Anti-ROR1 chimeric antigen receptor (CAR), cells expressing CAR, and related methods

Abstract

The present invention comprises anti-ROR1 chimeric antigen receptor (CAR) T cells (CAR-T cells), natural killer cells (CAR-NK cells), compositions comprising the cells and methods of making and using the same, including methods of treating tumors expressing ROR1.

Description

Technical Field

[0001] The present invention relates to the field of oncology, and more specifically to cell therapy utilizing immune cells targeted to genetically engineered tumors.

[0002] Statement Regarding Federally Funded Research

[0003] none.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The .XML copy created on August 29, 2023 is named CBI049_30_SL.xml and is 61,699 bytes in size. Background Art

[0006] Chimeric antigen receptor (CAR)-T cell therapy has shown significant potential against hematological malignancies.

[0007] CAR-T cells have not shown such robust and reproducible antitumor activity against solid malignancies. Natural killer (NK) cells express the receptor NKG2D, which promotes their innate ability to detect and kill tumors expressing stress ligands. NK cells are also more active than T cells in the immunosuppressive tumor microenvironment.

[0008] ROR1 is expressed in a variety of tumors, including hematologic malignancies, ovarian cancer, lung cancer, pancreatic cancer, and breast cancer. Innovative treatments for hematologic and solid tumors that express ROR1 are needed, such as treatment with engineered cells that target tumor-specific ROR1 expression. Summary of the invention

[0009] In one embodiment, the present invention is a chimeric antigen receptor (CAR) comprising: an anti-ROR1 scFv; a transmembrane domain; a hinge domain and a cytoplasmic domain. In some embodiments, the cytoplasmic domain comprises a CD3ζ domain and a 4-1BB domain. In some embodiments, the anti-ROR1 scFv comprises a light chain (V L ) and heavy chain (V H ), consisting essentially of, and said V L comprising a sequence selected from SEQ ID NO: 3, 7 and 11, and consisting essentially of the sequence, wherein V H The invention relates to a method for treating a ROR1 scFv comprising a sequence selected from the group consisting of SEQ ID NO: 2, 6 and 10, and consisting essentially of the sequence. In some embodiments, the anti-ROR1 scFv comprises a light chain (V L ) and the heavy chain (V H). In some embodiments, the linker comprises a linker of formula (G x S y ) n , wherein G is glycine and S is serine, e.g., G4S. In some embodiments, the anti-ROR1 scFv comprises the light chain (V L ) in the complementarity determining regions CDR1, CDR2 and CDR3, and the heavy chain (V H ) in CDR1, CDR2 and CDR3, and comprising: said V H SEQ ID NO: 29 in the CDR1, the V H SEQ ID NO:30 in the CDR2, the V H SEQ ID NO:31 in the CDR3, the V L SEQ ID NO:32 in the CDR1, the V L SEQ ID NO: 33 in the CDR2 and the V L In some embodiments, the V H The CDR1 consists of SEQ ID NO: 29, the V H The CDR2 consists of SEQ ID NO: 30, the V H The CDR3 consists of SEQ ID NO: 31, the V L The CDR1 consists of SEQ ID NO: 32, the V L The CDR2 consists of SEQ ID NO: 33, and the V LThe CDR3 of is composed of SEQ ID NO:34. In some embodiments, the cytoplasmic domain comprises a CD3 ζ domain. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain consists essentially of SEQ ID NO:22. In some embodiments, the CD8 transmembrane domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:21. In some embodiments, the hinge domain comprises a CD8 hinge domain. In some embodiments, the CD8 hinge domain consists essentially of SEQ ID NO:20. In some embodiments, the CD8 hinge domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:19. In some embodiments, the CD3 ζ domain consists essentially of SEQ ID NO:24. In some embodiments, the CD3 ζ domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:23. In some embodiments, the 4-1BB domain consists essentially of SEQ ID NO:26. In some embodiments, the 4-1BB domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:25. In some embodiments, the CAR further comprises a signal sequence. In some embodiments, the signal sequence is selected from a CD8 signal sequence and a CD28 signal sequence. In some embodiments, the cytoplasmic domain comprises a binding motif for an intracellular signal transduction protein. In some embodiments, the binding motif is present in the CD3ζ domain. In some embodiments, the intracellular signal transduction protein is a STAT protein or a JAK protein. In some embodiments, the JAK binding domain comprises SEQ ID NO:43. In some embodiments, the STAT binding domain is selected from SEQ ID NO:39-42. In some embodiments, the cytoplasmic domain further comprises an IL-2Rb cytoplasmic domain. In some embodiments, the IL-2Rb cytoplasmic domain consists essentially of SEQ ID NO:50. In some embodiments, the IL-2Rb cytoplasmic domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:51.

[0010] In some embodiments, the CAR comprises or consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, and 27. In some embodiments, the CAR is encoded by a nucleic acid comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 36, and 38.

[0011] In one embodiment, the present invention is a separated nucleic acid comprising a vector sequence and a sequence encoding a chimeric antigen receptor (CAR) described herein. In some embodiments, the separated nucleic acid further comprises a promoter selected from the group consisting of: PGK1 promoter, MND promoter, Ubc promoter, CAG promoter, CaMKIIa promoter, SV40 early promoter, SV40 late promoter, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus long terminal repeat (Rous sarcoma virus long terminal repeat, RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, beta-interferon promoter, hsp70 promoter, EF-1α promoter and beta-actin promoter. In some embodiments, the promoter comprises a CAG promoter. In some embodiments, the promoter comprises an MND promoter. In some embodiments, the promoter comprises an EF-1α promoter. In some embodiments, the vector comprises a plasmid. In some embodiments, the vector comprises a viral vector derived from a virus selected from the group consisting of adenovirus type 2 and adenovirus type 5, retrovirus, lentivirus, adeno-associated virus (AAV), simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (Epstein-Barr virus, EBV) and herpes simplex virus (HSV). In some embodiments, the isolated nucleic acid further comprises a coding sequence for a cytokine. In some embodiments, the cytokine is IL-36γ. In some embodiments, IL-36γ is encoded by a nucleic acid comprising a sequence selected from SEQ IDNO: 44, 46 and 48. In some embodiments, the isolated nucleic acid comprises a sequence selected from SEQ IDNO: 14, 16, 18, 36 and 38.

[0012] In one embodiment, the present invention is an immune cell comprising a chimeric antigen receptor (CAR) described herein. In some embodiments, the cell is selected from T cells, natural killer (NK) cells and induced natural killer (iNK) cells. In some embodiments, the cell comprises a sequence selected from 4, 8, 12 and 27 selected from SEQ ID NO:. In some embodiments, the cell further comprises an armored genome modification. In some embodiments, the armored genome modification comprises one or more or two or more of the immune checkpoint genes or regulatory genes selected from the group consisting of: PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, CISH, CBLB and 2B4. In some embodiments, the armored genome modification comprises the inactivation of CISH. In some embodiments, the armored genome modification comprises the inactivation of CBLB. In some embodiments, the armored genome modification comprises the inactivation of PDCD1. In some embodiments, the armored genome modification comprises the inactivation of Tim3. In some embodiments, the armored genome modification comprises the inactivation of LAG3. In some embodiments, the armored genome modification comprises inactivation of TIGIT. In some embodiments, the armored genome modification comprises inactivation of B2M. In some embodiments, the armored genome modification further comprises inserting an HLA-E-B2M fusion construct into the B2M gene. In some embodiments, the cell is engineered to express a cytokine. In some embodiments, the cytokine is membrane-bound. In some embodiments, the cytokine is expressed as a cytokine-receptor fusion protein. In some embodiments, the cytokine is selected from IL-15 and IL-21. In some embodiments, the cytokine is selected from mbIL-15 and mbIL-21.

[0013] In one embodiment, the present invention is a method for preparing an immune cell described herein, the method comprising introducing a nucleic acid comprising a sequence selected from SEQ ID NO: 14, 16, 18, 36 and 38 into a cell. In some embodiments, the cell is selected from T cells, NK cells and induced pluripotent stem cells (iPSC). In some embodiments, the cell is iPSC, and the method further comprises differentiating the iPSC into an immune cell. In some embodiments, the introduction step comprises introducing a sequence-dependent nuclease into the cell. In some embodiments, the introduction step comprises introducing a CRISPR system into the cell, the CRISPR system comprising a nucleic acid-guided nuclease and a nucleic acid-targeting nucleic acid (NATNA) guide. In some embodiments, the nucleic acid-guided nuclease is selected from Cas9, Cas12a and CASCADE. In some embodiments, the endonuclease comprises a catalytically inactivated CRISPR endonuclease conjugated to the cleavage domain of the restriction endonuclease Fok I. In some embodiments, the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN-Fok I fusions, transcription activator-like effector nucleases (TALENs), and TALEN-Fok I fusions. In some embodiments, the endonuclease cleaves the genome of the cell at a locus selected from the group consisting of TRAC, CBLB, PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, and B2M. In some embodiments, the nucleic acid comprising a sequence selected from SEQ ID NOs: 14, 16, 18, 36, and 38 comprises a vector. In some embodiments, the vector is a viral vector derived from a virus selected from the group consisting of adenovirus type 2 and adenovirus type 5, retrovirus, lentivirus, adeno-associated virus (AAV), simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV).

[0014] In one embodiment, the present invention is a composition comprising the immune cells described herein and a pharmaceutically acceptable excipient. In some embodiments, the immune cells are between 1×10 6 With 2×10 8 In some embodiments, the immune cells are between 1×10 7 With 2×10 9The amount of CAR-NK cells between cells. In some embodiments, the immune cells are a mixture of CAR-T cells and CAR-NK cells present in a ratio of about 1:10 CAR-T to CAR-NK. In some embodiments, the pharmaceutically acceptable excipients include one or more of the following: carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, water, alcohols, polyols, glycerol, vegetable oils, phospholipids, surfactants, sugars, derived sugars, sugar alcohols, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, inositol, aldonic acid, esterified sugars, sugar polymers, monosaccharides, fructose, maltose, galactose, glucose, D-mannose, sorbose, disaccharides, lactose, sucrose, trehalose, cellobiose, polysaccharides, raffinose, melezitose, maltodextrin, dextran, starch, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate and sodium phosphate. In some embodiments, the antibacterial agent comprises one or more of the following: benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal. In some embodiments, the composition further comprises an antioxidant selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium pyrosulfite. In some embodiments, the composition further comprises a surfactant selected from polysorbate, sorbitan ester, lecithin, phosphatidylcholine, phosphatidylethanolamine, fatty acid, fatty acid ester and cholesterol. In some embodiments, the composition further comprises a refrigerant selected from 3% to 12% dimethyl sulfoxide (DMSO) and 1% to 5% human albumin. In some embodiments, the composition further comprises a preservative selected from one or more of the following: methylparaben, propylparaben, sodium benzoate, benzalkonium chloride, antioxidants, chelating agents, parabens, chlorobutanol, phenol, and sorbic acid.

[0015] In one embodiment, the present invention is a method of inhibiting tumor growth in a patient, the method comprising administering a composition described herein to a patient suffering from the tumor. In some embodiments, the tumor is a solid tumor selected from the following: ovarian cancer, triple-negative breast cancer, colorectal cancer, non-small cell lung cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, melanoma and endometrial cancer or selected from the following hematological tumors: MCL, CLL, SLL, B-ALL, B-NHL and AML. In some embodiments, the administration is selected from the group consisting of: systemic delivery, parenteral delivery, intramuscular delivery, intravenous delivery, subcutaneous delivery and intradermal delivery. In some embodiments, the composition further comprises a timed delivery component, which enables the composition to be released, delayed release or sustained release by time. In some embodiments, the timed delivery component is selected from monostearate, gelatin, semi-permeable matrix and solid hydrophobic polymer. In some embodiments, the method further comprises administering a cytokine to the patient. In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18 and IL-21. In some embodiments, the method further comprises applying quality control measures to the immune cells before administration to the patient, the quality control measures comprising evaluating one or more characteristics selected from the following: the presence of anti-ROR1 CAR in the cell genome, the surface expression of the anti-ROR1 CAR, ROR1-dependent lysis of target cells expressing ROR1, proliferation in the presence of target cells expressing ROR1, cytokine or chemokine secretion in the presence of target cells expressing ROR1, reduction of tumor burden in experimental animals carrying tumors expressing ROR1, and persistence in the circulation of the animal carrying tumors expressing ROR1 after administering the immune cells to the experimental animals. In some embodiments, the presence of the anti-ROR1 CAR in the cell genome is evaluated by a method selected from the following: nucleic acid hybridization, nucleic acid sequencing, polymerase chain reaction (PCR), quantitative PCR (qPCR), real-time PCR (rtPCR) and droplet digital PCR (ddPCR). In some embodiments, the surface expression of the anti-ROR1 CAR is evaluated by flow cytometry, fluorescence activated cell sorting (FACS), microfluidics-based screening, ELISA or Western blot. In some embodiments, the surface expression of the anti-ROR1 CAR is assessed by flow cytometry. In some embodiments, the immune cell population with the highest surface expression of the anti-ROR1 CAR is selected for administration to the patient. In some embodiments, the ROR1-dependent lysis of target cells carrying ROR1 is assessed by: co-culturing immune cells containing the CAR with target cells expressing ROR1 at an effector: target ratio between about 0.1 and about 10 and assessing target cell lysis.In some embodiments, the immune cell population with the highest lysis rate of target cells carrying ROR1 is selected for administration to the patient. In some embodiments, the ROR1-dependent proliferation is evaluated by co-culturing the immune cells with target cells expressing ROR1 and evaluating the proliferation of the immune cells. In some embodiments, the immune cell population with the highest proliferation rate in the presence of target cells expressing ROR1 is selected for administration to the patient. In some embodiments, the cytokine or chemokine is selected from IFN-γ, TNF-α, GM-CSF, IL-10, IL-5 and IL-13, MIP-1α, MIP-1β, IL-8 and RANTES. In some embodiments, the cytokine secretion is evaluated by co-culturing the immune cells with target cells expressing ROR1 and measuring the amount of cytokines in the co-culture supernatant. In some embodiments, the immune cell population with the highest cytokine secretion is selected for administration to the patient. In some embodiments, the reduction of the tumor burden of experimental animals carrying tumors expressing ROR1 is measured in the form of changes in the bioluminescence of bioluminescent tumor cells within a certain period of time after the animal has been injected with the immune cells. In some embodiments, the bioluminescence change is expressed as an area under the curve (AUC). In some embodiments, the immune cell population with the smallest AUC is selected for administration to the patient. In some embodiments, the persistence in the circulation of an experimental animal carrying a tumor expressing ROR1 after the immune cell is administered to the animal is measured by counting cells expressing human CD56 in the circulation of the animal. In some embodiments, the immune cell population with the highest count of cells expressing human CD56 in the circulation of the animal is selected for administration to the patient. In some embodiments, the immune cell comprises an armored genome modification. In some embodiments, the genome modification comprises the inactivation of one or more of the following: CISH, CBLB, B2M, PDCD1, Tim3, LAG3 and TIGIT. In some embodiments, the immune cell is engineered to express a cytokine. In some embodiments, the cytokine is selected from mbIL-15 and mbIL-21. In some embodiments, the method further comprises applying quality control measures to the immune cells prior to administration to the patient, the quality control measures comprising evaluating one or more characteristics selected from the group consisting of: surface expression of the cytokine, ROR1-dependent lysis of target cells expressing ROR1, and reduction of tumor burden in experimental animals carrying tumors expressing ROR1. In some embodiments, the immune cell population with the highest surface expression of the cytokine is selected for administration to the patient. In some embodiments, the immune cell population with the highest ROR1-dependent lysis of target cells expressing ROR1 is selected for administration to the patient.In some embodiments, the immune cell population that has the highest rate of reducing tumor burden in an experimental animal bearing a tumor expressing ROR1 is selected for administration to the patient. In some embodiments, the composition comprises between 1×10 . 7 With 2×10 8 CAR-T cells between 1×10 8 With 2×10 9 The invention can be used to treat CAR-NK cells in an amount between 1:10 cells or a mixture of CAR-T cells and CAR-NK cells in a ratio of approximately 1:10 CAR-T to CAR-NK.

[0016] In one embodiment, the present invention is a method for making an anti-ROR1 immune cell, the method comprising introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into a cell population, the CAR comprising: anti-ROR1 scFv; a transmembrane domain; a hinge domain; and a cytoplasmic domain, wherein the nucleic acid comprises a sequence selected from SEQ ID NO: 4, 8, 12 and 27. In some embodiments, the cell population is selected from T cells, natural killer (NK) cells and cells capable of differentiating into NK cells. In some embodiments, the cells capable of differentiating into NK are selected from induced pluripotent stem cells (iPSC), hematopoietic progenitor cells (HPC) and lymphoid progenitor cells. In some embodiments, the introduction is introduced into iPSC, thereby forming CAR-iPSC. In some embodiments, the method further comprises inducing the CAR-iPSC to differentiate into an induced CAR-NK (CAR-iNK). In some embodiments, the induced differentiation comprises: contacting the CAR-iPSC with one or more cytokines selected from the following to produce hematopoietic progenitor cells (HPC): BMP4, VEGF, SCF, IL3, IL6 and TPO; by selecting CD34 -cells to enrich the HPCs; in the presence of feeder cells, the HPCs are contacted with one or more cytokines selected from the following to produce inducible natural killer cells (iNK): IL3, IL15, IL7, SCT, FLT3L. In some embodiments, the method further comprises amplifying the iNKs by a method comprising culturing the iNKs in the presence of feeder cells and cytokines. In some embodiments, the feeder cells secrete cytokines of cell membranes or express the cytokines. In some embodiments, the feeder cells express 4-1BB ligands (4-1BBL) and membrane-bound IL21 (mbIL21). In some embodiments, the nucleic acid is introduced into a precursor cell population by chemical or electrochemical means. In some embodiments, the nucleic acid is introduced into a precursor cell population by a vector selected from a plasmid vector and a viral vector. In some embodiments, the viral vector is derived from a virus selected from the group consisting of: adenovirus type 2 and adenovirus type 5, retrovirus, lentivirus, adeno-associated virus (AAV), simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (EBV) and herpes simplex virus (HSV). In some embodiments, the introducing step comprises introducing a CRISPR system into the cell, the CRISPR system comprising a nucleic acid-guided endonuclease and a nucleic acid-targeting nucleic acid (NATNA) guide. In some embodiments, the nucleic acid-guided endonuclease is selected from Cas9, Cas12a and CASCADE. In some embodiments, the endonuclease comprises a catalytically inactive CRISPR endonuclease conjugated to the cleavage domain of the restriction endonuclease Fok I. In some embodiments, the endonuclease is selected from the group consisting of: zinc finger nuclease (ZFN), ZFN-Fok I fusion, transcription activator-like effector nuclease (TALEN) and TALEN-Fok I fusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the anti-ROR1 chimeric antigen receptor (CAR).

[0018] Figure 2 This is a workflow for generating inducible anti-ROR1 CAR NK cells (iNK cells) from induced pluripotent stem cells (iPSCs).

[0019] Figure 3 Shows the Figure 1 The iNK pathway transformed by the anti-ROR1 CAR construct shown in ( Figure 2 CAR expression in cells at different stages of ).

[0020] Figure 4Shown is the expression of ROR1 in the cell lines SKOV3 and JeKo-1.

[0021] Figure 5 Shown is the lysis of SKOV3 tumor cells (proportion of viable tumor cells) by anti-ROR1 CAR iNK cells.

[0022] Figure 6 Lysis of JeKo1 tumor cells by anti-ROR1 CAR iNK cells (specific lysis of tumor cells) is shown.

[0023] Figure 7 Shown are repeated lysis of JeKo1 tumor cells by anti-ROR1 CAR iNK cells in a serial rechallenge assay.

[0024] Figure 8 Shown is the tumor burden (shown as area under the curve, AUC) of NGS mice after administration of anti-ROR1 CAR iNK cells.

[0025] Fig. 9 Shown is the tumor burden of IL15 transgenic mice after administration of anti-ROR1 CAR iNK cells (AUC).

[0026] Fig.10 shows that through the expression of human CD56 + The persistence of human iNK cells in the mouse circulation was determined by quantification of cells.

[0027] Fig.11 iNK cells (hCD45 + CD56 + cells).

[0028] Fig.12 Cytokine secretion by CBLB-deficient iNK cells is shown.

[0029] Fig.13 Evaluation of the phenotype of CBLB-deficient iNK cells is shown.

[0030] Fig.14 In vitro cytotoxicity of CBLB-deficient iNK cells against SKOV3 tumor cells is shown.

[0031] Fig.15 Degranulation of CBLB-deficient iNK cells in co-culture with SKOV3 tumor cells is shown.

[0032] Fig.16 In vivo antitumor activity of CBLB-deficient iNK cells in a mouse xenograft model is shown.

[0033] Fig.17 In vitro cytotoxicity of CBLB-deficient iNK cells and CISH-deficient iNK cells against SKOV3 tumor cells is shown.

[0034] FIG. 18 shows effector molecule secretion by CBLB-deficient iNK cells and CISH-deficient iNK cells in co-culture with SKOV3 tumor cells (IFNγ and TNFα).

[0035] FIG. 19 shows effector molecule secretion (perforin and granzyme B) by CBLB-deficient iNK cells and CISH-deficient iNK cells in co-culture with SKOV3 tumor cells.

[0036] Fig. 20 Shown is the in vivo antitumor activity (measured as AUC of tumor bioluminescence) of CBLB-deficient iNK cells and CISH-deficient iNK cells in a mouse xenograft model.

[0037] Fig.21 The in vivo antitumor activity of CBLB-deficient iNK cells and CISH-deficient iNK cells in a mouse xenograft model (measured as the survival probability of tumor-transplanted animals) is shown.

[0038] Fig. 22 Shown are the cytotoxicity and survival rates of co-cultures with iNK T cells protected from immune challenge by the B2M-HLA-E fusion construct.

[0039] Fig.23 The proportions of CD8+ cells in the CD56+ cell populations of T cells, iNK cells, and co-cultures are shown.

[0040] Fig.24 is a diagram of an expression construct for expressing an IL-15 cytokine-receptor fusion.

[0041] Fig.25 The cytotoxic properties of iNKs engineered to constitutively express cytokine-receptor fusions are shown.

[0042] Fig.26 are diagrams of the original and optimized anti-ROR1 CAR expression constructs.

[0043] Fig.27A , Fig.27B and Fig.27C Specific target cell lysis (in vitro cytotoxicity) by anti-ROR1 CAR-T cells is shown.

[0044] Fig.28A and Fig.28BCytokine secretion by anti-ROR1 CAR-T cells in co-culture with target cells expressing ROR1 is shown.

[0045] Fig.29A and Fig.29B The in vivo antitumor activity of anti-ROR1 CAR-T cells is shown, as measured by body weight and survival probability ( Fig.29A ) and bioluminescence intensity ( Fig.29B ) is measured in the form of changes in the

[0046] Fig. 30A and Fig. 30B Lysis of specific target cells by checkpoint-deficient anti-ROR1 CAR-T cells (in vitro cytotoxicity) is shown.

[0047] Fig.31A , Fig.31B , Fig.31C and Fig.31D Shown is cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells.

[0048] Fig.32 Shown are the results of serial re-stimulation of checkpoint-deficient anti-ROR1 CAR-T cells with ROR1-expressing tumor cells.

[0049] Fig.33 Lysis of specific target cells by multiple checkpoint-deficient anti-ROR1 CAR-T cells (in vitro cytotoxicity) is shown.

[0050] Fig.34 Specific target cell lysis (in vitro cytotoxicity) of anti-ROR1 CAR-T cells pCB7432, pCB7437, and pCB7438 (Table 3) against ROR1-positive and ROR1-negative cell lines and B-CLL primary cells was shown.

[0051] Fig.35A and Fig.35B Specific target cell lysis (in vitro cytotoxicity) of anti-ROR1 CAR-T cells pCB7429, pCB7430, pCB7431, pCB7433, pCB7434 and pCB7436 (Table 3) against ROR1-positive and ROR1-negative cell lines and B-CLL primary cells is shown, expressed as AUC.

[0052] Fig.36A and Fig.36BCytokine secretion by anti-ROR1 CAR-T cells pCB7429, pCB7430, pCB7431, pCB7433, pCB7434 and pCB7436 (Table 3) in co-culture with ROR1-positive and ROR1-negative target cells is shown.

[0053] Fig.37A and Fig.37B Cytokine secretion by anti-ROR1 CAR-T cells pCB7432, pCB7437, and pCB7438 (Table 3) in co-culture with ROR1-positive and ROR1-negative target cells is shown.

[0054] Fig.38 The results show that in the presence of ROR1-positive JeKo-1 cells, anti-ROR1 CAR-T cells pCB7306 ( Fig.26 ) or pCB7436, pCB7437 and pCB7438 (Table 3) secreted interleukin 36γ (IL36γ).

[0055] Fig.39A and Fig.39B The in vivo anti-tumor efficacy of anti-ROR1 CAR-T cells pCB7430, pCB7436, and pCB7438 (Table 3) is shown.

[0056] Fig.40 Shown is a comparison of anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in terms of surface CAR expression and CAR-T cell counts during in vitro serial re-challenge with ROR1-expressing target tumor cells.

[0057] Fig.41 Shown is a comparison of anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in terms of target cell lysis (expressed as area under the curve, AUC) during in vitro serial re-challenge with target tumor cells expressing ROR1.

[0058] Fig.42 Shown is a comparison of anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in reducing tumor burden (expressed as tumor bioluminescence and AUC of bioluminescence measurements) in animals transplanted with Jeko-1 ROR1-expressing tumors.

[0059] Fig.43 Shown is a comparison of anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in prolonging overall survival of animals transplanted with tumors expressing Jeko-1 ROR1.

[0060] Fig.44Reduction of tumor burden (expressed as tumor bioluminescence) in animals transplanted with ROR1 -expressing tumors is shown by anti-ROR1 CAR-iNK cells lacking CBLB and B2M expression but expressing B2M-HLA-E fusion and IL15 receptor fusion.

[0061] Fig.45 It is shown that tumor burden (expressed as AUC of tumor bioluminescence measurements) was reduced in animals transplanted with ROR1-expressing tumors by anti-ROR1 CAR-iNK cells lacking CBLB and B2M expression but expressing B2M-HLA-E fusion and IL15 receptor fusion.

[0062] Fig.46 Shown are the results of in vitro serial re-challenge of anti-ROR1 CAR-iNK cells lacking CBLB and B2M expression but expressing B2M-HLA-E fusion and IL15 receptor fusion with SKOV3, a tumor cell line with low ROR1 antigen density.

[0063] Fig.47 Shown are the results of in vitro serial re-challenge of anti-ROR1 CAR-iNK cells lacking CBLB and B2M expression but expressing B2M-HLA-E fusion and IL15 receptor fusion with Hs746t, a tumor cell line with high ROR1 antigen density.

[0064] Fig.48A and Fig.48B Shown are the results of multiple rounds of treatment with anti-ROR1 iNK cells in animals transplanted with luciferase-expressing SKOV3, a tumor cell line with low ROR1 antigen density. Fig.48A : Bioluminescence; Fig.48B : Area under the curve (AUC) calculated from bioluminescence.

[0065] Fig.49A and Fig.49B Shown are the results of multiple rounds of treatment with anti-ROR1 iNK cells in animals transplanted with luciferase-expressing Hs746t, a tumor cell line with high ROR1 antigen density. Fig.49A : Bioluminescence; Fig.49B : Area under the curve (AUC) calculated from bioluminescence. DETAILED DESCRIPTION

[0066] definition

[0067] The following definitions are provided to aid in understanding the present disclosure. Unless defined in this section, technical and scientific terms used in the present disclosure have the meanings commonly understood by those of ordinary skill in the art. See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 4th ed. Cold Spring Harbor Lab. Press (2012).

[0068] The term "activation" refers to the state of T cells, including one or both of cell proliferation and cell secretion of cytokines.

[0069] The term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. The term also refers to antibody fragments, including Fv, Fab and F(ab)2, scFv and other forms, such as described in "Antibodies: A Laboratory Manual", 2nd edition Greenfield, E., ed., Cold Spring Harbor Laboratory Press, NY (2013).

[0070] The term "costimulatory domain" refers to a part of a chimeric T cell receptor (CAR), and CAR is a binding partner specifically bound to a co-stimulatory ligand, thereby mediating the co-stimulatory response, proliferation and cytokine secretion of T cells. The example of co-stimulatory ligands includes CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB and HVEM. The example of a co-stimulatory domain includes CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C and B7-H3.

[0071] The term "therapeutic benefit" refers to the effect of improving a condition in a subject relative to a medical treatment of the condition. The term includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treatment of cancer can involve, for example, reducing tumor size, reducing tumor invasiveness, reducing tumor growth rate, or preventing metastasis, or prolonging overall survival (OS) or progression-free survival (PFS) of a subject with cancer.

[0072] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other harmful reactions in patients. For example, pharmaceutically and pharmacologically acceptable preparations should meet the standards set forth by the FDA Office of Biological Standards.

[0073] The terms "pharmaceutically acceptable carrier" and "excipient" refer to aqueous solvents (e.g., water, aqueous alcohol solutions, saline solutions, sodium chloride, Ringer's solution, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters), as well as dispersion media, coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, stabilizers, binders, disintegrants, lubricants, sweeteners, flavoring agents, and dyes. The concentration and pH of the various components in the pharmaceutical composition are adjusted according to the well-known parameters of each component.

[0074] The term "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

[0075] The term "effector function" refers to the specialized functions of differentiated cells, such as NK cells.

[0076] The term "adoptive cells" refers to cells that can be genetically modified for use in cell therapy treatment. Examples of adoptive cells include T cells, macrophages, and natural killer (NK) cells.

[0077] The term "cell therapy" refers to the treatment of a disease or condition using genetically modified cells. The term "adoptive cell therapy (ACT)" refers to a therapy using genetically modified adoptive cells. Examples of ACT include T cell therapy, CART cell therapy, natural killer (NK) cell therapy, and CAR NK cell therapy.

[0078] The term "lymphocyte" refers to a white blood cell that is part of the vertebrate immune system. Lymphocytes include T cells, such as CD4 + and / or CD8 + Cytotoxic T cells, α / β T cells, γ / δ T cells and regulatory T cells. Lymphocytes also include natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells and antigen presenting cells (APCs), such as dendritic cells. Lymphocytes also include tumor infiltrating lymphocytes (TILs).

[0079] The terms "effective amount" and "therapeutically effective amount" of a composition (eg, a cell therapy composition) refer to an amount of the composition sufficient to provide a desired response in a patient to whom the composition is administered.

[0080] The terms "peptide", "polypeptide" and "protein" are interchangeable and refer to polymers of amino acids, including natural amino acids and synthetic (non-natural) amino acids, as well as amino acids not found in naturally occurring proteins, such as peptidomimetics and D optical isomers. Polypeptides may be branched or linear and may be interrupted by non-amino acid residues. The terms also encompass amino acid polymers that have been modified by acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, cross-linking or conjugation (e.g., with a label). Although a polypeptide need not include the full-length amino acid sequence of a reference molecule, only as much of the reference molecule may be included as needed so that the polypeptide retains its desired activity. For example, the terms "protein" and "polypeptide" encompass polypeptides comprising full-length proteins, fragments thereof, polypeptides with amino acid deletions, additions and substitutions, as long as the desired activity is retained. For example, polypeptides having 95%, 90%, 80% or less sequence identity to a reference polypeptide are included, as long as the polypeptide retains the desired activity.

[0081] The terms "CRISPR" (clustered regularly interspaced short palindromic repeats), "Cas" (CRISPR associated protein), "CRISPR-Cas" and "CRISPR system" refer to a genome editing tool derived from a prokaryotic organism and comprising a nucleic acid guide molecule and a sequence-specific nucleic acid-guided nuclease capable of cleaving the target nucleic acid strand at a site complementary to the sequence in the nucleic acid guide.

[0082] The term "NATNA" (nucleic acid targeting nucleic acid) refers to the nucleic acid guide molecule of the CRISPR system. NATNA can include two nucleic acids ("dual guides") targeting a polynucleotide, including CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). NATNA can include a single nucleic acid ("single guide") targeting a polynucleotide, which includes crRNA and tracrRNA connected by a fusion region (joint). The crRNA may include a targeting region and an activation region. The tracrRNA may include a region that can hybridize with the activation region of the crRNA. The term "targeting region" refers to a region that can hybridize with a sequence in a target nucleic acid. The term "activation region" refers to a region that interacts with a polypeptide (e.g., CRISPR nuclease).

[0083] Chimeric antigen receptor (CAR)-T cell therapy has shown significant potential against hematological malignancies (see, e.g., U.S. Pat. No. 9,464,140). However, CAR-T cells have not shown such potent and reproducible antitumor activity against solid malignancies. Natural killer (NK) cells express the receptor NKG2D, which promotes their innate ability to detect and kill tumors expressing stress ligands.

[0084] ROR1 (receptor tyrosine kinase-like orphan receptor 1, also known as NTRKR1, neurotrophic tyrosine kinase receptor-related 1) is an orphan receptor tyrosine kinase-like antigen. ROR1 is a protein composed of 937 amino acids, of which amino acids 30-406 comprise the extracellular domain. ROR1 is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. Binding of ROR1 to its specific ligand WNT5A activates the downstream Rac / Rho pathway responsible for cell motility and invasion.

[0085] ROR1 is expressed in a variety of tumors, including hematological malignancies (chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL)), ovarian cancer, lung cancer, pancreatic cancer, and triple-negative breast cancer. ROR1 expression drives tumor cell growth, survival, and metastasis. Specifically, it has been shown that ROR1 is expressed on ovarian cancer stem cells and promotes migration, invasion, and cancer stem cell sphere formation. Overexpression of ROR1 has been detected in both hematological cancers and solid tumors. Only low levels of ROR1 expression have been detected in normal human tissues such as adipose tissue, endocrine tissue, and the gastrointestinal tract, making it a promising target for cellular immunotherapy against cancer.

[0086] Disclosed herein is an engineered T cell and T cell composition, as well as NK cells and NK cell compositions, wherein the T cells and NK cells are engineered to express ROR1-specific CAR (anti-ROR1-CAR). The T cells and NK cells of the present invention have enhanced specificity and function for solid tumors and exhibit transport to ROR1+ tumors. Engineered T cells and NK cells can detect tumor cells by anti-ROR1-CAR. In addition, engineered NK cells can detect tumor cells by their natural NKG2D receptors.

[0087] Disclosed herein are methods and compositions for treating tumors with engineered cells, including T cells and natural killer (NK) cells, that target ROR1.

[0088] In some embodiments, the present invention includes adoptive cells and uses of adoptive cells in cellular immunotherapy. The adoptive cells of the present invention include T cells, CAR-T cells, natural killer (NK) cells, induced natural killer (iNK) cells, CAR-NK cells, CAR-iNK cells and precursors thereof.

[0089] In some embodiments, the cells of the invention are allogeneic cells, ie, cells isolated from a donor individual, such as a healthy human donor, regardless of sex.

[0090] In certain embodiments, cells are separated from healthy donors using standard techniques. For example, lymphocytes can be separated from blood (including peripheral blood and umbilical cord blood) or from lymphoid organs (such as thymus, bone marrow, lymph nodes and mucosa-associated lymphoid tissue (MALT)). The techniques for separating lymphocytes from such tissues are well known in the art, see, for example, Smith, JW (1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1: 203-206.

[0091] In some embodiments, the isolated lymphocytes are characterized in terms of specificity, frequency and function. In some embodiments, the isolated lymphocyte population is enriched for a specific subset of cells such as T cells or NK cells.

[0092] In some embodiments, the isolated lymphocyte population is enriched for a specific subset of T cells, such as CD4 + 、CD8 + 、CD25 + CD62L + See, e.g., Wang et al., Mol. Therapy-Oncolytics (2016) 3:16015. In some embodiments, the isolated lymphocyte population is enriched for CD56 indicative of NK cells. + Phenotype. In some embodiments, after separation, lymphocytes are activated to promote proliferation and differentiation into specialized lymphocytes. For example, T cells can be activated using soluble CD3 / 28 activators or magnetic beads coated with anti-CD3 / anti-CD28 monoclonal antibodies.

[0093] In some embodiments, NK cells are generated by differentiating stem cells such as embryonic stem cells (ESCc) and induced pluripotent stem cells (iPSC). See, e.g., T. Cheng (ed.), Hematopoietic Differentiation of Human Pluripotent Stem Cells, Springer Briefs in Stem Cells, DOI 10.1007 / 978-94-017-7312-6_5, Hermanson et al., Chapter 5, Human Pluripotent Stem Cells as a Renewable Source of Natural Killer Cells; Woll et al., (2016) Differentiation of Human Embryonic Stem Cells into Homogeneous Populations of Natural Killer Cells with Potent In Vivo Antitumor Activity, Blood, 113(24)6094; and Denman et al., (2012) Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells. Killer Cells, PLOS ONE, 7(1): e30264.

[0094] The differentiation process generally comprises two steps and optionally three steps: differentiating stem cells into hematopoietic progenitor cells (HPCs), differentiating HPCs into natural killer cells (NKs), and optionally expanding NK cells. The differentiation (or expansion) products can also be tested for the presence of NK phenotypes, such as NK-specific gene expression patterns, including the expression of NK-specific cell surface markers, such as CD45 and CD56.

[0095] In some embodiments, the step of forming hematopoietic progenitor cells (HPCs) comprises forming spheres by allowing the cells to aggregate (optionally assisted by low speed centrifugation) and incubating the spheres in the presence of one or more cytokines selected from the group consisting of BMP4, VEGF, SCF, IL3, IL6, and TPO. In some embodiments, a combination of BMP4, VEGF, and SCF is used. In some embodiments, a combination of BMP4, VEGF, SCF, IL3, IL6, and TPO is used. In some embodiments, HPCs are formed by incubation in the presence of feeder cells such as bone marrow stromal cells.

[0096] In some embodiments, the step of forming NK cells comprises incubating the spheroids in the presence of one or more cytokines selected from the group consisting of IL-3, IL-15, IL-7, SCT, and FLT3L. In some embodiments, a combination of IL-3, IL-15, IL-7, SCT, and FLT3L is used. In some embodiments, feeder cells (stromal cells) are used. In some embodiments, fetal liver stromal cells are used as feeders. In some embodiments, prior to initiating the NK differentiation step, the HPC fraction is enriched for CD34 + In some embodiments, upon completion of the differentiation stage, a fraction of cells is tested for surface expression of NK-specific markers, such as CD45 and CD56.

[0097] In certain embodiments, the step of NK cell amplification is included in the presence of cytokines and antigen presenting cells (APC) and incubate NK cells. In certain embodiments, APC is engineered to express cytokines on the cell surface. In certain embodiments, APC overexpresses 41BBL and membrane-bound IL-21. In certain embodiments, APC overexpresses membrane-bound IL-15. In certain embodiments, APC overexpresses cytokine receptor fusions. In certain embodiments, APC overexpresses IL-15-IL-15R fusions. In certain embodiments, APC overexpresses IL-21-IL-21R fusions. In certain embodiments, exogenous cytokines are also added. In certain embodiments, IL-2 is added. In certain embodiments, the iNK product of final amplification is evaluated in in vitro and in vivo functional assays.

[0098] The present invention comprises allogeneic engineered immune cells, including T cells and NK cells (including NK cells and induced NK (iNK) cells). In some embodiments, the cells described herein are genetically modified to express a chimeric antigen receptor (CAR). In some embodiments, the cells are CAR-T cells or CAR-NK cells. In some embodiments, the cells are CAR-iNK cells. In some embodiments, the present invention comprises a combination of CAR-T cells and CAR-NK cells for administration to a patient. In some embodiments, CAR-T cells and CAR-NK cells are administered to a patient simultaneously as a single formulation. In some embodiments, CAR-T cells and CAR-NK cells are administered to a patient sequentially.

[0099] A typical chimeric antigen receptor (CAR) comprises an extracellular domain comprising an antigen binding region, a transmembrane domain, and one or more intracellular activation (costimulation) domains. In some embodiments, CAR further comprises a hinge domain. In some embodiments, CAR further comprises a leader peptide that guides CAR to the cell membrane.

[0100] CAR disclosed herein comprises an extracellular domain comprising an antigen binding region targeting ROR1. In some embodiments, the antigen binding region is derived from an antibody. In some embodiments, the antigen binding region is derived from a monoclonal antibody. In some embodiments, the antigen binding region comprises a mouse (mouse) sequence. In some embodiments, the antigen binding region comprises a human sequence. In some embodiments, the antigen binding region comprises a humanized mouse (mouse) sequence.

[0101] In some embodiments, the antigen binding region comprises a single chain variable fragment (scFv). scFv comprises an antibody light chain (V L ) and antibody heavy chain (V H ) is connected to a variable region of a variable region. In some embodiments, V L Through a peptide linker and V H connect.

[0102] The peptide linker generally comprises about 5 to about 40 amino acids. The linker can be a naturally occurring sequence or an engineered sequence. For example, in some embodiments, the linker is derived from a human protein, for example, an immunoglobulin selected from IgG, IgA, IgD, IgE or IgM. In some embodiments, the linker comprises 5-40 amino acids from the CH1, CH2 or CH3 domains of an immunoglobulin heavy chain. In some embodiments, the linker is a linker having the sequence (G x S y ) n Glycine- and serine-rich linkers. Additional linker examples and sequences are disclosed in: U.S. Pat. No. 5,525,491 Serine-rich peptide linkers, U.S. Pat. No. 5,482,858 Polypeptide linkers for production of biosynthetic proteins, and WO2014087010 Improved polypeptides directed against IgE.

[0103] In some embodiments, the sequence of the scFv is further optimized for binding to the ROR1 antigen. In some embodiments, the optimization process comprises changing the heavy chain (V H ) and light chain (V L). It has been reported that the linker sequence can affect antigen-scFv binding (Navabi et al., (2021) Design and optimization of a single-chain fragment variable (scFv) antibody against IL2Rα(CD25): An in silico and in vitro study, Iran J Basic Med Sci 24:1.). Specifically, it has been shown that designing the amino acid sequence of the linker by computer simulation in the context of the known crystal structure of the scFv antibody complex can produce a linker sequence that does not confer better antigen affinity to the scFv. It has also been shown that the binding of a chimeric antigen receptor (CAR) containing an scFv to an antigen can be optimized simply by changing the length of the linker, where the linker contains a variable number of repeats of the same sequence. Singh et al., (2021) Antigen-independent activation enhances the efficacy of 41BB co-stimulated CD22 CAR T cells, Nat. Med. 27(5):842. In some embodiments, the sequence is G x S y , and the joint has the general formula (G x S y ) n .

[0104] In some embodiments, the CAR comprises an scFv described in international application serial number PCT / US2023 / 067314 filed on May 22, 2023, an anti-ROR1 antibody and ROR1-targeting engineered cells.

[0105] In some embodiments, the CAR comprises a scFv selected from clones 857, 858, and 862 described in PCT / US2023 / 067314. In some embodiments, the CAR comprises a scFv comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9. In some embodiments, the CAR comprises a scFv consisting of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9. In some embodiments, the scFv comprising the sequence of SEQ ID NO: 9 is further optimized to comprise SEQ ID NO: 27 containing a linker G4S. In some embodiments, the optimized scFv consists of SEQ ID NO: 27.

[0106] In some embodiments, the scFv is encoded by a nucleic acid sequence comprising: SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 17, or SEQ ID NO: 28. In some embodiments, the scFv is encoded by a nucleic acid sequence consisting of: SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 17, or SEQ ID NO: 28.

[0107] In some embodiments, the antigen binding region comprises a heavy chain (V H ), which comprises a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10. In some embodiments, the antigen binding region comprises a heavy chain (V H ): SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:10.

[0108] In some embodiments, the antigen binding region comprises a light chain (V L ), which comprises a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11. In some embodiments, the antigen binding region comprises a heavy chain (V H ): SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:11.

[0109] Mouse antibodies and scFv, humanized antibodies and scFv derived therefrom, and human antibodies and scFv in heavy antibody chain and light antibody chain (V H and V L ) each contain three complementarity determining regions (CDRs).

[0110] In some embodiments, the CDRs are identified using a crystal structure of an antigen-antibody complex. In some embodiments, the CDRs are identified using an in vitro method such as phage display. In some embodiments, the CDRs are identified using an in silico method, such as IMGT (Lefranc et al., (2009) International Immunogenetics Information System ( The international immunogenetics information system), "Nucl. Acids Res." 37: D1006) and Kabat (Kabat et al., (1987) Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services National Institutes of Health (USHHS, NIH)). In some embodiments, CDRs are identified using the IMGT system. In some embodiments, CDRs are identified using the Kabat tool. In some embodiments, the smallest portion of a CDR is identified as the overlap of a sequence located by the IMGT tool with a sequence located by the Kabat tool.

[0111] In some embodiments, the scFv included in the CAR of the present invention comprises a V H In some embodiments, the scFv comprises V H In some embodiments, the scFv comprises V H In some embodiments, the scFv comprises V L In some embodiments, the scFv comprises V L In some embodiments, the scFv comprises V L SEQ ID NO:34 in CDR3 of.

[0112] In some cases, the scFv included in the CAR of the present invention comprises a V H SEQ ID NO: 29 in CDR1 of H SEQ ID NO: 30 in CDR2 of H SEQ ID NO: 31 in CDR3 of L SEQ ID NO: 32 of CDR1, and VL SEQ ID NO: 33 of CDR2 and V L SEQ ID NO:34 of CDR3.

[0113] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is derived from a CD8 protein. In some embodiments, the hinge domain comprises SEQ ID NO: 20. In some embodiments, the hinge domain consists of SEQ ID NO: 20. In some embodiments, the hinge domain is encoded by a nucleic acid comprising SEQ ID NO: 19. In some embodiments, the hinge domain is encoded by a nucleic acid consisting of SEQ ID NO: 19.

[0114] In some embodiments, CAR comprises a signal peptide (signal sequence) capable of transporting CAR to a cell membrane. In some embodiments, the signal sequence comprises a CD28 signal sequence. In some embodiments, the signal sequence consists essentially of a CD28 signal sequence. In some embodiments, the signal sequence comprises a CD8 signal sequence. In some embodiments, the signal sequence consists essentially of a CD8 signal sequence.

[0115] In some embodiments, the transmembrane domain of CAR is derived from a membrane-bound protein or transmembrane protein. In some embodiments, the transmembrane domain is derived from the same protein as the protein of the costimulatory domain described below. For example, the transmembrane domain of CAR can be the following transmembrane domain: T cell receptor alpha chain or beta chain, CD3 ζ chain, CD28, CD3 ε chain, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, DNAM1, NKp44, NKp46, NKG2A, NKG2C, NKG2D, 2B4, TMIG2, TLR1, TLR2, TLR4, TLR5, TLR6 or GITR. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises SEQ ID NO: 22. In some embodiments, the transmembrane domain consists of SEQ ID NO: 22. In some embodiments, the transmembrane domain is encoded by a nucleic acid comprising SEQ ID NO: 21. In some embodiments, the transmembrane domain is encoded by a nucleic acid consisting of SEQ ID NO: 21.

[0116] The cytoplasm or intracellular signaling domain is also referred to as the costimulatory domain of CAR, which is responsible for activating one or more effector functions of immune cells expressing CAR. In some embodiments, the costimulatory domain of CAR includes a portion or all of the following sequences: TCR ζ chain, CD3 ζ link, CD3 ε chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12, DNAM1, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MyD88, IL18R, CD40 or a combination thereof. In some embodiments, the cytoplasmic domain of CAR includes a CD3 ζ costimulatory domain. In some embodiments, the cytoplasmic domain of CAR includes a 4-1BB costimulatory domain. In some embodiments, the cytoplasmic domain of CAR consists of CD3 ζ and 4-1BB costimulatory domains.

[0117] In some embodiments, the CD3 zeta costimulatory domain comprises SEQ ID NO: 24. In some embodiments, the CD3 zeta costimulatory domain consists of SEQ ID NO: 24. In some embodiments, the CD3 zeta costimulatory domain is encoded by a nucleic acid comprising SEQ ID NO: 23. In some embodiments, the CD3 zeta costimulatory domain is encoded by a nucleic acid consisting of SEQ ID NO: 23.

[0118] In some embodiments, the 4-1BB co-stimulatory domain comprises SEQ ID NO: 26. In some embodiments, the 4-1BB co-stimulatory domain consists of SEQ ID NO: 26. In some embodiments, the 4-1BB co-stimulatory domain is encoded by a nucleic acid comprising SEQ ID NO: 25. In some embodiments, the 4-1BB co-stimulatory domain is encoded by a nucleic acid consisting of SEQ ID NO: 25.

[0119] In some embodiments, the cytoplasmic domain of the anti-ROR1 CAR comprises one or more additional elements that enhance the intracellular signal transduction of the CAR. In some embodiments, the cytoplasmic domain comprises one or more protein association motifs that bind to and recruit one or more intracellular signal transduction proteins. For example, U.S. Patent No. 10,336,810 discloses an increase in the cytotoxic activity of anti-CD19 CAR-T cells, which have CARs enhanced with JAK and STAT binding motifs in the cytoplasmic region.

[0120] In certain embodiments, CAR comprises two or more intracellular (or cytoplasmic) domains and two or more protein association motifs, wherein each protein association motif is located in a separate intracellular domain of CAR. In certain embodiments, two or more protein association motifs are located in the same intracellular domain of CAR.

[0121] In some embodiments, the intracellular signal transduction protein is signal transducer and activator of transcription 3 or STAT3. In some embodiments, the STAT3 association motif is YXXQ (SEQ ID NO: 39). In some embodiments, the STAT3 association motif is YRHQ (SEQ ID NO: 40). The STAT3 association motif is naturally present in IL-6 and IL-10. In some embodiments, the STAT3 association motif is engineered into the CD3ζ intracellular signaling domain of the CAR.

[0122] In some embodiments, the intracellular signal transduction protein is signal transducer and activator of transcription 5 or STAT5. In some embodiments, the STAT5 association motif is YXXL (SEQ ID NO: 41). In some embodiments, the STAT5 association motif is YLSL (SEQ ID NO: 42). The STAT5 association motif is naturally present in the IL-2Rβ chain. In some embodiments, the STAT5 association motif is engineered into the CD3ζ intracellular signaling domain of the CAR.

[0123] In some embodiments, the intracellular signal transduction protein is a Janus kinase, such as JAK1. In some embodiments, the JAK association motif is LKCNTPDPS (SEQ ID NO: 43). In some embodiments, the JAK association motif is selected to be present in IL2Rγ (IL2RG), erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), granulocyte macrophage colony stimulating factor receptor (GM-CSFR) or growth hormone receptor (GHR). In some embodiments, the JAK association motif is engineered into the CD3ζ intracellular signaling domain of the CAR.

[0124] In some embodiments, the chimeric antigen receptor (CAR) comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 35, and 37. In some embodiments, the CAR consists of a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 35, and 37. In some embodiments, the CAR is encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 36, and 38. In some embodiments, the CAR is encoded by a nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 36, and 38.

[0125] In some embodiments, the CAR is fully human or humanized to reduce immunogenicity in human patients. In some embodiments, the CAR sequence is optimized for codon usage in human cells.

[0126] Example 1 of the present disclosure and Figure 1 Examples of anti-ROR1 CAR constructs are described in. Examples 12 and Fig.26 Other examples of anti-ROR1 CAR constructs are described in.

[0127] Nucleic acids encoding CAR (e.g., nucleic acids comprising SEQ ID NO: 14, 16, 18, 36 or 38) can be introduced into cells as genomic DNA sequences or cDNA sequences. The cDNA sequence comprises an open reading frame for translating a protein (e.g., CAR), and in some embodiments, the cDNA further comprises a non-translated element that improves, for example, the stability of CAR mRNA or the translation rate.

[0128] In some embodiments, the CAR coding sequence is inserted into the cell genome of T cells or NK cells at the endogenous T cell receptor alpha chain (TRAC) locus. In some embodiments, the TRAC locus is targeted by CRISPR nuclease (e.g., Cas9, Cas12a or CASCADE) and guide polynucleotides. In some embodiments, the guide polynucleotide is a CRISPR hybrid DNA-RNA polynucleotide (chRDNA).

[0129] In some embodiments, the insertion of CAR depends on the endogenous homologous recombination system of the cell. In such embodiments, the CAR coding sequence can be introduced into the cell by chemical or electrochemical means (such as lipid nanoparticles or electroporation). The CAR coding sequence can also be introduced into the cell using a vector such as a lentiviral vector, as discussed in detail elsewhere in this disclosure.

[0130] In some embodiments, the cells used in the present invention are engineered to express CAR, and further include genomic modifications, thereby causing the cells to defend against attacks by the immune system of allogeneic immune cells (immune cells derived from donors) receptors. In some embodiments, the armored modification includes protecting the host from being recognized by cytotoxic T cells. Cytotoxic T cells recognize MHC class I antigens. MHC class I molecules are cell surface molecules containing beta-2 microglobulin (B2M) associated with the heavy chain of HLA-I proteins (selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G). The B2M / HLA-I complex on the surface of allogeneic cells is cytotoxic CD8 +T cells recognize, and if HLA-I is recognized as non-self, the allogeneic cells are killed by T cells. In some embodiments, the cells of the invention comprise armored genome modifications comprising disruption of the B2M gene and thus disruption of the MHC class I cell surface-bound complex. This disruption eliminates MHC class I antigen recognition that normally stimulates cytotoxic T cell attack.

[0131] In some embodiments, the armored genome modification comprises the destruction of the recognition of the CAR-T cells or CAR-NK cells of the natural killer (NK) cells of the host. NK cells recognize cells without MHC-I proteins as "missing self" and kill such cells. NK cells are inhibited by HLA-I proteins, and the HLA-I molecules include HLA-E, a HLA-I protein with minimal polymorphism. In some embodiments, the cell of the present invention comprises a first armored genome modification, the first armored genome modification comprises the destruction of the B2M gene and the destruction of the complex bound to the surface of the MHC class I cell and the destruction of the MHC class I antigen recognition that stimulates the attack of cytotoxic T cells, and further comprises a second armored genome modification, the second armored genome modification comprises the insertion of the HLA-E gene fused to the β-2-microglobulin (B2M) gene and the expression of the B2M-HLA-E construct designed to make the cell invisible from the attack of NK cells. See, e.g., Gornalusse et al. (2017) HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells, Nat. Biotechnol. (2017) 35:765-772.

[0132] The present inventors have successfully achieved immune cloaking of iNK cells using B2M-HLA-E fusion constructs. An example of such immune cloaking is shown in Example 10.

[0133] In some embodiments, the armor modification comprises transcriptional silencing or disruption of one or more immune checkpoints or regulatory genes. In some embodiments, the checkpoint genes are selected from PD1 (encoded by the PDCD1 gene), CBLB, CISH (ISH), ADAM17, PRDM1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M and 2B4.

[0134] In some embodiments, the silenced or disrupted immune checkpoint gene is CBLB. The CBLB protein is an immune checkpoint that controls the activity of NK cells. CBLB binds to activated NK cell downstream effectors after phosphorylation and downregulates them through proteasome-mediated degradation. CBLB has a negative impact on the cytotoxicity, cytokine production and persistence of NK cells, and inhibiting CBLB activity in NK cells can enhance the anti-tumor function of NK cells. See Lu et al. (2021) Cbl-b Is Upregulated and Plays a Negative Role in Activated Human NK Cells, J Immunol. 206(4):677–685; Lametschwandtner et al. (2015) Cbl-b silenced human NK cells respond stronger to cytokine stimulation, J ImmunoTher. of Cancer, 3(Suppl. 2):P230; Guo et al. (2021) CBLB ablation with CRISPR / Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy. Journal of Cancer Immunotherapy 9:e001975.

[0135] In some embodiments, the destruction of the CBLB gene by the methods described herein occurs in NK cells. In some embodiments, the NK cells are primary NK cells. In some embodiments, the NK cells are iNK cells (NK cells differentiated from iPSC in vitro).

[0136] In some embodiments, the disruption of the CBLB gene by the methods described herein occurs in iNK cell precursors, such as iPSCs. In some embodiments, the disruption of the CBLB gene in iPSCs does not negatively affect their ability to differentiate into iNK cells.

[0137] The inventors herein demonstrate that iNK cells lacking CBLB expression (CBLB KO) exhibit enhanced in vitro degranulation and cytotoxicity, as well as enhanced in vivo anti-tumor activity. The inventors also demonstrate that CBLB-destroyed iPSCs efficiently differentiate into iNKs, and the resulting iNKs express standard NK cell markers at levels similar to those of wild-type iNKs. (See Examples 8 and 9).

[0138] In some embodiments, the silenced or disrupted regulatory gene is CISH. CISH or CIS (cytokine-induced SH2 protein) is a negative regulator of T cells and NK cells. Inhibition of CISH has been shown to enhance the effectiveness of NK cells against certain cancers and infections (U.S. Patent No. 11,104,375).

[0139] In some embodiments, the destruction of CISH gene by the methods described herein occurs in NK cells. In some embodiments, NK cells are primary NK cells. In some embodiments, NK cells are iNK cells (NK cells differentiated from iPSC in vitro).

[0140] In some embodiments, the disruption of the CISH gene by the methods described herein occurs in iNK cell precursors, such as iPSCs. In some embodiments, the disruption of the CISH gene in iPSCs does not negatively affect their ability to differentiate into iNK cells.

[0141] The inventors herein demonstrate that iNK cells lacking CISH expression (CISH KO) exhibit enhanced in vitro cytotoxicity. (See Example 9.)

[0142] In some embodiments, the silenced or disrupted immune checkpoint gene is LAG3. Lymphocyte activation gene 3 (LAG3, also known as CD223) is an immune checkpoint receptor expressed on activated or exhausted T cells. LAG3 interacts with MHC class II molecules to inhibit T cell function and lead to T cell exhaustion. Chronic lymphocytic leukemia (CLL) cells both express and secrete LAG3, which is thought to contribute to CLL tumor growth and evasion of T cell attack. Shapiro et al., (2017) Lymphocyte activation gene 3: a novel therapeutic target in chronic lymphocytic leukemia, Haematologica, 102(5):874.

[0143] In some embodiments, disruption of the LAG3 gene by the methods described herein occurs in T cells. The inventors herein demonstrate that T cells lacking LAG3 expression exhibit enhanced in vitro cytotoxicity. (See Examples 17 and 18.)

[0144] In some embodiments, the silenced or disrupted immune checkpoint gene is Tim3. T cell immunoglobulin mucin 3 (Tim3) is a negative regulator of T cell function. Tim3 binds to its receptor galectin-9 on the surface of T cells and inhibits the expression of proteins including CD4 + T cells, CD8 + Functions of various types of T cells, T cells, and T helper cells. In hematological malignancies including chronic lymphocytic leukemia (CLL), high levels of Tim3 upregulate suppressive Treg cells, inhibit T helper cell function, and are associated with poor prognosis. Pang et al., (2021) Activated Galectin-9 / Tim3 promotes Treg and suppresses Th1 effector function in chronic lymphocytic leukemia, FASEB J. 35:e21556.

[0145] In some embodiments, disruption of the Tim3 gene by the methods described herein occurs in T cells. The inventors demonstrate herein that T cells lacking Tim3 expression exhibit enhanced in vitro cytotoxicity (see Examples 17 and 18).

[0146] In some embodiments, the silenced or disrupted immune checkpoint gene is TIGIT. T cell immune receptor with immunoglobulin and ITIM domains (TIGIT) is activated by CD8 + T and CD4 +T cells, natural killer (NK) cells, regulatory T cells (Tregs), and follicular T helper cells express. High levels of TIGIT expression in CAR-T cells are associated with poor responses to CAR-T cell therapy. Jackson et al., (2022) Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma, Cancer Discov. 12(8):1886.

[0147] In some embodiments, disruption of the Tim3 gene by the methods described herein occurs in T cells. The inventors herein demonstrate that T cells lacking TIGIT expression exhibit enhanced in vitro cytotoxicity. (See Examples 17 and 18).

[0148] In some embodiments, multiple immune checkpoint genes are inactivated. In some embodiments, two or more immune checkpoint genes are inactivated in the same cell. In some embodiments, the multiple checkpoint genes include a combination of PD1 and Tim3, PD1 and LAG3, PD1 and TIGIT, and Tim3 and LAG3. The inventors found that, surprisingly, multiple checkpoint inactivation in CAR-T cells had the most significant synergistic effect on the duration of the cytotoxic potential of CAR-T cells (see Fig.32 and 33 ).

[0149] In certain embodiments, an endonuclease of a nucleic acid chain in a target sequence of a gene to be destroyed is used to specifically cut an immune checkpoint gene or a regulatory gene. The chain cutting performed by a sequence-specific endonuclease results in a nucleic acid chain break that can be repaired by non-homologous end joining (NHEJ). NHEJ is an imperfect repair process, which may result in direct reconnection, but more generally results in the deletion, insertion or substitution of one or more nucleotides in the target sequence. Such deletion, insertion or substitution of one or more nucleotides in the target sequence may result in missense or nonsense mutations in a protein coding sequence, and eliminates the production of any protein or causes the production of non-functional protein.

[0150] In some embodiments, the immune checkpoint gene is disrupted by contacting the cell with a sequence-specific endonuclease and triggering the NHEJ process within the cell, resulting in gene mutation and elimination of protein expression of the immune checkpoint gene.

[0151] In some embodiments, the sequence-specific endonuclease is selected from a rare-cutting restriction enzyme, a TALEN, a zinc finger nuclease (ZFN), and a CRISPR endonuclease.

[0152] In some embodiments, the sequence-specific endonuclease is a CRISPR endonuclease selected from Cas9 and Cas12a. In some embodiments, the CRISPR endonuclease is part of a nucleoprotein complex comprising a CRISPR endonuclease and a CRISPR guide RNA (nucleic acid targeting nucleic acid or NATNA). In some embodiments, NATNA comprises one or more DNA nucleotides and is a CRISPR hybrid RNA-DNA or chRDNA. In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. In some embodiments, NATNA is selected from the embodiments described in the following: International Application Publication No. WO 2022086846, DNA-containing polynucleotides and guides for CRISPR Type V systems, and methods of making and using the same.

[0153] In some embodiments, the present invention includes a method for producing an anti-ROR1 chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encoding CAR is introduced into the target cell where the desired CAR is expressed. In some embodiments, the nucleic acid introduced is selected from an expression vector containing a sequence encoding CAR, an mRNA encoding CAR, and a delivery vector containing a CAR encoding donor sequence to be inserted into the cell genome. In some embodiments, the target cell is contacted with a nucleic acid encoding CAR in vitro, in vivo, or ex vivo.

[0154] In some embodiments, the vector for delivering the nucleic acid encoding CAR is a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector). In some embodiments, the vector is a lentiviral vector. The lentiviral vector packaging system is available from multiple commercial suppliers, such as ThermoFisher Scientific, LabCorp, etc. Such systems include host cells, wherein one or more plasmids encoding sequences of interest (e.g., CAR encoding sequences) are introduced together with lentiviral vector encoding genes. Then, the transfected host cells produce lentiviral vectors with CAR sequence payloads.

[0155] In some embodiments, the coding sequence of CAR is introduced into the cell by a viral vector. Suitable vectors are non-replicative in target cells. In some embodiments, the vector is selected from SV40, EBV, HSV or BPV, or is designed based thereon. In some embodiments, the vector is a lentiviral vector or any other suitable viral vector capable of delivering a payload of sufficient size. In some embodiments, in order to promote homologous recombination, the coding sequence is connected to the homology arms of 5' (upstream) and 3' (downstream) of the insertion site in the desired insertion site in the genome. In some embodiments, the homology arms are about 500bp long. See Eyquem J. et al., (2017) Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection, "Nature", 543: 113-117. In some embodiments, the sequence encoding CAR is cloned into a viral vector plasmid together with the homology arms. Plasmids are used to package sequences into viruses.

[0156] The vector incorporates a protein expression sequence. In some embodiments, the expression sequence is codon-optimized for expression in mammalian cells. In some embodiments, the vector also incorporates a regulatory sequence, including a transcription activator binding sequence, a transcription repressor binding sequence, an enhancer, an intron, etc. In some embodiments, the viral vector supplies a constitutive promoter or an inducible promoter. In some embodiments, the promoter is selected from EF1α, PGK1, MND, Ubc, CAG, CaMKIIa and β-actin promoter. In some embodiments, the promoter is selected from SV40 early and late promoters, cytomegalovirus (CMV) immediate early promoter and Rous sarcoma (Rous sarcoma) virus long terminal repeat sequence (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat sequence (MMTV-LTR) promoter, β-interferon promoter, hsp70 promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is an MND promoter. In some embodiments, the promoter is a CAG promoter.

[0157] In some embodiments, the viral vector supplies a transcription terminator.

[0158] In some embodiments, the vector comprises a nucleic acid selected from SEQ ID NOs: 14, 16, 18, 36, and 38.

[0159] In some embodiments, the nucleic acid comprises more than one coding sequence and encodes a polycistronic transcript. In some embodiments, the first coding sequence in the polycistronic transcript encodes CAR (e.g., SEQ ID NO), and the second coding sequence in the polycistronic transcript encodes a cytokine gene selected from the following: IL-2, IL-12, IL-15, IL-18, IL-21 and IL36. In some embodiments, the cytokine is IL36. In some embodiments, the cytokine is IL36 with an IL36 signal peptide. In some embodiments, the cytokine is IL36 with an IL36 signal peptide. In some embodiments, the cytokine is IL36, wherein the signal peptide of the IL2 mutation is described in the following: Zhang et al., (2005), Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo, Journal of Gene Medicine, 7: 354. The signal peptide of the IL-2 mutation contains modifications in the following two domains of the signal peptide: a basic domain and a hydrophobic domain. In some embodiments, the coding sequence in the polycistronic transcript is separated from the coding sequence for the P2A polypeptide to allow separation of the nascent peptide during translation.

[0160] In some embodiments, the vector is a plasmid selected from a prokaryotic plasmid, a eukaryotic plasmid, and a shuttle plasmid.

[0161] In some embodiments, the CAR is expressed in eukaryotic cells, such as mammalian or human NK cells (or precursors thereof), and the vector is a plasmid comprising a eukaryotic promoter active in the desired cell type, a secretion signal, a polyadenylation signal, and a stop codon, and optionally one or more regulatory elements, such as an enhancer element.

[0162] In some embodiments, the expression vector comprises one or more selection markers. In some embodiments, the selection marker is an antibiotic resistance gene or other negative selection marker. In some embodiments, the selection marker comprises a protein whose mRNA is transcribed together with the fusion protein mRNA and the polycistronic transcript is cut before translation.

[0163] In some embodiments, the expression vector comprises a polyadenylation signal. In some embodiments, the polyadenylation site is the SV-40 polyadenylation signal.

[0164] In some embodiments, NK cells or their precursors are contacted with viral vectors so that the genetic material delivered by the vector is integrated into the genome of the target cell and then expressed in the cell or on the cell surface. In such embodiments, methods well known in the art (such as fluorescence activated cell sorting (FACS), microfluidic-based screening, ELISA or protein blotting (Western blot)) can be used to test transduced and transfected cells to confirm transgenic expression on the cell surface. For example, cells can be tested by staining or flow cytometry with antibodies or labeled antigens specific to a part of CAR (for example, ROR1 in the case of engineered anti-ROR1 CAR-T and CAR-NK cells).

[0165] The present invention relates to the manipulation of nucleic acids isolated or extracted from a sample, including genomic DNA and plasmid DNA. Methods for nucleic acid extraction are well known in the art. See J. Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), 2nd edition, (Cold Spring Harbor Laboratory Press: New York, NY). Various reagents and kits for extracting nucleic acids (DNA or RNA) from biological samples are commercially available, including products from BD Biosciences (San Jose, Cal.), Clontech (TaKaRa Bio); Epicentre Technologies (Madison, Wisc.); Gentra Systems (Minneapolis, Minn.); Qiagen (Valencia, Calif.); Ambion (Austin, Texas); BioRad Laboratories (Hercules, Cal.); KAPA Biosystems (Roche Sequencing Solutions, Pleasanton, Calif.), etc.

[0166] In some embodiments, the present invention relates to intermediate purification or separation steps of nucleic acids, such as removing unused reactants from DNA. Purification or separation can be performed by a size selection method selected from gel electrophoresis, affinity chromatography, and size exclusion chromatography. In some embodiments, size selection can be performed using a solid phase reversible immobilization (SPRI) technology from Beckman Coulter (Brea, Cal.).

[0167] In some embodiments, a nucleic acid sequence encoding an exogenous protein (e.g., a sequence encoding a CAR) is introduced into a cell, such as a T cell or a NK cell, or a NK cell precursor, such as an induced pluripotent stem cell (iPSC). In some embodiments, a "naked" nucleic acid is introduced into a lymphocyte by electroporation, as described in U.S. Patent No. 6,410,319.

[0168] In some embodiments, the cell comprises a CRISPR system. In some embodiments, the CRISPR system comprises a nucleic acid-guided endonuclease and a nucleic acid-targeting nucleic acid (NATNA) guide (e.g., a CRISPR guide RNA selected from tracrRNA, crRNA, or a single guide RNA incorporating elements of tracrRNA and crRNA in a single molecule). In some embodiments, the components of the CRISPR system are introduced into a cell (e.g., a T cell, a NK cell, or a NK cell precursor) in the form of a nucleic acid.

[0169] In some embodiments, the components of the CRISPR system are introduced into a cell (e.g., a T cell, a NK cell, or a NK cell precursor) in the form of DNA encoding a nucleic acid-guided endonuclease and a NATNA guide. In some embodiments, a gene encoding a nucleic acid-guided endonuclease (e.g., a CRISPR nuclease selected from Cas9 and Cas12a) is inserted into a plasmid capable of propagation in a target cell. In some embodiments, a gene encoding a NATNA guide is inserted into a plasmid capable of propagation in a target cell.

[0170] In some embodiments, the nucleic acid-guided endonuclease and the NATNA guide are introduced into a target cell (eg, a T cell, a NK cell, or a NK cell precursor) in the form of RNA (eg, mRNA encoding the nucleic acid-guided endonuclease and the NATNA guide).

[0171] In some embodiments, the nucleic acid-guided endonuclease and NATNA guide are introduced into a target cell (e.g., a T cell, a NK cell, or a NK cell precursor) as a preassembled nucleoprotein complex. In some embodiments, the nucleic acid-guided endonuclease and NATNA guide are introduced into a target cell (e.g., a T cell, a NK cell, or a NK cell precursor) by any combination of different means, such as the endonuclease is introduced as DNA via a plasmid containing a gene encoding the endonuclease, while the guide is introduced in its final format as RNA (or RNA containing DNA nucleotides).

[0172] In some embodiments, nucleic acids encoding nucleic acid-guided endonucleases and NATNA guides are introduced into cells by electroporation.

[0173] In some embodiments, nucleic acid encoding a nucleic acid-guided endonuclease is introduced into cells in the form of mRNA as described, for example, in U.S. Patent No. 10,584,352 by electroporation with viral pseudotransduction as described herein.

[0174] In some embodiments, one or more of the coding sequences described herein are introduced into the genome of the cell with the help of a sequence-specific endonuclease. In some embodiments, the endonuclease is an endonuclease guided by a nucleic acid encoded by the CRISPR locus. The CRISPR (clustered regularly spaced short palindromic repeats) genomic locus is present in many prokaryotic genomes and resists invasion of foreign nucleic acids. In Makarova et al., Evolution and classification of the CRISPR-Cas systems, Nature Reviews: Microbiology (Nature Reviews Microbiology) June 2011; 9 (6): The structure, naming and classification of the CRISPR locus are reviewed in 467-477.

[0175] In short, a typical CRISPR locus includes many short repetitive sequences regularly spaced with spacers. The CRISPR locus also includes the coding sequence of CRISPR-related (Cas) genes. The spacer repeat sequence unit encodes CRISPR RNA (crRNA). In vivo, mature crRNA is processed by a polycistronic transcript called pre-crRNA or pre-crRNA array. The repetitive sequences in the pre-crRNA array are recognized by the protein encoded by Cas, and the protein encoded by Cas binds and cuts the repetitive sequences to release mature crRNA. The CRISPR system performs the cutting of target nucleic acids, wherein Cas protein and crRNA form CRISPR ribonucleoprotein (crRNP). The crRNA molecule guides crRNP to the target nucleic acid (e.g., foreign nucleic acid invading bacterial cells), and the Cas nuclease protein cuts the target nucleic acid.

[0176] Type I CRISPR systems include devices for processing pre-crRNA arrays, including a multiprotein complex called Cascade (CRISPR-associated complex for antiviral defense) comprising subunits CasA, B, C, D, and E. The Cascade-crRNA complex recognizes the target nucleic acid by hybridization of the target nucleic acid with crRNA. The bound nucleoprotein complex recruits Cas3 helicase / nuclease to promote the cutting of the target nucleic acid.

[0177] Type II CRISPR systems include trans-activated CRISPR RNA (tracrRNA). TracrRNA hybridizes with crRNA repeats in the pre-crRNA array and recruits endogenous RNase III to cut pre-crRNA sequences. The tracrRNA / crRNA complex can be combined with a nuclease, such as Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid by hybridization of the target nucleic acid with crRNA. Hybridization of crRNA with the target nucleic acid activates the Cas9 nuclease for target nucleic acid cutting.

[0178] Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre-crRNA array with the help of one or more CRISPR polymerase-like proteins.

[0179] The VI CRISPR system contains a diverse set of Cas-like genes, including Csf1, Csf2, Csf3, and Csf4, which are distant homologs of the Cas genes in type I-III CRISPR systems.

[0180] The V-type CRISPR system is classified into several different subtypes, including, for example, VA, VB, VC, VD, Ve, VF, Vg, VH, VI, VJ, VK and VU. See, for example, Makarova et al., (National Microbiology Review (Nat. Rev. Microbiol.), 2020, 18: 67-83) and Pausch et al., (Science, 2020, 369 (6501): 333-337). The VA subtype encodes Cas12a protein (formerly known as Cpf1). Cas12a has a RuvC-like nuclease domain homologous to the corresponding domain of Cas9 but lacks the HNH nuclease domain present in the Cas9 protein. The V-type system can include a single crRNA sufficient to target Cas12 to a target site, or a crRNA-tracrRNA guide pair for targeting Cas12 to a target site.

[0181] CRISPR endonucleases require a nucleic acid targeting nucleic acid (NATNA), also known as a guide RNA. The endonuclease is capable of forming a ribonucleoprotein complex (RNP) with one or more guide RNAs. In some embodiments, the endonuclease is a type II CRISPR endonuclease, and the NATNA comprises a tracrRNA and a crRNA.

[0182] In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, NATNA may comprise a spacer extension, a spacer, a minimal CRISPR repeat sequence, a single guide connector, a minimal tracrRNA, a 3'tracrRNA sequence, and a tracrRNA extension in a 5' to 3' order. In some cases, the nucleic acid targeting nucleic acid may comprise a tracrRNA extension, a 3'tracrRNA sequence, a minimal tracrRNA, a single guide connector, a minimal CRISPR repeat sequence, a spacer, and a spacer extension in any order.

[0183] In some embodiments, the nucleic acid of the targeting guide nucleic acid may comprise a single guide NATNA. NATNA comprises a spacer sequence that can be engineered to hybridize with a target nucleic acid sequence. NATNA further comprises a CRISPR repeat sequence that comprises a sequence that can hybridize with a tracrRNA sequence. Optionally, NATNA may have a spacer extension and a tracrRNA extension. These elements may include elements that contribute to the stability of NATNA. CRISPR repeat sequences and tracrRNA sequences may interact to form a double-stranded structure of base pairing. The structure may promote the binding of endonucleases to NATNA.

[0184] In some embodiments, the single-guide NATNA comprises a spacer sequence positioned at the 5' position of the first duplex, the first duplex comprising a hybridization region between the minimum CRISPR repeat sequence and the minimum tracrRNA sequence. The first duplex may be interrupted by a protrusion. The protrusion promotes the recruitment of endonucleases to the NATNA. The protrusion may be followed by a first stem comprising a joint connecting the minimum CRISPR repeat sequence and the minimum tracrRNA sequence. The last pair of nucleotides at the 3' end of the first duplex may be connected to a second joint connecting the first duplex to the mid-tracrRNA. The mid-tracrRNA may comprise one or more additional hairpins.

[0185] In some embodiments, the NATNA may comprise a dual-guide nucleic acid structure. The dual-guide NATNA comprises a spacer extension, a spacer, a minimal CRISPR repeat sequence, a minimal tracrRNA sequence, a 3'tracrRNA sequence, and a tracrRNA extension. The dual-guide NATNA does not include a single-guide connector. Instead, the minimal CRISPR repeat sequence comprises a 3'CRISPR repeat sequence, and the minimal tracrRNA sequence comprises a 5'tracrRNA sequence, and the dual-guide NATNA can hybridize through the minimal CRISPR repeat sequence and the minimal tracrRNA sequence.

[0186] In some embodiments, NATNA is an engineered guide RNA (CRISPR hybrid RNA-DNA or chRDNA) comprising one or more DNA residues. In some embodiments, NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. In some embodiments, NATNA is selected from the embodiments described in the following: International Application Publication No. WO 2022086846, DNA-containing polynucleotides and guides for CRISPR V-type systems and methods for their preparation and use. In brief, some chRDNA used with a type II CRISPR system can be composed of two chains forming a secondary structure, the secondary structure comprising an activation region consisting of an upper duplex region, a lower duplex region, a protrusion, a targeting region, a connection, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain different proportions of DNA and RNA. Other chRDNAs may be single guide D (R) NAs used with a type II CRISPR system, the system comprising a targeting region and an activation region, the activation region consisting of a lower duplex region, an upper duplex region, a fusion region, a protrusion, a connection, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region can comprise DNA and RNA in different ratios. For example, the targeting region can comprise DNA or a mixture of DNA and RNA, and the activation region can comprise RNA or a mixture of DNA and RNA.

[0187] In some embodiments, the endonuclease used to introduce one or more of the genetic modifications described herein (e.g., gene inactivation or insertion of a sequence encoding a CAR) into the genome of the cell is a restriction endonuclease, such as a Type II restriction endonuclease.

[0188] In some embodiments, the endonuclease used to introduce one or more of the genetic modifications described herein is a catalytically inactive CRISPR endonuclease (e.g., catalytically inactive Cas9 or Cas12a) conjugated to the cleavage domain of the restriction endonuclease Fok I (see, e.g., Guilinger, JP et al., (2014). Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nature Biotechnology, 32(6), 577-582.

[0189] In some embodiments, the endonuclease endonuclease used to introduce one or more genetic modifications described herein is a zinc finger nuclease (ZFN) or a ZFN-Fok I fusion. In such embodiments, the target sequence is about 22-52 bases in length and comprises a pair of ZFN recognition sequences, each sequence is 9-18 nucleotides in length, separated by a spacer of 4-18 nucleotides in length. (See, e.g., Kim YG et al., (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proc Natl Acad Sci USA. 93(3): 1156–1160.

[0190] In some embodiments, the endonuclease endonuclease used to introduce one or more genetic modifications described herein is a transcription activator-like effector nuclease (TALEN) or a TALEN-Fok I fusion. In such embodiments, the target sequence is about 48-85 nucleotides in length and comprises a pair of ZFN recognition sequences, each sequence is 18-30 bases in length, separated by a spacer of 12-25 bases in length. (See, e.g., Christian M. et al., (2010) Targeting DNA double-strand breaks with TAL effector nucleases, Genetics. 186(2):757-61.

[0191] In some embodiments, quality control measures that assess one or more properties of the engineered anti-ROR1 CAR-T cells and CAR-NK cells are applied to the cells prior to administration of the cells to a patient.

[0192] In some embodiments, the assessed characteristics of CAR-T cells and CAR-NK cells are the presence of CAR nucleic acids in the cell genome. The presence of CAR in the cell genome can be assessed by a method selected from the following: nucleic acid hybridization, nucleic acid sequencing, and specific amplification, including polymerase chain reaction (PCR), quantitative PCR (qPCR), real-time PCR (rtPCR), and droplet digital PCR (ddPCR). In some embodiments, the presence of CAR in the cell genome is assessed by ddPCR with amplification primers specific to one or two CAR insertion sites.

[0193] In some embodiments, the assessed properties of CAR-T cells and CAR-NK cells are surface expression of CAR. The surface expression of CAR can be assessed by fluorescence activated cell sorting (FACS), microfluidic-based screening, ELISA or Western blot. In some embodiments, the surface expression of CAR is assessed by flow cytometry with anti-Fab2 antibodies or labeled purified antigens. In some embodiments, the surface expression of CAR is assessed by flow cytometry with ROR1. In some embodiments, the CAR-T cells or CAR-NK cell populations with the highest surface expression of CAR are selected for administration to patients.

[0194] In some embodiments, the fraction of cells in a cell population that carry a CAR in the genome, or the fraction of cells in a cell population that express a CAR on the cell surface, is used to determine the total number of cells in the cell population that constitutes a therapeutically effective dose.

[0195] In some embodiments, the properties of CAR-T cells and CAR-NK cells are assessed in vitro and are selected from the following: antigen-dependent lysis of target cells expressing the antigen (antigen-specific lysis); proliferation in the presence of target cells expressing the antigen (antigen-dependent proliferation); and cytokine secretion, cell exhaustion, and the presence of a memory cell phenotype in the presence of target cells expressing the antigen.

[0196] In some embodiments, the in vitro evaluation of anti-CAR-T cells and CAR-NK cells utilizes target cells or target cell lines. In some embodiments, the target cells are tumor cells selected from primary tumor cells and established tumor cell lines. In some embodiments, the tumor cells are known to express specific antigens of CAR-T cells or CAR-NK cells, i.e., the tumor cells express ROR1 recognized by anti-ROR1 CAR-T cells and CAR-NK cells. In some embodiments, the tumor cells are from the tumor cell lines SKOV3, REC1, Mino, and JeKo-1. In some embodiments, the target cells are first evaluated for ROR1 expression. Examples of evaluating ROR1 expression in target cells are disclosed in Examples 3 and 4 of the present invention. Figure 4 The presented.

[0197] In some embodiments, the property evaluated is antigen-dependent lysis of target cells carrying an antigen. ROR1-dependent cell lysis can be evaluated by co-culturing a population comprising engineered anti-ROR1 CAR-T cells or CAR-NK cells (effector cells or effectors) with a target cell (target) expressing ROR1. In some embodiments, the target cell is a tumor cell line expressing ROR1. In some embodiments, the tumor cell line expressing ROR1 is selected from JeKo1 (B cell lymphoma, BCL), REC1 (mantle cell lymphoma, MCL) and Mino (MCL). In some embodiments, the target cell is a primary cell from a patient with a tumor known to express ROR1. In some embodiments, the primary cell is from a lymphoma patient, such as a B cell lymphoma patient. The co-culture can be established with different effector: target ratios (E: T ratios). In some embodiments, the E: T ratio is in the range of about 0.1 to about 10. In some embodiments, two or more E: T ratios within a selected range are evaluated. In some embodiments, the target cell is pre-cultured with a cell tracking dye (e.g., CellTrace TM Violet dye (CTV)) to label target cells expressing ROR1 and co-culture with anti-ROR1 CAR-T or CAR-NK effector cells at a range of E:T ratios. At the analysis time point, a dead cell stain (e.g., 7-aminoactinomycin (7AAD) stain) is added to the culture. The number of live target cells is then counted, for example, by flow cytometry as the remaining trace stain / no dead stain (e.g., CTV + 7AAD - ) cells. In some embodiments, the number of live target cells is normalized to the number of live target cells in a control culture lacking anti-ROR1 CAR-T or CAR-NK cells. Figure 5 and 6 Examples of measuring in vitro target cell lysis by anti-ROR1 CAR-NK cells are presented. Figures 27A-27C An example of measuring in vitro target cell lysis by anti-ROR1 CAR-T cells is presented.

[0198] In some embodiments, the property evaluated is repeated antigen-dependent lysis of antigen-bearing target cells in a serial re-challenge assay. Antigen-dependent cell lysis can be evaluated by serially stimulating the same population of engineered anti-ROR1 CAR-T cells or CAR-NK cells with two or more fresh aliquots of target cells. Figure 7 Examples of measuring repeated in vitro target cell lysis by anti-ROR1 CAR-NK cells are presented. Fig.32An example of measuring repeated in vitro target cell lysis by anti-ROR1 CAR-T cells is presented.

[0199] In some embodiments, the CAR-T or CAR-NK cell population that affects the highest percentage of lysis of target cells expressing ROR1 is selected for administration to a patient. In some embodiments, the CAR-T or CAR-NK cell population that affects the highest percentage of lysis of target cells expressing ROR1 but has low non-specific target cell lysis is selected for administration to a patient. In some embodiments, the CAR-T or CAR-NK cell population that affects the highest percentage of lysis of target cells expressing ROR1 repeatedly is selected for administration to a patient.

[0200] In some embodiments, the property evaluated is antigen-dependent proliferation of CAR-T or CAR-NK cells. Proliferation can be evaluated by co-culturing a population comprising engineered anti-ROR1 CAR-T or CAR-NK cells (effectors, E) with target cells expressing ROR1 (targets, T). In some embodiments, the E:T ratio of the co-culture is about 1. In some embodiments, cell proliferation is assessed by staining with a cell-permeable stable fluorescent dye (e.g., CellTrace TM Violet) to label CAR-T or CAR-NK cells and measure the dilution of the dye within the CAR-T or CAR-NK cell population for detection.

[0201] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the highest proliferation rate in the presence of target cells expressing ROR1 is selected for administration to a patient.

[0202] In some embodiments, the evaluated characteristic is a cytokine or chemokine secreted by CAR-T or CAR-NK cells. In some embodiments, the secretion of one or more cytokines or chemokines is evaluated. The one or more cytokines are selected from IFN-γ, TNF-α, GM-CSF, IL-10, IL-5 and IL-13, and chemokines such as MIP-1α, MIP-1β, IL-8 and RANTES. Cytokine or chemokine secretion can be evaluated by co-culturing a population comprising engineered anti-ROR1CAR-T or CAR-NK cells (effector, E) with a target cell (target, T) expressing ROR1. In some embodiments, the E:T ratio of co-culture is about 1. In some embodiments, the cytokine or chemokine in the supernatant of co-culture can be detected or quantitatively detected by the following method: antibody-based or antibody-conjugate-based assays (such as Western blot or ELISA) and similar secondary antibody-based methods and colorimetric methods or fluorescence detection methods are used to detect.

[0203] As in Example 9 and Figures 18A-18B Examples of repeated in vitro target cell lysis (in a serial rechallenge assay) measured by anti-ROR1 CAR-NK cells are presented in Examples 20 and 19A-19B of the present disclosure. Fig.32 An example of measuring repeated in vitro target cell lysis by anti-ROR1 CAR-T cells is presented.

[0204] In some embodiments, a population of CAR-T or CAR-NK cells that exhibits the highest level of cytokine or chemokine secretion in the presence of target cells expressing ROR1 is selected for administration to a patient.

[0205] In some embodiments, the properties of the CAR-T or CAR-NK cells are evaluated in vivo and selected from properties that affect the experimental animal carrying the target tumor cells. In some embodiments, the target cells are tumor cells known to express ROR1, and the experimental animals are mice transplanted with tumor cells before administering a dose of anti-ROR1 CAR-T or CAR-NK cells. In some embodiments, the experimental animals are NGS mice transplanted with JeKo-1 tumor cells. In some embodiments, the evaluation of CAR-T or CAR-NK cells comprises monitoring the weight, overall survival, and tumor burden of mice transplanted with tumor cells and administered a dose of anti-ROR1 CAR-T or CAR-NK cells.

[0206] As in Example 5 and Figure 8 And Example 6 and Fig. 9 An example of measuring the in vivo reduction of tumor burden in animals transplanted with JeKo-1 tumors by administration of anti-ROR1 CAR-NK cells is presented.

[0207] As in Example 16 and Figures 29A-29B An example of measuring the in vivo reduction of tumor burden in animals transplanted with JeKo-1 tumors by administration of anti-ROR1 CAR-T cells is presented.

[0208] In some embodiments, animals are transplanted with fluorescently labeled tumor cell lines, and tumor burden is assessed by measuring in vivo fluorescence (and other mouse measurements). In some embodiments, the experimental animals are immunodeficient NGS mice transplanted with tumor cells expressing JeKo-1-GFPluc luciferase. In some embodiments, the results are expressed as changes in the fluorescence of the tumor (or animal) over time and are assessed as the area under the curve (AUC).

[0209] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the greatest reduction in tumor burden in experimental animals transplanted with tumors expressing ROR1 and injected with anti-ROR1 CAR-T or CAR-NK cells is selected for administration to a patient. In some embodiments, the CAR-T or CAR-NK cell population that exhibits the smallest area under the curve (AUC) is selected for administration to a patient.

[0210] In some embodiments, the property evaluated is the persistence of anti-ROR1 CAR-T or CAR-NK cells in the circulation of an experimental animal transplanted with a tumor expressing ROR1 and injected with anti-ROR1 CAR-T or CAR-NK cells. In some embodiments, the persistence of anti-ROR1 CAR-NK cells is measured by CD56 in the blood of the animal. + In some embodiments, the persistence of anti-ROR1 CAR-T cells is assessed by the presence and / or number of CD8 + In some embodiments, the presence and / or number of anti-ROR1 CAR-NK cells or CAR-T cells in a blood sample is assessed. In some embodiments, the qualitative and / or quantitative assessment of anti-ROR1 CAR-NK cells or CAR-T cells in a blood sample is performed by flow cytometry using an anti-CD56 antibody or an anti-CD8 antibody, respectively. In some embodiments, the antibodies are anti-human CD56 antibodies and anti-human CD8 antibodies.

[0211] As in Example 7 and Fig.10 An example of measuring the in vivo persistence of anti-ROR1 CAR-NK cells in circulation is presented.

[0212] In some embodiments, the population of CAR-T or CAR-NK cells that exhibit the highest persistence in the circulation of experimental animals transplanted with a tumor expressing ROR1 and injected with anti-ROR1 CAR-T or CAR-NK cells is selected for administration to a patient.

[0213] In some embodiments, the evaluated characteristic is the sustained expression of the anti-ROR1 CAR in the CAR-T or CAR-NK cells in the circulation of experimental animals transplanted with tumors expressing ROR1 and injected with anti-ROR1 CAR-T or CAR-NK cells. In some embodiments, the expression of CAR is evaluated by flow cytometry, and the expression is compared with or normalized to the expression of other NK-related genes. In some embodiments, the additional genes are one or more genes selected from CD45 (or hCD45) and CD56.

[0214] As in Example 7 and Fig.11 An example of measuring the expression of anti-ROR1 CAR in CAR-NK cells is presented.

[0215] In some embodiments, a population of CAR-T or CAR-NK cells that exhibits the highest anti-ROR1 CAR expression levels among cells recovered from the circulation of an experimental animal transplanted with a tumor expressing ROR1 and injected with anti-ROR1 CAR-T or CAR-NK cells is selected for administration to a patient.

[0216] In some embodiments, CAR-NK cell clones or populations are selected for inclusion in the therapeutic compositions described herein. The inventors found that, surprisingly, anti-ROR1 CAR-NK cells with similar in vitro tumor cell killing abilities exhibited significant differences in in vivo anti-tumor activity. (See Figure 5-6 , 9-10).

[0217] In some embodiments, the CAR-T cells of the present invention are compared with existing anti-ROR1 CAR-T cells. An example of the most advanced ROR1 targeted therapy is LYL797 (Spigel, D. et al., (2022, September 9-13), a ROR1-targeted CAR T-cell therapy with genetic and epigenetic reprogramming for the treatment of advanced solid tumors, poster presentation at the 2022 ESMO Congress in Paris, France). LYL797 CAR-T cells express CAR, which includes a single-chain variable fragment (scFv) derived from a high-affinity monoclonal antibody R12 that binds to the amino-terminal Ig-like / curled domain of ROR1. (Hudecek et al. (2013) Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells. Clin. Cancer Res. 19:3153).

[0218] In some embodiments, the CAR-T cells of the present invention are superior to T cells expressing benchmark CAR (R12) in terms of surface CAR expression, in vitro antigen-dependent cellular cytotoxicity, in vivo tumor control, and prolonged survival of experimental animals transplanted with tumors expressing ROR1, see Example 26.

[0219] In some embodiments, the present invention comprises a composition comprising an anti-ROR1 CAR-T or CAR-NK cell exhibiting anti-tumor properties. In some embodiments, the present invention comprises a composition comprising both anti-ROR1 CAR-T and CAR-NK cells exhibiting anti-tumor properties. In some embodiments, the present invention comprises a composition comprising an anti-ROR1 CAR-T or CAR-NK cell, which is evaluated for satisfactory properties or satisfactory parameter levels selected from one or more of the following: the presence of CAR in the cell genome, surface expression of CAR, in vitro antigen-dependent cytotoxicity, in vivo anti-tumor activity, in vivo or in vitro antigen-dependent proliferation, and in vivo or in vitro cytokine secretion.

[0220] Once produced and (optionally) evaluated for desired properties, engineered cells can be formulated into compositions for delivery to human subjects to be treated. Compositions include engineered lymphocytes and one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobials, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohol, polyols, glycerol, vegetable oils, phospholipids, and surfactants. Carbohydrates (such as sugars), derived sugars (such as sugar alcohols), aldonic acids, esterified sugars, and / or glycopolymers can exist as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, etc. Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, sodium dihydrogen phosphate, etc. and combinations thereof.

[0221] In some embodiments, the composition further comprises an antimicrobial agent for preventing or stopping the growth of microorganisms. In some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, etc. and combinations thereof.

[0222] In some embodiments, the composition further comprises an antioxidant added to prevent lymphocyte deterioration. In some embodiments, the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium pyrosulfite, etc. and combinations thereof.

[0223] In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates; sorbitan esters; lipids, such as phospholipids (lecithin and other phosphatidylcholines); phosphatidylethanolamine; fatty acids and fatty esters; steroids, such as cholesterol, etc.

[0224] In some embodiments, the composition further comprises a cryogen, such as 3% to 12% dimethyl sulfoxide (DMSO) or 1% to 5% human albumin.

[0225] The number of anti-ROR1 CAR-T and / or CAR-NK cells in the composition will vary depending on many factors but will optimally contain a therapeutically effective dose per vial. The therapeutically effective dose can be experimentally determined by repeated administration of increasing amounts of a composition containing anti-ROR1 CAR-T and / or CAR-NK cells to determine which amount produces a clinically desired endpoint.

[0226] In some embodiments, when the subject is a human, the number of anti-ROR1 CAR-T cells per dose is less than about 2×10 7 cells, and the number of anti-ROR1 CAR-NK cells per dose was less than approximately 2×10 8 In some embodiments, the dose comprises 4×10 7 , 8×10 7 , 1.2×10 8 cells or 1×10 6 -2×10 8 In some embodiments, the dose comprises 4×10 8 , 8×10 8 , 1.2×10 9 cells or 1×10 7 -2×10 9 In some embodiments, the dosage comprises a combination of anti-ROR1 CAR-T cells and anti-ROR1 CAR-NK cells. In some embodiments, the anti-ROR1 CAR-T cells and anti-ROR1 CAR-NK cells in the combination are present at a ratio of about 1:10.

[0227] In some embodiments, the total number of cells in the dosage is adjusted based on the percentage of cells expressing CAR in all cells in the cell composition. In some embodiments, the total number of cells administered is multiplied by 100 / N, where N is the percentage of cells expressing CAR in the cell composition. Multiplication produces the total number of cells that must be administered to the patient in order to administer the desired number of cells expressing CAR.

[0228] In some embodiments, the present invention is a method for treating, preventing, or ameliorating a disease associated with ROR1 expression, the method comprising administering an immune cell population of an anti-ROR1 CAR-T cell, an anti-ROR1 CAR-NK cell, or a combination thereof described herein.

[0229] In some embodiments, the immune cell population administered to the patient has been evaluated for a satisfactory property or a satisfactory parameter level selected from one or more of the following: presence of CAR in the cell genome, surface expression of CAR, in vitro antigen-dependent cellular cytotoxicity, in vivo anti-tumor activity, antigen-dependent proliferation, and cytokine secretion.

[0230] In some embodiments, diseases or conditions that can be treated by the immune cells of the present invention include various malignancies, including solid tumors selected from the following: ovarian cancer, triple-negative breast cancer, colorectal cancer, non-small cell lung cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, melanoma and endometrial cancer or selected from the following hematological tumors: CLL, SLL, B-ALL, B-NHL, MCL and AML. ROR1 is a tumor antigen that is expressed in large quantities in these types of tumors, see Balakrishnan et al., (2017) Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues, Clinical Cancer Research, 23 (12): 3061-3071 and Zhao et al., (2021) Tyrosine kinase ROR1 as a target in cancer therapies, Frontiers in Oncology, 11: 680834.

[0231] In some embodiments, the invention is a method of inhibiting tumor growth in a patient.

[0232] In some embodiments, the present invention comprises a method of administering to a subject or patient a therapeutically effective number of T cells, NK cells, or a combination of T cells and NK cells expressing an anti-ROR1 CAR described herein. In some embodiments, the immune cells are pre-activated and amplified before administration. In some embodiments, administration of immune cells according to the present invention treats, prevents, or improves a disease or condition in a subject or patient. In some embodiments, the disease or condition is selected from cancer or a tumor and an infection that can be treated by administering immune cells that induce an immune response.

[0233] In some embodiments, the administration comprises repeated administration of a therapeutically effective number of T cells, NK cells, or a combination of T cells and NK cells expressing an anti-ROR1 CAR described herein. In some embodiments, repeated administration occurs 1, 2, 3 or more times, with one, two, or three or more days between administrations.

[0234] Pharmaceutical compositions comprising T cells, NK cells, or a combination of T cells and NK cells expressing an anti-ROR1 CAR of the present disclosure can be delivered by various routes and delivery methods, such as local or systemic delivery, including parenteral delivery, intramuscular delivery, intravenous delivery, subcutaneous delivery, or intradermal delivery.

[0235] In some embodiments, the method comprises administering a composition of the invention to a subject that has been pretreated with an immunodepleting (eg, lymphocyte depleting) therapy. In some embodiments, pretreatment is with a lymphocyte depleting agent, including a combination of cyclophosphamide and fludarabine.

[0236] In some embodiments, the composition or formulation for administration to a patient is a pharmaceutical composition or formulation that allows the biological activity of the active ingredient and contains only non-toxic additional components (such as pharmaceutically acceptable carriers). In some embodiments, pharmaceutically acceptable carriers include buffers, excipients, stabilizers, and preservatives.

[0237] In some embodiments, a preservative is used. In some embodiments, the preservative comprises one or more of: methylparaben, propylparaben, sodium benzoate, benzalkonium chloride, antioxidants, chelating agents, parabens, chlorobutanol, phenol, and sorbic acid. In some embodiments, the preservative is present in about 0.0001% to about 2% by weight of the total composition.

[0238] In some embodiments, a carrier is used. In some embodiments, the carrier comprises a buffer, an antioxidant, including ascorbic acid and methionine; a protein, such as serum albumin, gelatin, or immunoglobulin; a hydrophilic polymer, such as polyvinylpyrrolidone; an amino acid, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a carbohydrate, such as a monosaccharide, a disaccharide, glucose, mannose, or dextran; a chelating agent, such as EDTA; a sugar, such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion, such as sodium; a metal complex (e.g., a Zn protein complex); and / or a nonionic surfactant, such as polyethylene glycol (PEG).

[0239] In some embodiments, the carrier comprises a buffer. In some embodiments, the buffer comprises citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, the buffer is present in about 0.001% to about 4% by weight of the total composition.

[0240] In some embodiments, the method comprises administering a pharmaceutical composition comprising a delivery system such that delivery of the composition occurs over time. In such embodiments, the pharmaceutical composition comprises a time-release component. In some embodiments, the pharmaceutical composition comprises aluminum monostearate or gelatin. In some embodiments, the pharmaceutical composition comprises a semipermeable matrix of a solid hydrophobic polymer. In some embodiments, the matrix is ​​in the form of a film or microcapsule.

[0241] In some embodiments, the method comprises administering a pharmaceutical composition comprising a sterile liquid, such as an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which may be buffered to a selected pH. In some embodiments, the pharmaceutical composition is a sterile injectable solution prepared by incorporating cells into a solvent such as sterile water, saline or solution or glucose, dextran, etc. In some embodiments, depending on the route of administration and the desired formulation, the pharmaceutical composition further comprises a dispersant or emulsifier, a pH buffer, a gelling or viscosity enhancing additive, a preservative, a flavoring agent, a coloring agent, etc.

[0242] In some embodiments, T cells or NK cells expressing the anti-ROR1 CAR described herein are co-administered with cytokines. In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18, and IL-21. In some embodiments, the cytokine is administered at a dose per kg of human body weight equivalent to 10 ng / mouse for IL-15, 100,000 units / mouse for IL-2, and 10 μg / mouse for IL-21.

[0243] In some embodiments, the T cells or NK cells expressing the anti-ROR1 CAR described herein are engineered to constitutively express cytokines. In some embodiments, the cytokine is a human cytokine. In some embodiments, the constitutively expressed cytokine is membrane-bound. In some embodiments, the constitutively expressed membrane-bound cytokine is selected from IL-15 (mbIL-15) and IL-21 (mbIL-21). In some embodiments, the constitutively expressed membrane-bound cytokine comprises a fusion of a cytokine and its receptor (cytokine-receptor fusion). In some embodiments, the membrane-bound cytokine-receptor fusion is selected from IL-15-IL-15 receptor fusion (IL-15-IL15RA fusion) and IL-21-IL-21 receptor fusion (IL-21-IL-21RA fusion). In some embodiments, the fusion further comprises a signal peptide. In some embodiments, the leader peptide is selected from IL-2 signal peptide and CD2 signal peptide.

[0244] Using a mouse model, Rowley, J. et al. ((2009) Expression of IL-15RA or an IL-15 / IL-15RA fusion on CD8 T cells modifies adoptively transferred T cell function in cis. Eur. J. Immunol. 39:491) successfully fused IL-15 to IL-15R via a serine-glycine linker, expressed the fusion in T cells, and demonstrated that mice expressing the fusion CD8 + The survival and proliferation of T cells were improved.

[0245] Fusion of human IL-21 with its receptor is described in international application serial number PCT / US23 / 67427, filed on May 24, 2023, “Cytokine-receptor fusions for immune cell stimulation.”

[0246] In some embodiments, the coding sequence of the membrane-bound cytokine is introduced into the cell by chemical or electrochemical means (such as lipid nanoparticles or electroporation). In some embodiments, the coding sequence of the membrane-bound cytokine is introduced into the cell using a vector such as a lentiviral vector.

[0247] In some embodiments, the lentiviral vector includes an expression construct comprising a promoter and a coding sequence of a cytokine and its receptor. In some embodiments, the cytokine and its receptor are connected by a serine-glycine linker. In some embodiments, the promoter is selected from EF1α, PGK1, MND, Ubc, CAG, CaMKIIa, β-actin, SV40 early and late promoters, cytomegalovirus (CMV) immediate early promoter and Rous sarcoma (Rous sarcoma) virus long terminal repeat sequence (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat sequence (MMTV-LTR) promoter, β-interferon promoter and hsp70 promoter. In some embodiments, the promoter is an EF-1α promoter.

[0248] In some embodiments, the lentiviral construct described herein is introduced into NK cells. In some embodiments, the NK cells are primary NK cells. In some embodiments, the NK cells are iNK cells (NK cells differentiated from iPSC in vitro).

[0249] In some embodiments, the lentiviral constructs described herein are introduced into iNK cell precursors, such as iPSCs. In some embodiments, the introduction of the lentiviral constructs described herein and the presence of cytokine receptor fusions do not negatively affect the ability of iPSCs to differentiate into iNK cells.

[0250] In certain embodiments, the surface expression of the membrane-bound cytokine of T cell or NK (or iNK) cell is assessed. The surface expression of the membrane-bound cytokine can be assessed by fluorescence activated cell sorting (FACS), microfluidic-based screening, ELISA or Western blot. In certain embodiments, the surface expression of the membrane-bound cytokine is assessed by flow cytometry with anti-cytokine antibodies. In certain embodiments, the highest T cell or NK (or iNK) cell colony of the surface expression of the membrane-bound cytokine is selected for administration to a patient. In certain embodiments, the highest iPSC cell colony of the surface expression of the membrane-bound cytokine is selected for differentiation into iNK cells.

[0251] In some embodiments, the cytotoxicity of T cells or NK (or iNK) cells expressing membrane-bound cytokines is evaluated. In some embodiments, the cytotoxicity property is evaluated by co-culturing with human tumor cells. In some embodiments, the NK (or iNK) cell colony expressing membrane-bound cytokines with the highest cytotoxic activity is selected for administration to a patient.

[0252] Examples

[0253] Example 1. Anti-ROR1 CAR Constructs

[0254] CAR constructs such as Figure 1 As shown. CAR contains 3 single-chain variable regions (scFv) described in international application serial number PCT / US2023 / 067314, anti-ROR1 antibody and ROR1-targeting engineered cells, filed on May 22, 2023. CAR contains CD8 hinge and transmembrane domains, and the cytoplasmic domain consists of 4-1BB and CD3ζ domains. The expression construct further includes a minimal CAG (mCAG) CMV-derived promoter.

[0255] Example 2. Preparation of iNK cells expressing anti-ROR1 CAR

[0256] In this experiment, according to Figure 2 The protocol shown prepares differentiated (induced) CAR-NK cells (CAR-iNK cells). CAR insertion is performed before the initial differentiation stage.

[0257] CAR insertion

[0258] Briefly, the CAR construct ( Figure 1 ) were introduced into induced pluripotent stem cells (iPSCs) by lentiviral transduction (HIV-1 derived virus, (SignaGen Laboratories, Frederick, Md.) MOI 30) and selection with zeocin. Successful transduction was obtained by sorting (FACS) based on CAR staining and differentiation into iNKs. CAR expression and NK (iNK) differentiation stage were confirmed by co-staining of anti-ROR1 CAR and CD56.

[0259] Differentiating iPSCs into NK cells

[0260] Briefly, to initiate differentiation of iPSC clones, spheres of defined size were generated and seeded onto a surface coated with LN-511. The spheres were grown in a medium containing a specific set of cytokines (BMP4, VEGF, SCF, IL3, IL6, and TPO) to induce differentiation into hematopoietic cells (HPCs). HPC differentiation had two phases (each consisting of a one-week interval) involving different cytokine cocktails in the culture medium.

[0261] Cells were collected from the HPC differentiation stage, the CD34 positive fraction of the enriched cells was transferred to the culture vessel of the first feeder cell line (AFT024-mouse fetal liver stromal cell line) cultured on the surface (inoculated one day in advance). This transfer to the irradiated feeder line #1, combined with a cytokine mixture (IL3, IL15, IL7, SCT, FLT3L), initiated the iNK differentiation stage. The differentiated iNKs were re-plated on a fresh feeder every week. The culture medium changed after 3 days. The iNK differentiation period lasted for four weeks. Cell identification was performed by in-process flow cytometry every week to test the positive rate of CD45 and CD56.

[0262] After 4 weeks in the iNK differentiation stage, the iNK expansion stage lasted for two weeks. iNK cells were co-cultured with an irradiated suspension of a second feeder cell line (engineered to overexpress 41BBL and membrane-bound IL-21 K562 cell line). After 7 days of amplification, the cultured cells were characterized by flow cytometry. At this point, the amplified iNK was cryopreserved. For in vitro and in vivo functional evaluation, the cryopreserved iNK was thawed, and fresh feeders were added to start the second week of amplification. The medium was replaced every 2-3 days. After 2 weeks in the amplification stage, the final amplified iNK product was evaluated in in vitro and in vivo functional assays.

[0263] During the differentiation process, anti-ROR1 CAR expression was measured by flow cytometry and stained with ROR1-Avi tag (ACRO Biosystems, Newark, Del.). Figure 3 Shown using Figure 1 The percentage of cells expressing CAR during each stage of iNK differentiation for cells transduced with each of the indicated CAR constructs.

[0264] Example 3. In vitro anti-ROR1 cytotoxicity.

[0265] In this experiment, the cytotoxicity of the anti-ROR1 iNK cells of Example 2 was tested in vitro against the tumor cell lines SKOV3 expressing ROR1 and JeKo-1 engineered to express GFPluc. The expression of ROR1 in SKOV3 and JeKo-1 was assessed by flow cytometry. Figure 4 shown.

[0266] In vitro cytotoxicity was assessed by co-culturing anti-ROR1 CAR iNK cells with tumor cells at effector:target (E:T) ratios ranging from E:T=0.03:1 to E:T=10:1.

[0267] Results for the SKOV3 target cell line Figure 5 The proportion of live target cells remaining after E:T = 10:1 co-culture is calculated by Live Cell Analysis System (Sartorius, AG, Göttingen, Germany) Germany)) was evaluated as a + Image analysis system for SKOV3 cells.

[0268] JeKo-1-GFPluc target cells were pre-labeled with cell trace violet dye (CTV) and co-cultured with anti-ROR1CAR iNK effector cells at a range of E:T ratios. At the time points analyzed, 7AAD dead cell stain was added to the cultures. Assay analysis was based on flow cytometry analysis of the remaining CTV. + 7AAD - The counts of live target cells were normalized to those of target-only (no effector) wells. Figure 6 shown.

[0269] Example 4. Repetitive cytotoxic activity of anti-ROR1 iNK cells.

[0270] In this experiment, a repeated stimulation ("serial re-stimulation") assay was performed on the JeKo-1 cell line expressing GFP. Briefly, plates seeded with target cells were co-cultured with anti-ROR1 CAR iNK cells. After one to two days, additional target cells were added to the selected plates to test whether the iNK cells were still able to generate a cytotoxic response. The results were Figure 7 Specific lysis of target cells at different E:T ratios is shown in FIG. The three lines in each graph represent the first, second, and third encounters of iNK cells with target cells. The dotted line represents the cytotoxic activity level of control (no CAR) iNK cells when stimulated with target cells for the first time.

[0271] Example 5. Anti-tumor activity of anti-ROR1 CAR-iNK cells in NGS mice

[0272] In this experiment, the in vivo antitumor activity of anti-ROR1 CAR iNK cells was evaluated in NGS mice.

[0273] The assay design is shown in Table 1:

[0274] Table 1. Assay setup for in vivo antitumor activity (NGS mice).

[0275]

[0276] * iNK without CAR or indicating CAR

[0277] ** iNK injection day after tumor transplantation

[0278] In brief, JeKo-1-GFPluc cells were injected intravenously (IV). On the 3rd day after transplantation, effector cells were also injected IV. IL-2 (10,000U / animal) was administered intraperitoneally (IP) to animals on the iNK injection day, and thereafter every 3-4 days until the 21st day. On the iNK injection day, animals also received IP administration of IL-15 (10ng / animal), and thereafter once a day until the 7th day. Before iNK transplantation, the magnetic beads used for CAR enrichment were not removed.

[0279] The results are Figure 8 Shown in Figure 3 are changes in tumor burden for each treatment group measured as the area under the curve (AUC).

[0280] Example 6. Anti-tumor activity of anti-ROR1 CAR-iNK cells in NOG mice expressing human IL-15

[0281] In this experiment, the in vivo antitumor activity of anti-ROR1 CAR iNK cells was evaluated in NOG mice expressing transgenic human IL-15. The assay design is shown in Table 2.

[0282] Table 2. Assay setup for in vivo antitumor activity (IL-15 mice).

[0283]

[0284] * iNK without CAR or indicating CAR

[0285] ** iNK injection day after tumor transplantation

[0286] Mice engineered to express transgenic human IL-15 (hIL-15) were used. In short, JeKo-1-GFPluc cells were injected intravenously (IV). On the 3rd day after transplantation, effector cells were also injected IV. Animals were given IL-2 (10,000U / animal) intraperitoneally (IP) on the iNK injection day, and every 3-4 days thereafter until the 21st day, except for Group 6, which did not receive IL2. Unlike Example 5, exogenous IL15 was not used. Unlike Example 5, the magnetic beads used for CAR enrichment were removed before iNK transplantation.

[0287] Example 7. Persistence of circulating anti-ROR1 CAR-iNK cells in vivo.

[0288] In this example, the characteristics of iNK cells were evaluated in treated NOG-hIL15 animals. Blood samples were collected on day 7 after transplantation and iNKs were quantified by flow cytometry by co-staining with CD45, CD56 and using counting beads. The results are shown in Fig.10 Middle. iNK cell numbers were normalized to the volume of blood collected.

[0289] The cells were further evaluated for sustained expression of anti-ROR1 CAR. Surface expression of human CD45, human CD56, GFP, and anti-ROR1 CAR was detected by flow cytometry. The results are shown in Fig.11 middle.

[0290] Example 8. CBLB-deficient anti-ROR1 iNK cells

[0291] In this example, CRISPR Cas12a and a guide RNA containing DNA (chRDNA) were used to disrupt the CBLB gene in an iPSC cell line, essentially as described in International Application Publication No. WO 2022086846, DNA-containing polynucleotides and guides for CRISPR V-type systems and methods for their preparation and use. ChRDNA is designed to target the CBLB locus for cleavage with Cas12a, and subsequent DNA repair, resulting in disruption of the gene sequence and gene silencing ("knockout").

[0292] Next, CBLB-deficient iPSCs were differentiated into iNKs essentially as described in Example 2.

[0293] The resulting CBLB KO iNK cells were evaluated for perforin and granzyme B secretion phenotype by commercial immunoassays. Fig.12 The phenotype was further assessed by flow cytometry by measuring the expression levels of 11 NK cell markers relative to CD45 and CD56. Fig.13 ).

[0294] The cytotoxicity of CBLB KO iNKs against SKOV3 ovarian tumor target cells was further evaluated in in vitro co-culture, essentially as described in Example 3. The cytotoxic effect of CBLB KO iNKs is shown in Fig.14 The degranulation of iNKs from CBLB KOiNK-SKOV3 co-cultures was also assessed (measured as the release of CD107a). The results are shown in Fig.15 middle.

[0295] Still further, the in vivo antitumor activity of CBLB KO iNK was evaluated in SVOV3 tumor-implanted NSG mice, essentially as described in Example 5, and the results are shown in Fig.16 middle.

[0296] Example 9. CBLB-deficient anti-ROR1 iNK cells and CISH-deficient anti-ROR1 iNK cells

[0297] The destruction of the CBLB gene is described in Example 8. In this example, in a separate iPSC cell line, CRISPR Cas12a chRDNA is used to destroy the CISH gene, essentially as described in WO 2022086846. ChRDNA is designed to target the CISH locus for cutting with Cas12a, and then DNA repair, resulting in the destruction of the gene sequence and gene silencing ("knockout"). CBLB-deficient iPSCs and CISH-deficient iPSCs are differentiated into iNKs essentially as described in Example 2.

[0298] In vitro activities of CBLB-deficient iNK and CISH-deficient iNK

[0299] The resulting CBLB-deficient iNKs and the resulting CISH-deficient iNKs were evaluated for cytotoxicity against SKOV3 cells in co-culture, essentially as described in Example 3. CBLB KO iNKs or CISH KO iNKs were co-cultured with the SKOV3-GFPluc tumor cell line at an E:T ratio of 10:1. + SKOV3 target cells minus GFP + YO - PRO3 + Imaging of labeled dying target cells counts the number of remaining live SKOV3 cells at time zero relative to live SKOV3 GFP + YO - PRO3 - The counts are normalized. Fig.17 shown.

[0300] Effector molecule secretion by CBLB-deficient iNKs and CISH-deficient iNKs was assessed with or without co-culture with SKOV3 ovarian tumor target cells. IFNγ, TNFα, perforin, and granzyme B were measured after 24 h of culture of CISH KO iNKs or CBLB KO iNKs, or as a control, after primary NKs (pNKs) or wild-type iNKs co-cultured with or without SKOV3 target cells at a 1:1 E:T ratio. Effector molecules were measured using the ProcartaPlex immunoassay on a Luminex instrument (ThermoFisher Scientific, Waltham, Mass.). The results are shown in Figures 18 and 19.

[0301] In vivo antitumor activities of CBLB KO iNK and CISH KO iNK.

[0302] In this experiment, 3x 10 5 SKOV3-GFPluc tumor cells were injected intraperitoneally (IP) in NSG mice. On day -1, the tumors were visualized by fluorescence imaging (PerkinElmer, Waltham, MA). Animals with tumors were randomly assigned to different treatment groups. On day 0, 2x 10 7The iNKs (CBLB KO iNK and CISH KO iNK) were injected IP. The animals were imaged twice a week thereafter until day 45 to collect bioluminescence data. The area under the curve (AUC) analysis of bioluminescence intensity is shown in Fig. 20 AUC analysis covers only the time when all vehicle control animals survived. P value annotation * >0.05. The survival probability of vehicle and iNK-treated groups is as follows Fig.21 shown.

[0303] Example 10. Immune cloaking of anti-ROR1 iNK cells

[0304] In this example, the genome of iNK cells was edited by inserting a B2M-HLA-E fusion construct into the B2M locus. In control iNK cells, the B2M locus was destroyed by CRISPR Cas12a cleavage, but no construct was inserted ("B2MKO iNK").

[0305] The design and insertion of the B2M-HLA-E fusion construct into the B2M locus was performed essentially as described in WO 2022086846. In short, the fusion nucleic acid construct is encoded in the NC orientation, i.e., the N-terminal B2M secretion signal, the HLA-G derived peptide sequence, the first linker sequence, the B2M sequence, the second linker sequence, and the HLA-E sequence. The nucleic acid construct further contains the EF1α mammalian promoter sequence and the C-terminal BGH polyadenylation signal sequence.

[0306] The resulting edited iNK cells were tested for their cytotoxic properties and survival in co-culture with allogeneic T cells. CD8 + T cells. T cells were co-cultured with wild-type iNK, B2MKO iNK and B2M-HLA-E iNK at an effector: target (E:T) ratio of 4:1 for 6 days. In positive control cultures, T cells were stimulated by PMA / ionomycin. On day 6, T cells in all cultures were counted by CD8 staining and counting beads. The results are shown in Fig. 22 ("T cell count"). Also on day 6, iNK cells were counted in iNK-containing cultures by CD56 staining and counting beads. The results are shown in Fig. 22 (“NK cell count”). CD56 of iNK cells was also measured + CD8 in the population + The proportion of the group. Fig.23 ).

[0307] Example 11. Cytokine expression of anti-ROR1 iNK

[0308] This example describes iNKs engineered to constitutively express the membrane-bound cytokine IL-15 (mbIL-15).

[0309] a. Generation of iNK cells expressing mbIL-15

[0310] Lentiviral constructs were designed to transduce iPSCs and differentiate engineered iPSCs into iNK cells. The design of the lentiviral construct for expressing membrane-bound IL-15 (mbIL-15) is shown in Fig.24 The expression construct inserted into the lentiviral vector contains the EF-1α promoter, and the coding sequences of IL-15 and IL-15 receptor (IL-15RA) connected by a serine-glycine linker. The fusion also contains a CD2 signal sequence. As described in Example 2, iPSCs are transduced with lentiviral constructs. iPSCs are then selected to use a cell sorter (SH800, Sony Biotechnology, San Jose, Cal.), which uses an anti-IL-15 primary antibody and an anti-mouse AlexaFluor 488-conjugated secondary antibody to sort IL-15 positive cells.

[0311] Once selected, iPSC clones expressing mbIL-15 were differentiated into iNK cells following a three-stage differentiation protocol: a two-week stage to differentiate iPSCs into HPCs, a two-week stage to differentiate HPCs into cells that have already targeted CD34 + A four-week phase of positive selection of iNKs was performed, and a two-week expansion phase.

[0312] b. Cytotoxicity of iNK cells expressing mbIL-15

[0313] The cytotoxic properties of the engineered iNKs were tested in co-culture with SKOV-3 ovarian tumor cells expressing a firefly luciferase-GFP fusion protein as a target. Control co-cultures included wild-type iNKs. Cytotoxicity assays were performed in The co-cultures were performed on a live cell analysis system (Sartorius, Göttingen, Germany). The E:T ratios of the co-cultures were 10:1 and 3:1 and were imaged every 2 hours. The co-cultures included YO-PRO-3 vital dye (Thermo Fisher Scientific, Waltham, MA). The percentage of live SKOV-3 cells was calculated as an overlay of the percentage of total SKOV-3 cells from the start of the assay. The results are shown in Fig.25 middle.

[0314] Example 12. Optimization of anti-ROR1 scFv by varying linker length

[0315] In this experiment, a previously disclosed humanized single-chain variable fragment (scFv) (International Application Serial No. PCT / US2023 / 067314, filed on May 22, 2023, Anti-ROR1 Antibodies and Engineered Cells Targeting ROR1) was optimized by varying the length of the linker between the light chain and the heavy chain of the scFv. The amino acid sequence of scFv 862 disclosed herein as SEQ ID NO: 12 contains a linker consisting of four repeats of the glycine-serine linker G4S (G4S) 4. Variants of this sequence contain a single copy of the linker sequence (G4S) 1. The design of a CAR containing two scFvs is as follows Fig.26 As shown. CAR contains a CD8A-derived signal peptide (sp), hinge (h) and transmembrane domain (tm), and the cytoplasmic domain of CAR consists of 4-1BB and CD3ζ domains. The CAR expression construct includes the EF1α promoter. The original designed CAR is present in the plasmid construct pCB7306, and the short linker CAR is present in the plasmid pCB7339.

[0316] Example 13. Insertion of anti-ROR1 CAR into T cells

[0317] In this experiment, Fig.26 The CAR design shown in is introduced into T cells to be inserted into the TRAC locus. The TRAC locus is targeted using CRISPR Cas12a and CRISPR hybrid RNA-DNA guide (chRDNA), substantially as described below: International Application Publication WO 2022086846, DNA-containing polynucleotides and guides for CRISPR V-type systems and methods for their preparation and use. In order to target the TRAC locus, the exons were analyzed for the presence of PAM sequences suitable for V-type amino acid cocci (Acidaminococcus spp.) Cas12a. The target site is selected to be in the TRAC locus on human chromosome 14, between nucleotides 22547529 and 22547552. The segment of about 20 nucleotides 3' of the PAM sequence is used to design the targeting region of the Cas12a guide.

[0318] To design the inserted donor sequence, homology arms 5' and 3' with a length of 500 bp of the cleavage site were identified. The 5' and 3' homology arms were attached to the ends of the DNA donor (Example 12) containing the CAR in the opposite orientation (i.e., 3' to 5') relative to the homology arms. The nucleotide sequence of the DNA donor polynucleotide was provided to a commercial manufacturer for synthesis of a suitable recombinant AAV (rAAV) plasmid and packaging into AAV6 virus.

[0319] Transduction of primary T cells with rAAV

[0320] Primary activated T cells were obtained from healthy donor PBMCs and transfected with rAAV6 essentially as described in WO 2022086846. Briefly, T cells were electroporated with Cas12a chRDNA guide nucleoprotein complex targeting TRAC and transfected with rAAV6 virus packaged with CAR donor sequences at 4 x 10 5 The cells were infected with an MOI of 100 units / mL. After transduction, T cells were cultured for 24 hours in ImmunoCult-XF complete medium (STEMCELL Technologies, Cambridge, Mass.) supplemented with IL-2 (100 units / mL). The next day, the transduced T cells were transferred to a 50 mL conical tube and centrifuged at 300 x g for about 7-10 minutes to precipitate the cells. The supernatant was discarded, and the precipitate was gently resuspended, and the T cells were combined in an appropriate volume of ImmunoCult XF complete medium supplemented with IL-2 (100 units / mL). The enumerated T cells were cultured at 1 x 10 6 Cells / mL were resuspended in complete ImmunoCult-XF medium supplemented with IL-2 (100 units / mL) and plated into as many T-175 suspension flasks as needed (maximum volume per flask was 125 mL). Anti-ROR1 CAR expression was assessed by flow cytometry staining with PE-labeled human ROR1 protein His Tag (site-specific conjugation) (Biopsy Bio, Newark, Delaware).

[0321] Example 14. In vitro anti-ROR1 cytotoxicity of anti-ROR1 CAR-T cells.

[0322] In this experiment, the anti-ROR1 CAR-T cells of Example 13 were tested in vitro for cytotoxicity against target tumor cell lines JeKo-1, REC1 and Mino expressing ROR1, as well as primary B-cell chronic lymphocytic leukemia (B-CLL) cells (donor PBMC fraction). The TRAC locus of the control effector T cell population was disrupted, but there was no CAR insertion ("TRAC KO").

[0323] In vitro cytotoxicity was assessed by co-culturing anti-ROR1 CAR-T cells with tumor cells at effector:target (E:T) ratios ranging from E:T=0.1:1 to E:T=10:1. Target cells were pre-labeled with cell trace violet dye (CTV) and co-cultured with anti-ROR1 CAR-T effector cells for 48 h. At the analysis time points, propidium iodide (PI) dead cell stain was added to the cultures. Assay analysis was based on flow cytometry analysis of the remaining CTV. + PI - The counts of live target cells were normalized to the target-only (E:T = 0:1) wells. Fig.27A (Jeko-1 and REC1), Fig.27B (Mino and B-CLL, donor 1) and Fig.27C (B-LLL, Donor 2) Shown as percentage of specific cell lysis.

[0324] Example 15. In vitro cytokine secretion by anti-ROR1 CAR-T cells

[0325] In this experiment, cytokine secretion of co-cultures of CAR-T cells described in Example 14 and targets expressing ROR1 was evaluated. At the 24-hour time point, secretion of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), granzyme A, granzyme B, perforin, IL-2, IL-4, and IL-6 was measured by collecting supernatants of co-cultures (1:1 E:T ratio). Luminex-based multiplex assays were used to quantify secreted cytokine levels. Results are shown in Fig.28A (IFNγ and TNFα), Fig.28B (Granzyme A and Granzyme B), Fig.28C (IL-2 and perforin) and Fig.28D Shown are the concentrations of cytokines (IL-4 and IL-6) in the co-culture supernatants.

[0326] Example 16. Anti-tumor activity of anti-ROR1 CAR-T cells in NGS mice

[0327] In this experiment, the in vivo anti-tumor activity of anti-ROR1 CAR-T cells (Example 13) was evaluated in NGS mice. The assay design is shown in Table 1.

[0328] Table 1. In vivo antitumor activity (NSG mice) assay setup.

[0329]

[0330] Briefly, JeKo-1-GFPluc cells were injected intravenously (IV). On day 3 after transplantation, effector cells were also injected IV.

[0331] The in vivo anti-tumor activity of anti-ROR1 CAR-T cells was measured in the form of body weight change, survival rate, and tumor burden change, which was measured in the form of bioluminescence intensity and the area under the curve (AUC) of the bioluminescence intensity of each treatment group. The results are shown in Fig.29A (changes in body weight and probability of survival) and Fig.29B (Tumor burden measured as bioluminescence and AUC measurements of bioluminescence (BLI). Fig.29A : Kaplan-Meier survival curve comparisons were analyzed using the log-rank Mantel-Cox test. Vehicle control vs. pCB7306 p=0.0002, vehicle control vs. pCB7339 p=0.0002. Statistical differences between mouse body weights on day 28 were determined using the Kruskal-Wallis test, followed by multiple comparisons using the Dunn's post hoc test. Vehicle control vs. pCB7306 p=0.0218 ( *** ), vehicle control vs. pCB7339 p=0.0006 ( ** ). Fig.29B : To compare BLI for each treatment group from day 0 to day 28, the Kruskal-Wallis test was applied to AUC followed by Dunn's post hoc test. Vehicle control vs. pCB7306 p=0.0004, vehicle control vs. pCB7339 p=0.0042.

[0332] Example 17. Checkpoint-deficient anti-ROR1 CAR-T cells

[0333] In this experiment, immune checkpoint genes PDCD1, TIGIT, TIM3 and LAG3 were destroyed in the CAR-T cells of Example 13. Gene inactivation using CRISPR Cas12a and CRISPR hybrid RNA-DNA guide (chRDNA) is substantially as described below: International Application Publication WO 2022086846, DNA-containing polynucleotides and guides for CRISPR V-type systems and methods for their preparation and use. In short, CRISPR hybrid RNA-DNA guide (chRDNA) is designed to target loci for cutting with Cas12a, and then DNA repair is performed, resulting in the destruction of gene sequences and gene silencing ("knockout"). In order to target each gene, the exon was analyzed for the presence of a PAM sequence suitable for V-type amino acid coccus (Acidaminococcus spp.) Cas12a. The segment of about 20 nucleotides 3' of the PAM sequence is used to design the targeting region of the Cas12a guide for each gene.

[0334] For targeting the PDCD1 gene, the target site was selected to be in the PDCD1 locus on human chromosome 2, between nucleotides 241852860 and 241852883. For targeting the TIGIT gene, the target site was selected to be in the TIGIT locus on human chromosome 3, between nucleotides 114299581 and 114299604. For targeting the TIM3 gene, the target site was selected to be in the TIM3 locus on human chromosome 5, between nucleotides 157104679 and 157104702. For targeting the LAG3 gene, the target site was selected to be in the LAG3 locus on human chromosome 12, between nucleotides 6773330 and 6773353.

[0335] The Cas12a chRDNA guide nucleoprotein complex of the targeted gene is formed and introduced into anti-ROR1 CAR-T cells (Example 13), substantially as described in WO 2022086846, to produce gene knockout. The resulting cell colonies are named anti-ROR1 CAR+PD1 KO, anti-ROR1 CAR+TIM3 KO, anti-ROR1 CAR+LAG3 KO and anti-ROR1 CAR+TIGITKO. Cell colonies with multiple checkpoint inactivations are anti-ROR1CAR+PD1 KO+TIM3 KO, anti-ROR1 CAR+PD1 KO+LAG3 KO, anti-ROR1 CAR+PD1KO+TIGIT KO and anti-ROR1 CAR+TIM3 KO+LAG3 KO.

[0336] Example 18. In vitro cytotoxicity of checkpoint-deficient anti-ROR1 CAR-T cells

[0337] In this experiment, the checkpoint-deficient anti-ROR1 CAR-T cells of Example 17 were tested for in vitro cytotoxicity against the target JeKo-1 cell line expressing ROR1 and primary B-CLL donor cells, essentially as described in Example 14. For single checkpoint inactivation, the results are shown in Fig. 30A (JeKo-1 cells) and Fig. 30B (primary B-CLL cells). For dual checkpoint inactivation, the results are shown in Fig.33 (JeKo-1 cells and primary B-CLL cells).

[0338] Example 19. In vitro cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells

[0339] In this experiment, cytokine secretion of co-cultures of checkpoint-deficient anti-ROR1 CAR-T cells and targets expressing ROR1 described in Example 18 was evaluated. At the 24-hour time point, secretion of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), granzyme A, granzyme B, perforin, IL-2, IL-4, and IL-6 was measured by collecting the supernatant of the co-cultures. The levels of secreted cytokines were quantified using a Luminex-based multiplex assay. The results are shown in Fig.31A (IL-2 and IL-4), Fig.31B (IL-6 and IFNγ), Fig.31C (TNFα and perforin), Fig.31D (Granzyme A and Granzyme B).

[0340] Example 20. Serial re-stimulation of checkpoint-deficient anti-ROR1 CAR-T cells with ROR1-expressing tumor cells.

[0341] In this experiment, the in vitro cytotoxicity of the checkpoint-deficient anti-ROR1 CAR-T cells of Example 17 was evaluated in serial re-stimulation with the target cell line JeKo-1 expressing ROR1. Target cells engineered to express luciferase were added to effector cells at increasing E:T ratios ranging from 0:1 to 2:1. 15,000 fresh target cells were added to each culture every 3-4 days (up to 6 re-stimulations), and the number of viable cells was assessed by luminescence readings. Cytotoxicity (cell lysis) was measured after 1-6 stimulations with target cells expressing ROR1 and used to calculate the area under the curve AUC. The results are shown in Fig.32 shown.

[0342] Example 21. Alternative anti-ROR1 CAR designs.

[0343] Table 3. List of CAR constructs

[0344]

[0345] abbreviation:

[0346] <![CDATA[CD3ζ * ]]> CD3ζ binds to STAT3 with YRHQ motif Prom Promoter S Signal peptide scFv scFv PMC862 (SEQ ID NO:9) H Hinge domain TM Transmembrane domain CS1 Costimulatory domain 1 CS2 Costimulatory domain 2 SD Signaling domain IL36SP IL36 signal peptide IL2SP IL2 signal peptide IL2mutSP IL2 signal peptide with modifications in the basic and hydrophobic domains (SEQ ID NO: 48)

[0347] Example 22. Antigen-dependent cellular cytotoxicity of CAR-T cells designed as alternatives to the anti-ROR1 CAR against targets expressing ROR1.

[0348] In this example, the ROR1-specific cytotoxicity of the CAR-T cells of Example 21 was tested in co-culture with the ROR1-positive cell line JeKo1 and ROR1-positive primary B cells from patients with B-CLL. ROR1 positivity was determined by staining cells with phycoerythrin (PE)-conjugated anti-ROR1 antibody (BioLegend, San Diego, Cal., clone 2A2) and using BD PEQuantibrite according to the manufacturer's instructions. TM The amount of anti-ROR1 antibody bound to the beads per cell is quantified, and therefore the number of ROR1 molecules per cell is determined.

[0349] Using Cell Trace Violet TM (CTV) staining to mark target cells. Anti-ROR1 CAR-T and CTV-labeled target cells were co-cultured in triplicate for 48 hours at different effector: target (E: T) ratios. After 48 hours, propidium iodide (PI) dead cell staining was added to each well, and PI uptake was assessed by flow cytometry to quantify target cell death. The live target cell count was extracted, and the specific lysis relative to the 0: 1E: T control was calculated using the following formula:

[0350]

[0351] The area under the curve (AUC) for each effector was calculated to represent the overall specific cleavage for each ROR1-expressing target. The results are shown in Fig.34 (pCB7432, pCB7437 or pCB7438 with JeKo-1 or with B-CLL), Fig.35A (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, or pCB7436 with K562 or with B-CLL) and Fig.35B(pCB7429, pCB7430, pCB7431, pCB7433, pCB7434 or pCB7436 with JeKo-1).

[0352] Example 23. Cytokine secretion by CAR-T cells designed with alternative anti-ROR1 CARs in the presence of a target expressing ROR1.

[0353] In this example, cytokine secretion of the CAR-T cells of Example 21 in the presence of target cells expressing ROR1 was evaluated. TRAC KO T cells were used as a control. Co-culture with the ROR1 negative cell line K562 was used as a control. In addition, effector-only cultures were used to measure background cytokine secretion.

[0354] Effector and target cells were co-cultured at a 1:1 ratio for 24 hours. TM The supernatants were collected and analyzed for the presence of cytokines IFNγ, TNFα, IL-2, IL-4, IL-6, perforin, granzyme A, and granzyme B using the ELISA kit and manufacturer's protocol (Life Technologies, Waltham, Mass.). The results are shown in Fig.36A (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434 or pCB7436 secrete IL-2, IL-4, TNFα or perforin), Fig.36B (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434 or pCB7436 secrete IL-6, IFNγ, granzyme A or granzyme B), Fig.37A (pCB7432, pCB7437 or pCB7438 secrete IL-2, IL-4, TNFα or perforin) and Fig.37B (pCB7432, pCB7437 or pCB7438 secrete IL-6, IFNγ, granzyme A or granzyme B).

[0355] Example 24. IL-36γ secretion by CAR-T cells of alternative anti-ROR1 CAR design.

[0356] In this example, the expression of IL-36γ by anti-ROR1 CAR-T cells (pCB7306 ( Fig.26), pCB7436, pCB7437 and pCB7438, Example 21, Table 3) and negative T cell controls. Target JeKo-1 cells, ROR1 positive and engineered to express luciferase, were co-cultured with effector cells at increasing E:T ratios ranging from 0:1 to 2:1. Supernatants at a 2:1 E:T ratio were collected after each round before evaluating cytotoxicity (see Example 22). The supernatants from rounds 1 and 5 were assayed for IL-36γ concentration. The results are shown in Fig.38 shown.

[0357] Example 25. In vivo anti-tumor activity of CAR-T cells with alternative anti-ROR1 CAR designs.

[0358] 5x 10 5 JeKo-1-GFPluc tumor cells were injected intravenously (IV) into NGS female mice. On day 7 after transplantation, CAR-T cells (Example 21) were also injected at 1x 10 7 CAR + T cells were injected IV. Body weight and IVIS measurements were performed twice a week, and the anti-tumor activity of mice was evaluated by tracking changes in body weight, survival rate, and changes in tumor burden measured in the form of bioluminescence intensity. The results are shown in Fig.39A (weight and survival probability) and Fig.39B (Bioluminescence expressed in photons and AUC).

[0359] Example 26. Comparison of novel anti-ROR1 CAR-T cells with benchmark anti-ROR1 CAR-T cells.

[0360] In this example, Fig.26 The CAR construct (pCB7306) shown was compared to the same CAR, except that the scFv was replaced with the R12 scFv in the following publication: Hudecek et al., (2013) Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells, Clin. Cancer Res. 19: 3153. CAR-T cells expressing one of the two CARs were prepared as described in Example 13.

[0361] The resulting CAR-T cells expressing scFv PMC862 (present invention) or scFv R12 (Hudecek et al., supra) were tested in parallel to compare the anti-tumor properties in vitro and in vivo.

[0362] In vitro serial rechallenge with JeKo1 tumor cells

[0363] 24 hours before the first stimulation, CAR-T cells "PMC862" and "R12" and TRAC KO control T cells were thawed and recovered in IL-2-containing medium. Pre-stimulation (round zero, R0) CAR expression was assessed using PE-conjugated human recombinant ROR1 protein. Comparable surface CAR expression was observed between PMC862 and R12 CAR-T cells.

[0364] T cells were then plated with Jeko1-GFP:ffLuc target cells at different effector:target (E:T) ratios. Specific lysis of target cells was quantified by luciferase-based luminescence readout every 72-96 hours. CAR-T cell phenotype analysis was also performed by flow cytometry. BD Countbright TM Estimation of CAR by absolute bead counting + T cell counts. After each round of analyte measurement, the remaining CAR-T cells were re-stimulated with fresh JeKo1-GFP:ffLuc target cells. Serial re-stimulations were continued until one or two anti-ROR1 CAR-T cells were observed to lose specific lysis. Fig.40 and Fig.41 shown.

[0365] PMC862 CAR-T cells maintained high CAR expression through 5 rounds of re-challenge, whereas benchmark R12 CAR-T cells began to lose CAR expression at round 3, with undetectable expression at round 5. This loss in R12 CAR-T cells was associated with a complete loss of specific lysis at round 5 ( Fig.40 ). Loss of CAR expression in R12 CAR-T cells correlates with loss of antigen-specific target cell lysis ( Fig.41 ).

[0366] In vivo antitumor activity in NGS mice.

[0367] JeKo-1-GFP:ffLuc target cells were plated in PBS at a volume of 5 x 10 5 Seven days after transplantation, 10 7 PMC862 and R12 anti-ROR1 CAR were administered intravenously in X-Vivo serum-free medium. +T cells. Vehicle only (X-Vivo medium) was used as a negative control. The CAR positivity rates of PMC862 and R12 CAR-T cells were 89.6% and 88.4%, respectively.

[0368] Starting from the 5th day after CAR-T infusion, use Animals were imaged twice weekly using the Spectrum In Vivo Imaging System for bioluminescence imaging (BLI). The average BLI and area under the curve (AUC) were calculated from day 0 to day 22 using individual BLI measurements (n = 8 mice / group) ( Fig.42 ). The Mann-Whitney test comparing the AUC of PMC862 (CB-013CAR) and R12CAR showed that PMC862 CAR-T cells have superior tumor control ability ( *** indicates p<0.001).

[0369] Animals were euthanized after a weight loss of >20% compared to day 0 or after paralysis. Kaplan-Meier survival curves (Mantel-Cox log-rank test) were plotted for any time ( Fig.43 ). The median survival of the vehicle-treated group was 24 days, the median survival of the R12 CAR-T-treated group was 28 days, and the median survival of the PMC862 CAR-T-treated group was 39.5 days.

[0370] Example 27. In vivo anti-tumor activity of anti-ROR1 CAR-iNK cells with multiple genomic modifications

[0371] The in vivo experiment was designed basically as in Example 25. 5 SKOV3-GFPluc tumor cells were injected intraperitoneally (IP) into NGS female mice. On day 7 after transplantation, 2x 10 7 CAR-iNK cells.

[0372] CAR-iNK cells contain CAR from pCB7306 ( Fig.26 ), disrupted CBLB gene (Example 8), and B2M-HLA-E fusion peptide in the B2M locus (Example 10). CAR-iNK cells also express IL15 receptor fusion (Example 11). Unmodified iNK cells were used as controls.

[0373] Mice were measured by IVIS during 2-13 days after treatment, and changes in tumor burden were assessed based on bioluminescence intensity. The results are shown in Fig.44 (mean bioluminescence, BLI) and Fig.45 (Area under the curve, AUC, **Indicates p<0.01).

[0374] Example 28. Continuous re-stimulation of anti-ROR1 iNK cells with tumor cells

[0375] The experimental design was essentially the same as in Example 20. iNK cells were stimulated with tumor cells six times in succession. Two CAR iNK cells were tested: pCB7306 expressing Fig.26 , and pCB7447 expresses the same CAR except that an additional CD27 costimulatory domain is inserted between the CD3ζ domain and the 4-1BB domain (see Fig.26 ). Both types of CAR-iNK cells include the expression of the disrupted CBLB gene, the B2M-HLA-E fusion peptide in the B2M locus, and the IL15 receptor fusion (Example 27). The control iNK cells include the disrupted TRAC gene, and like the CAR iNK cells, also include the expression of the disrupted CBLB gene, the B2M-HLA-E fusion peptide in the B2M locus, and the IL15 receptor fusion described in Example 27, except that the control iNK cells lack CAR. Unmodified iNK cells and iNK cells expressing IL15 receptor fusions were used as two other controls.

[0376] Two types of target cells were used: SKOV3 is an ovarian tumor cell line with low ROR1 antigen density, and Hs764t is a gastric cancer cell line with high ROR1 antigen density.

[0377] Target cells engineered to express luciferase were added to effector cells at a 1:1 E:T ratio. Every 42 hours, 5,000 fresh target cells were added to each culture for an additional 5 times (6 excitations total). The proportion of remaining viable tumor cells was assessed by luminescence readings. The results are shown in Fig.46 (SKOV3 target) and Fig.47 (Hs746t target).

[0378] Example 29. Multiple Doses of Anti-ROR1 CAR-iNK Cells with Multiple Genomic Modifications

[0379] This in vivo experiment was designed essentially as in Example 27. The two CAR-iNK designs were identical to Example 28: the first CAR-iNK had CAR design pCB7306, and the second CAR-iNK had CAR-iNK design pCB7447, both sets of iNKs had disrupted CBLB genes, B2M-HLA-E fusion peptides in the B2M locus, and expressed IL15 receptor fusions (see Example 28). Unedited ("WT") iNKs were used as controls.

[0380] Two types of target tumor cell lines were used: SKOV3 is an ovarian tumor cell line with low ROR1 antigen density, and Hs764t is a gastric cancer cell line with high ROR1 antigen density.

[0381] 3x 10 5 SKOV3-GFPluc or 10 5 Hs746t-GFPluc tumor cells were injected intraperitoneally (IP) into NSG female mice (8 mice per treatment group, except for 7 mice in the Hs746t pCB7447 treatment group and 6 mice in the Hs746t pCB7306 treatment group). On day 7 after transplantation, 2 x 10 7 CAR-iNK cells. The second injection of 2x 10 7 CAR-iNK, and the third injection of 2x 10 7 A CARiNK.

[0382] Mice were measured by IVIS twice a week after initial iNK treatment, and changes in tumor burden were assessed based on bioluminescence intensity. The results are shown in Fig.48A (SKOV3 average bioluminescence, BLI), Fig.48B (Area under the curve of SKOV3, AUC calculated based on bioluminescence), Fig.49A (Hs746t average bioluminescence, BLI), Fig.49B (Area under the Hs746t curve, AUC) and Fig.45 (Area under the curve, AUC calculated from bioluminescence).

[0383] Although the present invention has been described in detail with reference to specific examples, it will be apparent to those skilled in the art that various modifications may be made within the scope of the present invention. Therefore, the scope of the present invention should not be limited to the examples described herein, but should be limited by the following claims.

[0384] Informal Sequence Listing

[0385] Informal Sequence Listing

[0386]

[0387]

[0388] Informal Sequence Listing

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

Claims

1. A chimeric antigen receptor (CAR), comprising: (i) anti-ROR1 scFv; (ii) transmembrane domain; (iii) hinge domain; (iv) Cytoplasmic domain.

2. The chimeric antigen receptor (CAR) of claim 1, wherein the cytoplasmic domain comprises a CD3ζ domain and a 4-1BB domain.

3. The chimeric antigen receptor (CAR) of claim 1, wherein the anti-ROR1 scFv comprises a light chain (V L ) and heavy chain (V H ), and the V L comprising a sequence selected from SEQ ID NO: 3, 7 and 11, wherein V H Comprising a sequence selected from SEQ ID NO: 2, 6 and 10.

4. The chimeric antigen receptor (CAR) of claim 3, wherein the anti-ROR1 scFv comprises a light chain (V L ) and heavy chain (V H ), and the V L Essentially consisting of a sequence selected from SEQ ID NO: 3, 7 and 11, and the V H Consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 2, 6 and 10.

5. The chimeric antigen receptor (CAR) of claim 1, wherein the anti-ROR1 scFv comprises a light chain (V L ) and the heavy chain (V H ) connector.

6. The chimeric antigen receptor (CAR) of claim 5, wherein the linker comprises the formula (G x S y ) n , wherein G is glycine and S is serine.

7. The chimeric antigen receptor (CAR) of claim 6, comprising G4S.

8. The chimeric antigen receptor (CAR) of claim 7, which consists of G4S.

9. The chimeric antigen receptor (CAR) of claim 1, wherein the anti-ROR1 scFv comprises the light chain (V L ) in the complementarity determining regions CDR1, CDR2 and CDR3, and the heavy chain (V H ) in CDR1, CDR2 and CDR3, and comprising: the V H SEQ ID NO: 29 in the CDR1, the V H SEQ ID NO:30 in the CDR2, the V H SEQ ID NO:31 in the CDR3, the V L SEQ ID NO:32 in the CDR1, the V L SEQ ID NO: 33 in the CDR2 and the V L SEQ ID NO:34 in the CDR3.

10. The chimeric antigen receptor (CAR) of claim 9, wherein in the anti-ROR1 scFv, the V H The CDR1 consists of SEQ ID NO: 29, the V H The CDR2 consists of SEQ ID NO: 30, the V H The CDR3 consists of SEQ ID NO: 31, the V L The CDR1 consists of SEQ ID NO: 32, the V L The CDR2 consists of SEQ ID NO: 33, and the V L The CDR3 consists of SEQ ID NO:

34.

11. The chimeric antigen receptor (CAR) of claim 1, wherein the cytoplasmic domain comprises a CD3 zeta domain.

12. The chimeric antigen receptor (CAR) of claim 1, wherein the transmembrane domain comprises a CD8 transmembrane domain.

13. The chimeric antigen receptor (CAR) of claim 12, wherein the CD8 transmembrane domain consists essentially of SEQ ID NO:

22.

14. The chimeric antigen receptor (CAR) of claim 12, wherein the CD8 transmembrane domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:

21.

15. The chimeric antigen receptor (CAR) of claim 1, wherein the hinge domain comprises a CD8 hinge domain.

16. The chimeric antigen receptor (CAR) of claim 15, wherein the CD8 hinge domain consists essentially of SEQ ID NO:

20.

17. The chimeric antigen receptor (CAR) of claim 15, wherein the CD8 hinge domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:

19.

18. The chimeric antigen receptor (CAR) of claim 1, wherein the CD3ζ domain consists essentially of SEQ ID NO:

24.

19. The chimeric antigen receptor (CAR) of claim 18, wherein the CD3 zeta domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:

23.

20. The chimeric antigen receptor (CAR) of claim 1, wherein the 4-1BB domain consists essentially of SEQ ID NO:

26.

21. The chimeric antigen receptor (CAR) of claim 20, wherein the 4-1BB domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:

25.

22. The chimeric antigen receptor (CAR) of claim 1, further comprising a signal sequence.

23. The chimeric antigen receptor (CAR) of claim 22, wherein the signal sequence is selected from a CD8 signal sequence and a CD28 signal sequence.

24. The chimeric antigen receptor (CAR) of claim 1, wherein the cytoplasmic domain comprises a binding motif for an intracellular signaling protein.

25. The chimeric antigen receptor (CAR) of claim 1, wherein the binding motif is present in the CD3ζ domain.

26. The chimeric antigen receptor (CAR) of claim 24, wherein the intracellular signal transduction protein is a STAT protein or a JAK protein.

27. The chimeric antigen receptor (CAR) of claim 26, wherein the JAK binding domain comprises SEQ ID NO:

43.

28. The chimeric antigen receptor (CAR) of claim 26, wherein the STAT binding domain is selected from SEQ ID NOs: 39-42.

29. The chimeric antigen receptor (CAR) of claim 1, wherein the cytoplasmic domain further comprises an IL-2Rb cytoplasmic domain.

30. The chimeric antigen receptor (CAR) of claim 29, wherein the IL-2Rb cytoplasmic domain consists essentially of SEQ ID NO:

50.

31. The chimeric antigen receptor (CAR) of claim 29, wherein the IL-2Rb cytoplasmic domain is encoded by a nucleic acid consisting essentially of SEQ ID NO:

51.

32. The chimeric antigen receptor (CAR) of claim 1, comprising a sequence selected from SEQ ID NO: 4, 8, 12 and 27.

33. The chimeric antigen receptor (CAR) of claim 1, consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, and 27.

34. The chimeric antigen receptor (CAR) of claim 1, encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 36 and 38.

35. The chimeric antigen receptor (CAR) of claim 1, encoded by a nucleic acid consisting essentially of a sequence selected from SEQ ID NOs: 14, 16, 18, 36 and 38.

36. An isolated nucleic acid comprising a vector sequence and a sequence encoding the chimeric antigen receptor (CAR) according to claim 1.

37. The isolated nucleic acid of claim 36, further comprising a promoter selected from the group consisting of: PGK1 promoter, MND promoter, Ubc promoter, CAG promoter, CaMKIIa promoter, SV40 early promoter, SV40 late promoter, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, beta-interferon promoter, hsp70 promoter, EF-1α promoter, and beta-actin promoter.

38. The isolated nucleic acid of claim 36, wherein the promoter comprises a CAG promoter.

39. The isolated nucleic acid of claim 36, wherein the promoter comprises the MND promoter.

40. The isolated nucleic acid of claim 36, wherein the promoter comprises an EF-1 alpha promoter.

41. The isolated nucleic acid of claim 36, wherein the vector comprises a plasmid.

42. The isolated nucleic acid of claim 36, wherein the vector comprises a viral vector derived from a virus selected from the group consisting of adenovirus type 2 and adenovirus type 5, retrovirus, lentivirus, adeno-associated virus (AAV), simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV).

43. The isolated nucleic acid of claim 36, further comprising a coding sequence for a cytokine.

44. The isolated nucleic acid of claim 43, wherein the cytokine is IL-36γ.

45. The isolated nucleic acid of claim 43, wherein IL-36γ is encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 44, 46 and 48.

46. ​​The isolated nucleic acid of claim 36, comprising a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 36 and 38.

47. An immune cell comprising the chimeric antigen receptor (CAR) according to claim 1.

48. The immune cell of claim 47, which is selected from the group consisting of a T cell, a natural killer (NK) cell, and an induced natural killer (iNK) cell.

49. An immune cell according to claim 47, wherein the chimeric antigen receptor (CAR) comprises a sequence selected from SEQ ID NO: 4, 8, 12 and 27.

50. The immune cell of claim 47, further comprising an armored genome modification.

51. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of an immune checkpoint gene or regulatory gene selected from the group consisting of: PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, CISH, CBLB, and 2B4.

52. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of CISH.

53. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of CBLB.

54. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of PDCD1.

55. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of Tim3.

56. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of LAG3.

57. An immune cell according to claim 50, wherein the armored genome modification comprises inactivation of TIGIT.

58. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of two or more genes from the group consisting of: PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, B2M, CISH, CBLB, and 2B4.

59. The immune cell of claim 50, wherein the armored genome modification comprises inactivation of B2M.

60. The immune cell of claim 50, wherein the armored genome modification further comprises inserting an HLA-E-B2M fusion construct into the B2M gene.

61. The immune cell of claim 47, which is engineered to express a cytokine.

62. The immune cell of claim 61, wherein the cytokine is membrane-bound.

63. The immune cell of claim 62, wherein the cytokine is expressed as a cytokine-receptor fusion protein.

64. The immune cell of claim 62, wherein the cytokine is selected from IL-15 and IL-21.

65. The method of claim 62, wherein the cytokine is selected from mbIL-15 and mbIL-21.

66. A method for preparing the immune cell according to claim 47, the method comprising introducing a nucleic acid comprising a sequence selected from SEQ ID NO: 14, 16, 18, 36 and 38 into the cell.

67. The method of claim 66, wherein the cell is selected from the group consisting of a T cell, a NK cell, and an induced pluripotent stem cell (iPSC).

68. The method of claim 67, wherein the cells are iPSCs, and the method further comprises differentiating the iPSCs into immune cells.

69. The method of claim 66, wherein the introducing step comprises introducing a sequence-dependent endonuclease into the cell.

70. The method of claim 69, wherein the introducing step comprises introducing a CRISPR system into the cell, the CRISPR system comprising a nucleic acid-guided endonuclease and a nucleic acid-targeting nucleic acid (NATNA) guide.

71. The method of claim 70, wherein the nucleic acid-guided endonuclease is selected from Cas9, Cas12a, and CASCADE.

72. The method of claim 70, wherein the endonuclease comprises a catalytically inactive CRISPR endonuclease conjugated to the cleavage domain of the restriction endonuclease FokI.

73. The method of claim 69, wherein the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN-Fok I fusions, transcription activator-like effector nucleases (TALENs), and TALEN-Fok I fusions.

74. The method of claim 69, wherein the endonuclease cleaves the genome of the cell at a locus selected from the group consisting of: TRAC, CBLB, PDCD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, and B2M.

75. The method of claim 66, wherein the nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 36, and 38 comprises a vector.

76. The method of claim 75, wherein the vector is a viral vector derived from a virus selected from the group consisting of adenovirus type 2 and adenovirus type 5, retrovirus, lentivirus, adeno-associated virus (AAV), simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV).

77. A composition comprising the immune cell of claim 47 and a pharmaceutically acceptable excipient.

78. The composition of claim 77, wherein the immune cells are between 1×10 6 With 2×10 8 The amount of CAR-T cells is between 1 and 2 cells.

79. The composition of claim 77, wherein the immune cells are between 1×10 7 With 2×10 9 The amount of CAR-NK cells is between 1 and 2 cells.

80. The composition of claim 77, wherein the immune cells are a mixture of CAR-T cells and CAR-NK cells present in a ratio of approximately 1:10 CAR-T to CAR-NK.

81. The composition of claim 77, wherein the pharmaceutically acceptable excipient comprises one or more of: carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, water, alcohols, polyols, glycerol, vegetable oils, phospholipids, surfactants, sugars, derivatized sugars, sugar alcohols, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, inositol, aldonic acids, esterified sugars, sugar polymers, monosaccharides, fructose, maltose, galactose, glucose, D-mannose, sorbose, disaccharides, lactose, sucrose, trehalose, cellobiose, polysaccharides, raffinose, melezitose, maltodextrin, dextran, starch, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, and sodium phosphate.

82. The composition of claim 77, wherein the antimicrobial agent comprises one or more of benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, and thimerosal.

83. The composition of claim 66, further comprising an antioxidant selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, and sodium metabisulfite.

84. The composition of claim 77, further comprising a surfactant selected from the group consisting of polysorbates, sorbitan esters, lecithin, phosphatidylcholine, phosphatidylethanolamine, fatty acids, fatty acid esters, and cholesterol.

85. The composition of claim 77, further comprising a cryogen selected from the group consisting of 3% to 12% dimethyl sulfoxide (DMSO) and 1% to 5% human albumin.

86. The composition of claim 77, further comprising a preservative selected from one or more of methylparaben, propylparaben, sodium benzoate, benzalkonium chloride, antioxidants, chelating agents, parabens, chlorobutanol, phenol, and sorbic acid.

87. A method of inhibiting tumor growth in a patient, the method comprising administering to a patient suffering from the tumor the composition of claim 77.

88. The method of claim 87, wherein the tumor is a solid tumor selected from the group consisting of ovarian cancer, triple-negative breast cancer, colorectal cancer, non-small cell lung cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, melanoma, and endometrial cancer or a hematological tumor selected from the group consisting of MCL, CLL, SLL, B-ALL, B-NHL, and AML.

89. The method of claim 87, wherein the administration is selected from the group consisting of systemic delivery, parenteral delivery, intramuscular delivery, intravenous delivery, subcutaneous delivery, and intradermal delivery.

90. The method of claim 87, wherein the composition further comprises a timed delivery component that enables timed release, delayed release, or sustained release of the composition.

91. The method of claim 90, wherein the timed delivery component is selected from the group consisting of monostearate, gelatin, a semipermeable matrix, and a solid hydrophobic polymer.

92. The method of claim 87, further comprising administering a cytokine to the patient.

93. The method of claim 92, wherein the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and IL-21.

94. The method of claim 87, further comprising applying quality control measures to the immune cells prior to administration to the patient, the quality control measures comprising evaluating one or more properties selected from the following: presence of an anti-ROR1 CAR in the cell genome, surface expression of the anti-ROR1 CAR, ROR1-dependent lysis of target cells expressing ROR1, proliferation in the presence of target cells expressing ROR1, cytokine or chemokine secretion in the presence of target cells expressing ROR1, reduction of tumor burden in an experimental animal carrying a tumor expressing ROR1, and persistence in the circulation of an animal carrying a tumor expressing ROR1 after administration of the immune cells to the experimental animal.

95. The method of claim 94, wherein the presence of the anti-ROR1 CAR in the cell genome is assessed by a method selected from the group consisting of nucleic acid hybridization, nucleic acid sequencing, polymerase chain reaction (PCR), quantitative PCR (qPCR), real-time PCR (rtPCR), and droplet digital PCR (ddPCR).

96. The method of claim 94, wherein the surface expression of the anti-ROR1 CAR is assessed by flow cytometry, fluorescence activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot.

97. The method of claim 94, wherein the surface expression of the anti-ROR1 CAR is assessed by flow cytometry.

98. The method of claim 94, wherein the immune cell population with the highest surface expression of the anti-ROR1 CAR is selected for administration to the patient.

99. The method of claim 94, wherein the ROR1-dependent lysis of a target cell carrying ROR1 is assessed by co-culturing the immune cell of claim 26 with a target cell expressing ROR1 at an effector:target ratio of between about 0.1 and about 10 and assessing target cell lysis.

100. The method of claim 94, wherein a population of immune cells that has the highest rate of lysis of target cells carrying ROR1 is selected for administration to the patient.

101. The method of claim 94, wherein the ROR1-dependent proliferation is assessed by co-culturing the immune cells with target cells expressing ROR1 and assessing proliferation of the immune cells.

102. The method of claim 94, wherein the immune cell population having the highest proliferation rate in the presence of target cells expressing ROR1 is selected for administration to the patient.

103. The method of claim 94, wherein the cytokine or chemokine is selected from IFN-γ, TNF-α, GM-CSF, IL-10, IL-5 and IL-13, MIP-1α, MIP-1β, IL-8 and RANTES.

104. The method of claim 94, wherein the cytokine secretion is assessed by co-culturing the immune cells with target cells expressing ROR1 and measuring the amount of cytokines in the co-culture supernatant.

105. The method of claim 94, wherein a population of immune cells having the highest cytokine secretion is selected for administration to the patient.

106. The method of claim 94, wherein said reducing tumor burden in an experimental animal bearing a tumor expressing ROR1 is measured in the form of a change in bioluminescence of bioluminescent tumor cells over a period of time after said animal has been injected with said immune cells.

107. The method of claim 106, wherein the change in bioluminescence is expressed as an area under the curve (AUC).

108. The method of claim 107, wherein the immune cell population with the smallest AUC is selected for administration to the patient.

109. The method of claim 94, wherein the persistence in the circulation of an experimental animal bearing a tumor expressing ROR1 following administration of the immune cells to the animal is measured by counting cells expressing human CD56 in the circulation of the animal.

110. The method of claim 94, wherein the immune cell population having the highest count of cells expressing human CD56 in the circulation of the animal is selected for administration to the patient.

111. The method of claim 94, wherein the immune cells comprise an armored genome modification.

112. The method of claim 111, wherein the genomic modification comprises inactivation of one or more of: CISH, CBLB, B2M, PDCD1, Tim3, LAG3, and TIGIT.

113. The method of claim 94, wherein the immune cells are engineered to express cytokines.

114. The method of claim 113, wherein the cytokine is selected from mbIL-15 and mbIL-21.

115. The method of claim 94, further comprising applying quality control measures to the immune cells prior to administration to the patient, the quality control measures comprising evaluating one or more properties selected from the group consisting of: surface expression of the cytokine, ROR1-dependent lysis of target cells expressing ROR1, and reduction of tumor burden in experimental animals carrying tumors expressing ROR1.

116. The method of claim 115, wherein a population of immune cells having the highest surface expression of the cytokine is selected for administration to the patient.

117. The method of claim 115, wherein a population of immune cells having the highest ROR1-dependent lysis of target cells expressing ROR1 is selected for administration to the patient.

118. The method of claim 115, wherein the immune cell population that has the highest rate of reducing tumor burden in an experimental animal bearing a tumor expressing ROR1 is selected for administration to the patient.

119. The method of claim 94, wherein the composition comprises between 1×10 7 With 2×10 8 CAR-T cells between 1×10 8 With 2×10 9 The invention can be used to treat CAR-NK cells in an amount between 1:10 cells or a mixture of CAR-T cells and CAR-NK cells in a ratio of approximately 1:10 CAR-T to CAR-NK.

120. A method for making an anti-ROR1 immune cell, the method comprising introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into a cell population, the CAR comprising: an anti-ROR1 scFv; a transmembrane domain; a hinge domain; and a cytoplasmic domain, wherein the nucleic acid comprises a sequence selected from SEQ ID NO: 4, 8, 12 and 27.

121. The method of claim 120, wherein the cell population is selected from T cells, natural killer (NK) cells, and cells capable of differentiating into NK cells.

122. The method of claim 121, wherein the cells capable of differentiating into NK are selected from induced pluripotent stem cells (iPSCs), hematopoietic progenitor cells (HPCs), and lymphoid progenitor cells.

123. The method of claim 122, wherein the introduction is into iPSCs, thereby forming CAR-iPSCs.

124. The method of claim 123, further comprising inducing the CAR-iPSC to differentiate into induced CAR-NK (CAR-iNK).

125. The method of claim 124, wherein said inducing differentiation comprises: (i) contacting the CAR-iPSC with one or more cytokines selected from the group consisting of BMP4, VEGF, SCF, IL3, IL6, and TPO to generate hematopoietic progenitor cells (HPCs); (ii) By selecting CD34 + cells to enrich the HPCs; (iii) contacting the HPCs with one or more cytokines selected from the group consisting of IL3, IL15, IL7, SCT, FLT3L in the presence of feeder cells to generate induced natural killer cells (iNK).

126. The method of claim 124, further comprising expanding the iNKs by a method comprising culturing the iNKs in the presence of feeder cells and cytokines.

127. The method of claim 124, wherein the feeder cells secrete cell membrane cytokines or express the cytokines.

128. The method of claim 124, wherein the feeder cells express 4-1BB ligand (4-1BBL) and membrane-bound IL21 (mbIL21).

129. The method of claim 120, wherein the nucleic acid is introduced into the precursor cell population chemically or electrochemically.

130. The method of claim 120, wherein the nucleic acid is introduced into the precursor cell population via a vector selected from the group consisting of a plasmid vector and a viral vector.

131. The method of claim 120, wherein the viral vector is derived from a virus selected from the group consisting of adenovirus type 2 and adenovirus type 5, retrovirus, lentivirus, adeno-associated virus (AAV), simian virus 40 (SV-40), vaccinia virus, Sendai virus, Epstein-Barr virus (EBV), and herpes simplex virus (HSV).

132. The method of claim 120, wherein the introducing step comprises introducing a CRISPR system into the cell, the CRISPR system comprising a nucleic acid-guided endonuclease and a nucleic acid-targeting nucleic acid (NATNA) guide.

133. The method of claim 132, wherein the nucleic acid-guided endonuclease is selected from Cas9, Cas12a, and CASCADE.

134. The method of claim 120, wherein the endonuclease comprises a catalytically inactive CRISPR endonuclease conjugated to the cleavage domain of the restriction endonuclease FokI.

135. The method of claim 120, wherein the endonuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN-Fok I fusions, transcription activator-like effector nucleases (TALENs), and TALEN-Fok I fusions.

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