;16(4):643-654.doi:10.1158 / 1541-7786.MCR-17-0539.Epub 2018 Feb 16.PMID:29453313 29.Spratt DE,Zhang J,Santiago-Jimenez M,Dess RT,Davis JW,Den RB,Dicker AP,Kane CJ,Pollack A,Stoyanova R,Abdollah F,Ross AE,Cole A,Uchio E,Randall JM,Nguyen H,Zhao SG,Meh,Glass LLC,Glass LLC J,du Plessis M,Choeurng V,Aranes M,Kolisnick T,Margrave J,Alter J,Jordan J,Buerki C,Yousefi K,Haddad Z,Davicioni E,Trabulsi EJ,Loeb S,Tewari A,Carroll PR,Weinmann S,Schaeffer EM,Klein Ea,Kenguyen RJN,FYN PL.Development and Validation of a Novel Integrated Clinical-Genomic Risk Group Classification for Localized Prostate Cancer.J Clin Oncol.2018 Feb 20;36(6):581-590.doi:10.1200 / JCO.2017.74.2940.Epu Nov 2017.74.2940. 29.PMID:29185869 30.Nguyen PL,Haddad Z,Ross AE,Martin NE,Deheshi S,Lam LLC,Chelliserry J,Tosoian JJ,Lotan TL,Spratt DE,Stoyanova RS,Punnen S,Ong K,Buerki C,Aranes M,Kolisnik T,Margrave J,Yousefi K,Choeurng V,Davicioni E,Trock BJ,Kane CJ,Pollack A,Davis JW,Feng FY,Klein EA.Ability of a Genomic Classifier to Predict Metastasis and Prostate Cancer-specific Mortality after Radiation or Surgery based on Needle Biopsy Specimens.Eur Urol.2017 Nov;72(5):845-852.doi:10.1016 / j.eururo.2017.05.009.Epub 2017 May 19.PMID:28528811 31.Klaassen Z,Howard LE,de Hoedt A,Amling CL,Aronson WJ,Cooperberg MR,Kane CJ,Terris MK,Freedland SJ.Factors predicting skeletal-related events in patients with bone metastatic castration-resistant prostate cancer.Cancer.2017 May 1;123(9):1528-1535.doi:10.1002 / cncr.30505.Epub 2016 Dec 27.PMID:28026865 32.Kohli M,Wang L,Xie F,Sicotte H,Yin P,Dehm SM,Hart SN,Vedell PT,Barman P,Qin R,Mahoney DW,Carlson RE,Eckel-Passow JE,Atwell TD,Eiken PW,McMenomy BP,Wieben ED,Jha G,Jimenez RE,Weinshilboum R,Wang L.Mutational Landscapes of Sequential Prostate Metastases and Matched Patient Derived Xenografts during Enzalutamide Therapy.PLoS One.2015 Dec 22;10(12):e0145176.doi:10.1371 / journal.pone.0145176.eCollection 2015.PMID:26695660 33.Lee GT,Kang DI,Ha YS,Jung YS,Chung J,Min K,Kim TH,Moon KH,Chung JM,Lee DH,Kim WJ,Kim IY.Prostate cancer bone metastases acquire resistance to androgen deprivation via WNT5A-mediated BMP-6 induction.Br J Cancer.2014 Mar 18;110(6):1634-44.doi:10.1038 / bjc.2014.23.Epub 2014 Feb 11.PMID:24518599 34.Lee GT,Kwon SJ,Kim J,Kwon YS,Lee N,Hong JH,Jamieson C,Kim WJ,Kim IY.WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages.Br J Cancer.2018 Mar 6;118(5):670-678.doi:10.1038 / bjc.2017.451.Epub 2018 Jan 30.PMID:29381686 35.Miyamoto DT,Zheng Y,Wittner BS,Lee RJ,Zhu H,Broderick KT,Desai R,Fox DB,Brannigan BW,Trautwein J,Arora KS,Desai N,Dahl DM,Sequist LV,Smith MR,Kapur R,Wu CL,Shioda T,Ramaswamy S,Ting DT,Toner M,Maheswaran S,Haber DA.RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance.Science.2015 Sep 18;349(6254):1351-6.doi:10.1126 / science.aab0917.PMID:26383955 36.F.Jin et al.,“Regulation of prostate cancer cell migration toward bone marrow stromal cell-conditioned medium by Wnt5a signaling.,”Mol.Med.Rep.,vol.8,no.5,pp.1486-92,Nov.2013. 37.Tamura K,Furihata M,Satake H,Hashida H,Kawada C,Osakabe H,Fukuhara H,Kumagai N,Iiyama T,Shuin T,Inoue K.SHISA2 enhances the aggressive phenotype in prostate cancer through the regulation of WNT5A expression.Oncol Lett.2017 Dec;14(6):6650-6658.doi:10.3892 / ol.2017.7099.Epub 2017 Sep 28.PMID:29344118 38.Huang L,Pu Y,Hu WY,Birch L,Luccio-Camelo D,Yamaguchi T,Prins GS.The role of Wnt5a in prostate gland development.Dev Biol.2009 Apr 15;328(2):188-99.doi:10.1016 / j.ydbio.2009.01.003.Epub 2009 Jan 14.PMID:19389372 39.M.Okamoto et al.,“Noncanonical Wnt5a enhances Wnt / β-catenin signaling during osteoblastogenesis.,”Sci.Rep.,vol.4,no.1,p.4493,Mar.2014. 40.Hosseini-Farahabadi S,Gignac SJ,Danescu A,Fu K,Richman JM.Abnormal WNT5A Signaling Causes Mandibular Hypoplasia in Robinow Syndrome.J Dent Res.2017 Oct;96(11):1265-1272.doi:10.1177 / 0022034517716916.Epub 2017 Jun 29.PMID:28662348 41.Choi MY,Widhopf GF 2nd,Wu CC,Cui B,Lao F,Sadarangani A,Cavagnaro J,Prussak C,Carson DA,Jamieson C,Kipps TJ.Pre-clinical Specificity and Safety of UC-961,a First-In-Class Monoclonal Antibody Targeting ROR1.Clin Lymphoma Myeloma Leuk.2015 Jun;15 Suppl:S167-9.doi:10.1016 / j.clml.2015.02.010.Review.PMID:26297272 42.Yu J,Chen L,Cui B,Wu C,Choi MY,Chen Y,Zhang L,Rassenti LZ,Widhopf Ii GF,Kipps TJ.Cirmtuzumab inhibits Wnt5a-induced Rac1 activation in chronic lymphocytic leukemia treated with ibrutinib.Leukemia.2017 Jun;31(6):1333-1339.doi:10.1038 / leu.2016.368.Epub 2016 Dec 1.PMID:27904138 43.Jamieson,CH,(PI),and Choi,M(Co-PI),Kipps,TJ(Responsible Party),ClinicalTrials.gov Identifier:NCT02222688,A Phase I Clinical Trial to Determine the Safety and Tolerability of UC-961(Cirmtuzumab),an Anti-ROR1 Monoclonal Antibody,for the Treatment of Patients With Relapsed or Refractory Chronic Lymphocytic Leukemia Who Are Ineligible for Chemotherapy.https: / / clinicaltrials.gov / ct2 / show / NCT02222688 44.Bauer M,Benard J,Gaasterland T,Willert K,Cappellen D.WNT5A encodes two isoforms with distinct functions in cancers.PLoS One.2013 8(11):e80526.doi:10.1371 / journal.pone.0080526.PMID 24260410. 45.Raheem,O.,A.A.Kulidjian,C.Wu,Y.B.Jeong,T.Yamaguchi,K.M.Smith,D.Goff,H.Leu,S.R.Morris,N.A.Cacalano,K.Masuda,C.H.Jamieson,C.J.Kane,and C.A.Jamieson,A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions.J Transl Med,2011.9:p.185. 46.Godebu,E.,M.Muldong,A.Strasner,C.N.Wu,S.C.Park,J.R.Woo,W.Ma,M.A.Liss,T.Hirata,O.Raheem,N.A.Cacalano,A.A.Kulidjian,and C.A.Jamieson,PCSD1,a new patient-derived model of bone metastatic prostate cancer,is castrate-resistant in the bone-niche.J Transl Med,2014.12:p.275. 47.Hirata,T.,S.C.Park,M.T.Muldong,C.N.Wu,T.Yamaguchi,A.Strasner,O.Raheem,H.Kumon,R.L.Sah,N.A.Cacalano,C.H.M.Jamieson,C.J.Kane,K.Masuda,A.A.Kulidjian,and C.A.M.Jamieson,Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer.Asian J Urol,2016.3(4):p.229-239 48.CAM Jamieson,Kulidjian,A.A.,Muldong,M,Wu,C.,Lee,S.,Mendoza,T.,Park,SC,Park,JS,Raheem,O.,Woo,JR,Godebu,E.,Strasner,A.,Kane,CJ,Gaasterland,T.Genomic analysis of a longitudinal series of surgical prostate cancer bone metastases and xenografts from the same patient with progressively therapy-resistant cancer revealed selection of metastatic clone.(Research completed,Manuscript in Preparation). 49.Ritter KE,Wang Z,Vezina CM,Bjorling DE,Southard-Smith EM1.Serotonin Receptor 5-HT3A Affects Development of Bladder Innervation and Urinary Bladder Function.Front Neurosci.2017 Dec 12;11:690.doi:10.3389 / fnins.2017.00690.eCollection 2017. 50.Hu WY,Hu DP,Xie L,Li Y,Majumdar S,Nonn L,Hu H,Shioda T,Prins GS.Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.Stem Cell Res.2017 Aug;23:1-12.doi:10.1016 / j.scr.2017.06.009.Epub 2017 Jun 16.PMID:28651114 51.Sarin S,Zuniga-Sanchez E,Kurmangaliyev YZ,Cousins H,Patel M,Hernandez J,Zhang KX,Samuel MA,Morey M,Sanes JR,Zipursky SL.Role for Wnt Signaling in Retinal Neuropil Development:Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis.Neuron.2018 Apr 4;98(1):109-126.e8.doi:10.1016 / j.neuron.2018.03.004.Epub 2018 Mar 22.PMID:29576390 52.Yu J,Chen L,Chen Y,Hasan MK,Ghia EM,Zhang L,Wu R,Rassenti LZ,Widhopf GF,Shen Z,Briggs SP,Kipps TJ.Wnt5a induces ROR1 to associate with 14-3-3ζ for enhanced chemotaxis and proliferation of chronic lymphocytic leukemia cells.Leukemia.2017 Dec;31(12):2608-2614.doi:10.1038 / leu.2017.132.Epub 2017 May 3.PMID:28465528 53.Zhang S,Zhang H,Ghia EM,Huang J,Wu L,Zhang J,Lam S,Lei Y,He J,Cui B,Widhopf GF 2nd,Yu J,Schwab R,Messer K,Jiang W,Parker BA,Carson DA,Kipps TJ.Inhibition of chemotherapy resistant breast cancer stem cells by a ROR1 specific antibody.Proc Natl Acad Sci U S A.2019 Jan 22;116(4):1370-1377.doi:10.1073 / pnas.1816262116.Epub 2019 Jan 8.PMID:30622177 54.Yu J,Chen L,Cui B,Widhopf GF 2nd,Shen Z,Wu R,Zhang L,Zhang S,Briggs SP,Kipps TJ.Wnt5a induces ROR1 / ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation.J Clin Invest.2016 Feb;126(2):585-98.PMID:26690702 55.Hasan MK,Rassenti L,Widhopf GF 2nd,Yu J,Kipps TJ.Wnt5a causes ROR1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells.Leukemia.2019 Mar;33(3):653-661.doi:10.1038 / s41375-018-0306-7.Epub 2018 Dec 19.PMID:30568170 56.Choi MY,Widhopf GF 2nd,Ghia EM,Kidwell RL,Hasan MK,Yu J,Rassenti LZ,Chen L,Chen Y,Pittman E,Pu M,Messer K,Prussak CE,Castro JE,Jamieson C,Kipps TJ.Phase I Trial:Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia.Cell Stem Cell.2018 Jun 1;22(6):951-959.e3.doi:10.1016 / j.stem.2018.05.018.PMID:29859176 57.Zhang S,Chen L,Wang-Rodriguez J,Zhang L,Cui B,Frankel W,Wu R,Kipps TJ.The onco-embryonic antigen ROR1 is expressed by a variety of human cancers.Am J Pathol.2012 Dec;181(6):1903-10.doi:10.1016 / j.ajpath.2012.08.024.Epub 2012 Oct 4.PMID:23041612 58.Li C,Wang S,Xing Z,Lin A,Liang K,Song J,Hu Q,Yao J,Chen Z,Park PK,Hawke DH,Zhou J,Zhou Y,Zhang S,Liang H,Hung MC,Gallick GE,Han L,Lin C,Yang L.A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis.Nat Cell Biol.2017 Feb;19(2):106-119.doi:10.1038 / ncb3464.Epub 2017 Jan 23.PMID:28114269 59.Woo JR, Liss MA, Muldong MT, Palazzi K, Strasner A, Ammirante M, Varki N, Shabaik A, Howell S, Kane CJ, Karin M, Jamieson CA. Tumor infiltrating B-cells are increased in prostate cancer tissue.J Transl Med.2014 Jan 30;12:30.doi:10.1186 / 1479-5876-12-30.PMID:24475900

[0316] Unofficial sequence listing UC961 VL: DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK (SEQ ID NO: 21)

[0317] Linker #3: GGGGSGSTSGSGKPGSGEGSTKGGGGGS (SEQ ID NO: 24)

[0318] UC961VH: QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS (SEQ ID NO: 27)

[0319] Spacer #3 (or "Hinge / Short"): VDESKYGPPCPPCP (SEQ ID NO:29)

[0320] Spacer CH3: VDESKYGPPCPPCPLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41)

[0321] Spacer CH2CH3 (or "long / full"): VDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 42)

[0322] UC961 CDR L1: KSISKY (SEQ ID NO: 43)

[0323] UC961 CDR L2: SGS (SEQ ID NO:44)

[0324] UC961 CDR L3: QQHDESPY (SEQ ID NO: 45)

[0325] UC961 CDR H1: GYAFTAYN (SEQ ID NO:46)

[0326] UC961 CDR H2: FDPYDGGS (SEQ ID NO:47)

[0327] UC961 CDR H3: GWYYFDY (SEQ ID NO:48)

[0328] 4A5 CDR L1: PDINSY (SEQ ID NO: 49)

[0329] 4A5 CDR L2: RAN (SEQ ID NO:50)

[0330] 4A5 CDR L3: LQYDEFPYT (SEQ ID NO:51)

[0331] 4A5 CDR H1: GFTFSSYA (SEQ ID NO:52)

[0332] 4A5 CDR H2: ISRGGTT (SEQ ID NO:53)

[0333] 4A5 CDR H3: YDYDGYYAMDY (SEQ ID NO:54)

[0334] Full length human ROR-1 protein (SEQ ID NO:55): MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWMEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL

[0335] A 21 amino acid stretch of human ROR-1 including glutamic acid at position 138 (SEQ ID NO:56): VATNGKEVVSSTGVLFVKFGP

[0336] A 15 amino acid stretch of human ROR-1 including glutamic acid at position 138 (SEQ ID NO: 57): EVVSSTGVLFVKFGP

Claims

(i) an antigen-binding region comprising a light chain variable domain and a heavy chain variable domain, (a) the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 43, CDR L2 as set forth in SEQ ID NO: 44, and CDR L3 as set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 46, CDR H2 as set forth in SEQ ID NO: 47, and CDR H3 as set forth in SEQ ID NO: 48; or (b) an antigen-binding region, wherein the light chain variable domain comprises CDR L1 as set forth in SEQ ID NO: 49, CDR L2 as set forth in SEQ ID NO: 50, and CDR L3 as set forth in SEQ ID NO: 51, and the heavy chain variable domain comprises CDR H1 as set forth in SEQ ID NO: 52, CDR H2 as set forth in SEQ ID NO: 53, and CDR H3 as set forth in SEQ ID NO: 54; (ii) a spacer domain; and (iii) a transmembrane domain; and (iv) an intracellular domain.

2. The chimeric antigen receptor of claim 1, wherein the light chain variable domain is covalently linked to the heavy chain variable domain via a polypeptide linker.

3. 2. The chimeric antigen receptor of claim 1, wherein the spacer domain comprises an immunoglobulin hinge domain, an immunoglobulin constant heavy 3 domain, an immunoglobulin constant heavy 2 domain, or a combination thereof.

4. 2. The chimeric antigen receptor of claim 1, wherein the spacer domain comprises the amino acid sequence of SEQ ID NO:29, SEQ ID NO:41, or SEQ ID NO:

42.

5. The chimeric antigen receptor of claim 1, wherein the light chain variable domain comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:

21.

6. The chimeric antigen receptor of claim 1, wherein the heavy chain variable domain comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:

27.

7. The chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises a CD8α transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a CD3ζ transmembrane domain, or any combination of two or more thereof.

8. 2. The chimeric antigen receptor of claim 1, wherein the intracellular domain comprises an intracellular costimulatory signaling domain, an intracellular T cell signaling domain, or a combination thereof.

9. The chimeric antigen receptor of claim 8, wherein the intracellular costimulatory signaling domain is a 4-1BB intracellular costimulatory signaling domain, a CD28 intracellular costimulatory signaling domain, an ICOS intracellular costimulatory signaling domain, an OX-40 intracellular costimulatory signaling domain, or any combination of two or more thereof.

10. The chimeric antigen receptor of claim 8, wherein the intracellular costimulatory signaling domain comprises a CD28 intracellular costimulatory signaling domain and a 41-BB intracellular costimulatory signaling domain.

11. The chimeric antigen receptor of claim 10, wherein the intracellular costimulatory signaling domain further comprises a CD3ζ intracellular T cell signaling domain.

12. A cell expressing the chimeric antigen receptor described in claim 1.

13. The cell of claim 12 , wherein the cell is a T lymphocyte.

14. The cell of claim 12 , wherein the cell is a CD4+ T lymphocyte or a CD8+ T lymphocyte.

15. The method of claim 12, wherein the cell is a natural killer cell.

16. A pharmaceutical composition comprising a cell expressing the chimeric antigen receptor of claim 1 and a pharma- ceutically acceptable excipient.

17. An isolated nucleic acid encoding the chimeric antigen receptor described in claim 1.

18. A cell comprising a nucleic acid encoding the chimeric antigen receptor described in claim 1.

19. A pharmaceutical composition for use in treating neuroendocrine cancer or preventing metastasis of neuroendocrine cancer, comprising a cell expressing a chimeric antigen receptor described in claim 1.

20. 20. The pharmaceutical composition of claim 19, wherein the neuroendocrine cancer is a carcinoid tumor, a pancreatic islet cell tumor, a medullary thyroid carcinoma, a pheochromocytoma, a neuroendocrine carcinoma of the skin, a small cell lung cancer, a large cell neuroendocrine carcinoma, a neuroendocrine prostate cancer, or a metastatic castration-resistant prostate cancer.

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