Anti-ROR1 Chimeric Antigen Receptors (CARs), Cells Expressing CARs, and Related Methods

Abstract

The present invention includes 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 them, including methods of treating ROR1-expressing tumors.

Description

[Technical Field]

[0001] The present invention relates to the field of oncology, and more specifically to cell therapy using genetically engineered tumor-targeting immune cells.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT none.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on August 29, 2023, is named CBI049_30_SL.xml, and is 61,699 bytes in size. [Background technology]

[0004] Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable potential for hematological malignancies.

[0005] CAR-T cells have not demonstrated such robust and reproducible antitumor activity against solid malignancies. Natural killer (NK) cells express the receptor NKG2D, promoting 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.

[0006] ROR1 is expressed in a wide range of tumors, including hematological malignancies, ovarian, lung, pancreatic, and breast cancer. There is a need for innovative therapies for hematological and solid tumors that express ROR1, such as therapies with engineered cells that target tumor-specific ROR1 expression. Summary of the Invention

[0007] In one embodiment, the 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 CD3 zeta domain and a 4-1BB domain. In some embodiments, the anti-ROR1 scFv comprises a light chain (V L ) and heavy chain (V H ) and consists essentially of V L comprises a sequence selected from SEQ ID NOs: 3, 7, and 11, and V H comprises or consists essentially of a sequence selected from SEQ ID NOs: 2, 6, and 10. In some embodiments, the anti-ROR1 scFv comprises a light chain (V L ) and 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 a light chain (V L ) and the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain (V H ) contains CDR1, CDR2, and CDR3, and V H CDR1 of SEQ ID NO: 29, V H CDR2 of SEQ ID NO: 30, V H CDR3 of SEQ ID NO: 31, V L CDR1 of SEQ ID NO: 32, V L SEQ ID NO: 33 in CDR2, and V L In some embodiments, the CDR3 of V comprises SEQ ID NO: 34. H CDR1 of V consists of SEQ ID NO: 29, H CDR2 of V consists of SEQ ID NO: 30, H CDR3 of V consists of SEQ ID NO: 31, L CDR1 of V consists of SEQ ID NO: 32, L CDR2 of V consists of SEQ ID NO: 33, Land the CDR3 of the CAR consists of SEQ ID NO: 34. In some embodiments, the cytoplasmic domain comprises a CD3 zeta 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 zeta domain consists essentially of SEQ ID NO: 24. In some embodiments, the CD3 zeta 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 signaling protein. In some embodiments, the binding motif is present in the CD3 zeta domain. In some embodiments, the intracellular signaling 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 NOs: 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.

[0008] In some embodiments, the CAR comprises or consists essentially of a sequence selected from 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 SEQ ID NOs: 14, 16, 18, 36, and 38.

[0009] In one embodiment, the invention is an isolated nucleic acid comprising a vector sequence and a sequence encoding a chimeric antigen receptor (CAR) described herein. In some embodiments, the isolated nucleic acid further comprises a promoter selected from the group consisting of a PGK1 promoter, an MND promoter, a Ubc promoter, a CAG promoter, a CaMKIIa promoter, an SV40 early promoter, an SV40 late promoter, a cytomegalovirus (CMV) immediate early promoter, a Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, a mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, a β-interferon promoter, an hsp70 promoter, an EF-1α promoter, and a β-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 (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-36 gamma. In some embodiments, IL-36 gamma is encoded by a nucleic acid comprising a sequence selected from SEQ ID NOs: 44, 46, and 48. In some embodiments, the isolated nucleic acid comprises a sequence selected from SEQ ID NOs: 14, 16, 18, 36, and 38.

[0010] In one embodiment, the invention is an immune cell comprising a chimeric antigen receptor (CAR) described herein. In some embodiments, the cell is selected from a T cell, a natural killer (NK) cell, and an inducible natural killer (iNK) cell. In some embodiments, the cell comprises a sequence selected from SEQ ID NOs: 4, 8, 12, and 27. In some embodiments, the cell further comprises an armoring genomic modification. In some embodiments, the armoring genomic modification comprises inactivation of one or more or two or more immune checkpoint 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 armoring genomic modification comprises inactivation of CISH. In some embodiments, the armoring genomic modification comprises inactivation of CBLB. In some embodiments, the armoring genomic modification comprises inactivation of PDCD1. In some embodiments, the armoring genomic modification comprises inactivation of Tim3. In some embodiments, the armed genome modification comprises inactivation of LAG3. In some embodiments, the armed genome modification comprises inactivation of TIGIT. In some embodiments, the armed genome modification comprises inactivation of B2M. In some embodiments, the armed genome modification further comprises insertion of an HLA-E-B2M fusion construct into the B2M gene. In some embodiments, the cells are 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.

[0011] In one embodiment, the present invention relates to a method of producing an immune cell as described herein, the method comprising introducing into the cell a nucleic acid comprising a sequence selected from SEQ ID NOs: 14, 16, 18, 36, and 38. In some embodiments, the cell is selected from a T cell, a NK cell, and an induced pluripotent stem cell (iPSC). In some embodiments, the cell is an iPSC, and the method further comprises differentiating the iPSC into an immune cell. In some embodiments, the introducing step comprises introducing into the cell a sequence-dependent endonuclease. In some embodiments, the introducing step comprises introducing into the cell a 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 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, a 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).

[0012] In one embodiment, the invention is a composition comprising the immune cells described herein and a pharmaceutically acceptable excipient. In some embodiments, the immune cells are present in a concentration of 1 x 10 6 ~2×10 8 In some embodiments, the immune cells are 1 x 10 CAR-T cells. 7 ~2×10 9In some embodiments, the immune cells are a mixture of CAR-T cells and CAR-NK cells, with a ratio of CAR-T to CAR-NK of approximately 1:10. In some embodiments, the pharmaceutically acceptable excipient comprises one or more of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, water, alcohols, polyols, glycerin, vegetable oils, phospholipids, surfactants, sugars, derivatized sugars, alditol, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, myo-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 antimicrobial agent comprises one or more of 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 disulfite. 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 cryogen 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 methylparaben, propylparaben, sodium benzoate, benzalkonium chloride, antioxidants, chelating agents, parabens, chlorobutanol, phenol, and sorbic acid.

[0013] In one embodiment, the present invention provides a method for inhibiting tumor growth in a patient, comprising administering a composition described herein to a patient having a tumor. In some embodiments, the tumor is a solid tumor selected from 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 MCL, CLL, SLL, B-ALL, B-NHL, and AML. In some embodiments, the administering 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 delivery timing component that allows for time-release, delayed release, or sustained release of the composition. In some embodiments, the delivery timing component is selected from monostearate, gelatin, a semipermeable matrix, and a 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 a quality control measure to the immune cells prior to administration to the patient, comprising assessing one or more characteristics selected from the presence of the anti-ROR1 CAR in the cellular genome, surface expression of the anti-ROR1 CAR, ROR1-dependent lysis of ROR1-expressing target cells, proliferation in the presence of ROR1-expressing target cells, cytokine or chemokine secretion in the presence of ROR1-expressing target cells, reduced tumor burden in an experimental animal bearing a ROR1-expressing tumor, and persistence in the circulation of an experimental animal bearing a ROR1-expressing tumor upon administration of the immune cells to the animal. In some embodiments, the presence of the anti-ROR1 CAR in the cellular genome is assessed by a method selected from 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, surface expression of the anti-ROR1 CAR is assessed by flow cytometry, fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. In some embodiments, 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, ROR1-dependent lysis of ROR1-bearing target cells is assessed by co-culturing immune cells containing a CAR with ROR1-expressing target cells at an effector:target ratio of about 0.1 to about 10 and assessing target cell lysis. In some embodiments, the immune cell population with the highest rate of lysis of ROR1-bearing target cells is selected for administration to the patient. In some embodiments, ROR1-dependent proliferation is assessed by co-culturing immune cells with ROR1-expressing target cells and assessing immune cell proliferation. In some embodiments, the immune cell population with the highest rate of proliferation in the presence of ROR1-expressing target cells 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, cytokine secretion is assessed by co-culturing immune cells with ROR1-expressing target cells and measuring the amount of cytokine 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, reducing tumor burden in experimental animals bearing ROR1-expressing tumors is measured as a change in bioluminescence of bioluminescent tumor cells over a period of time after the animal is injected with the immune cells. In some embodiments, the change in bioluminescence is expressed as the 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 of the immune cells in the circulation of an experimental animal bearing a ROR1-expressing tumor upon administration to the animal is measured by counting human CD56-expressing cells in the animal's circulation. In some embodiments, the immune cell population with the highest count of human CD56-expressing cells in the animal's circulation is selected for administration to the patient. In some embodiments, the immune cells comprise an armed genomic modification. In some embodiments, the genomic modification comprises inactivation of one or more of CISH, CBLB, B2M, PDCD1, Tim3, LAG3, and TIGIT. In some embodiments, the immune cells are 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 a quality control measure to the immune cells prior to administration to the patient, comprising assessing one or more properties selected from surface expression of a cytokine, ROR1-dependent lysis of ROR1-expressing target cells, and reduced tumor burden in an experimental animal bearing a ROR1-expressing tumor. In some embodiments, the immune cell population with the highest surface expression of a cytokine is selected for administration to the patient. In some embodiments, the immune cell population having the highest ROR1-dependent lysis of ROR1-expressing target cells is selected for administration to the patient. In some embodiments, the immune cell population having the highest rate of reducing tumor burden in experimental animals bearing ROR1-expressing tumors is selected for administration to the patient. In some embodiments, the composition comprises 1 x 10 immune cells. 7 ~2×10 8 CAR-T cells, or 1 x 10 cells 8 ~2×10 9 The CAR-NK cells may be present in an amount of 1:10 or a mixture of CAR-T and CAR-NK cells in a ratio of approximately 1:10 of CAR-T to CAR-NK.

[0014] In one embodiment, the present invention provides a method for producing anti-ROR1 immune cells, the method comprising introducing into a cell population a nucleic acid encoding a chimeric antigen receptor (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 NOs: 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 cells are selected from induced pluripotent stem cells (iPSCs), hematopoietic progenitor cells (HPCs), and lymphoid progenitor cells. In some embodiments, the introducing is into iPSCs, thereby forming CAR-iPSCs. In some embodiments, the method further comprises inducing differentiation of the CAR-iPSCs into inducible CAR-NKs (CAR-iNKs). In some embodiments, inducing differentiation includes contacting the CAR-iPSCs with one or more cytokines selected from BMP4, VEGF, SCF, IL3, IL6, and TPO to produce hematopoietic progenitor cells (HPCs); +The method includes enriching for HPCs by selecting the cells, and contacting the HPCs with one or more cytokines selected from IL3, IL15, IL7, SCT, and FLT3L in the presence of feeder cells to produce inducible natural killer cells (iNKs). In some embodiments, the method further includes expanding the iNKs by a method comprising culturing them in the presence of feeder cells and cytokines. In some embodiments, the feeder cells secrete cytokines or express cytokines on their cell membranes. In some embodiments, the feeder cells express 4-1BB ligand (4-1BBL) and membrane-bound IL21 (mbIL21). In some embodiments, the nucleic acid is introduced into the precursor cell population via chemical or electrochemical means. In some embodiments, the nucleic acid is introduced into the precursor cell population via 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 into the cell a 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 a zinc finger nuclease (ZFN), a ZFN-Fok I fusion, a transcription activator-like effector nuclease (TALEN), and a TALEN-Fok I fusion. [Brief explanation of the drawings]

[0015] [Figure 1] Diagram of anti-ROR1 chimeric antigen receptor (CAR). [Figure 2] This is a workflow for generating induced anti-ROR1 CAR NK cells (iNK cells) from induced pluripotent stem cells (iPSCs). [Figure 3] CAR expression in cells at various stages of the iNK pathway (FIG. 2) transformed with the anti-ROR1 CAR constructs shown in FIG. 1 is shown. [Figure 4] 1 shows ROR1 expression in cell lines SKOV3 and JeKo-1. [Figure 5] Shows SKOV3 tumor cell lysis (percentage of viable tumor cells) by anti-ROR1 CAR iNK cells. [Figure 6] JeKo1 tumor cell lysis (specific lysis of tumor cells) by anti-ROR1 CAR iNK cells. [Figure 7] Shows repeated JeKo1 tumor cell lysis by anti-ROR1 CAR iNK cells in serial re-challenge assays. [Figure 8] Shows tumor burden (shown as area under the curve, AUC) in NGS mice following administration of anti-ROR1 CAR iNK cells. [Figure 9] Shows tumor burden (AUC) in IL15 transgenic mice following administration of anti-ROR1 CAR iNK cells. [Figure 10] 1 shows the persistence of human iNK cells in the mouse circulation as determined by quantifying human CD56+ cells. [Figure 11] Shows persistence of anti-ROR1 CAR expression in iNK cells (hCD45+CD56+ cells). [Figure 12] Cytokine secretion by CBLB-deficient iNK cells is shown. [Figure 13] Phenotypic evaluation of CBLB-deficient iNK cells is shown. [Figure 14] Figure 1 shows the in vitro cytotoxicity of CBLB-deficient iNK cells against SKOV3 tumor cells. [Figure 15]Figure 1 shows degranulation of CBLB-deficient iNK cells in co-culture with SKOV3 tumor cells. [Figure 16] Figure 1 shows the in vivo antitumor activity of CBLB-deficient iNK cells in a mouse xenograft model. [Figure 17] 1 shows the in vitro cytotoxicity of CBLB-deficient and CISH-deficient iNK cells against SKOV3 tumor cells. [Figures 18A-18B] Figure 1 shows effector molecule secretion by CBLB-deficient and CISH-deficient iNK cells in co-culture with SKOV3 tumor cells (IFNγ and TNFα). [Figures 19A-19B] 1 shows effector molecule secretion (perforin and granzyme B) by CBLB-deficient and CISH-deficient iNK cells in co-culture with SKOV3 tumor cells. [Figure 20] Figure 1 shows the in vivo antitumor activity (measured as the AUC of tumor bioluminescence) of CBLB-deficient and CISH-deficient iNK cells in a mouse xenograft model. [Figure 21] Figure 1 shows the in vivo antitumor activity (measured as the survival probability of tumor-implanted animals) of CBLB-deficient and CISH-deficient iNK cells in a mouse xenograft model. [Figure 22] Figure 1 shows cytotoxicity and survival in co-culture with T cells of iNK protected from immune challenge by a B2M-HLA-E fusion construct. [Figure 23] The percentage of CD8+ cells in the CD56+ cell population of T cells, iNK cells, and co-cultures is shown. [Figure 24] FIG. 1 is a diagram of an expression construct for expressing an IL-15 cytokine-receptor fusion. [Figure 25] Figure 1 shows the cytotoxic properties of iNKs engineered to constitutively express cytokine-receptor fusions. [Figure 26] FIG. 1 is a diagram of the original optimized anti-ROR1 CAR expression construct. [Figure 27A] Specific target cell lysis (in vitro cytotoxicity) by anti-ROR1 CAR-T cells. [Figure 27B] Specific target cell lysis (in vitro cytotoxicity) by anti-ROR1 CAR-T cells. [Figure 27C] Specific target cell lysis (in vitro cytotoxicity) by anti-ROR1 CAR-T cells. [Figure 28A] 1 shows cytokine secretion by anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 28B] 1 shows cytokine secretion by anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 28C] 1 shows cytokine secretion by anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 28D] 1 shows cytokine secretion by anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 29A] Figure 29 shows the in vivo anti-tumor activity of anti-ROR1 CAR-T cells measured as changes in body weight and survival probability (Figure 29A) and bioluminescence intensity (Figure 29B). [Figure 29B] Figure 29 shows the in vivo anti-tumor activity of anti-ROR1 CAR-T cells measured as changes in body weight and survival probability (Figure 29A) and bioluminescence intensity (Figure 29B). [Figure 30A] Specific target cell lysis (in vitro cytotoxicity) by checkpoint-deficient anti-ROR1 CAR-T cells. [Figure 30B] Specific target cell lysis (in vitro cytotoxicity) by checkpoint-deficient anti-ROR1 CAR-T cells. [Figure 31A] Figure 1 shows cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 31B] Figure 1 shows cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 31C]Figure 1 shows cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 31D] Figure 1 shows cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells in co-culture with ROR1-expressing target cells. [Figure 32] 1 shows the results of serial rechallenge of checkpoint-deficient anti-ROR1 CAR-T cells with ROR1-expressing tumor cells. [Figure 33] Figure 1 shows specific target cell lysis (in vitro cytotoxicity) by multiple checkpoint-deficient anti-ROR1 CAR-T cells. [Figure 34] Specific target cell lysis (in vitro cytotoxicity) by anti-ROR1 CAR-T cells pCB7432, pCB7437, and pCB7438 (Table 3) against ROR1-positive and ROR1-negative cell lines and primary B-CLL cells. [Figure 35A] Specific target cell lysis (in vitro cytotoxicity), expressed as AUC, by anti-ROR1 CAR-T cells pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, and pCB7436 (Table 3) against ROR1-positive and ROR1-negative cell lines and primary B-CLL cells. [Figure 35B] Specific target cell lysis (in vitro cytotoxicity), expressed as AUC, by anti-ROR1 CAR-T cells pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, and pCB7436 (Table 3) against ROR1-positive and ROR1-negative cell lines and primary B-CLL cells. [Figure 36A] Figure 3 shows cytokine 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. [Figure 36B]Figure 3 shows cytokine 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. [Figure 37A] 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. [Figure 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. [Figure 38] Figure 26 shows interleukin-36 gamma (IL36γ) secretion by anti-ROR1 CAR-T cells pCB7306 (Figure 26), or pCB7436, pCB7437, and pCB7438 (Table 3) in the presence of ROR1-positive JeKo-1 cells. [Figure 39A] Figure 3 shows the in vivo anti-tumor efficacy of anti-ROR1 CAR-T cells pCB7430, pCB7436, and pCB7438 (Table 3). [Figure 39B] Figure 3 shows the in vivo anti-tumor efficacy of anti-ROR1 CAR-T cells pCB7430, pCB7436, and pCB7438 (Table 3). [Figure 40] 1 shows a comparison between anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in surface CAR expression and CAR-T cell enumeration during serial in vitro re-challenge with ROR1-expressing target tumor cells. [Figure 41] FIG. 1 shows a comparison between anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in target cell lysis (expressed as area under the curve, AUC) during serial in vitro re-challenge with ROR1-expressing target tumor cells. [Figure 42]1 shows a comparison between 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 implanted with Jeko-1 ROR1-expressing tumors. [Figure 43] 1 shows a comparison between anti-ROR1 CAR-T cells and benchmark anti-ROR1 CAR-T cells in extending overall survival of animals implanted with Jeko-1 ROR1-expressing tumors. [Figure 44] We show that anti-ROR1 CAR-iNK cells lacking CBLB and B2M expression but expressing B2M-HLA-E and IL15 receptor fusions reduce tumor burden (represented as tumor bioluminescence) in animals implanted with ROR1-expressing tumors. [Figure 45] We show that anti-ROR1 CAR-iNK cells lacking CBLB and B2M expression but expressing B2M-HLA-E and IL15 receptor fusions reduce tumor burden (expressed as AUC of tumor bioluminescence measurements) in animals implanted with ROR1-expressing tumors. [Figure 46] 1 shows the results of serial in vitro rechallenge of anti-ROR1 CAR-iNK cells, which lack CBLB and B2M expression but express a B2M-HLA-E fusion and an IL15 receptor fusion, with the tumor cell line SKOV3, which has low ROR1 antigen density. [Figure 47] 1 shows the results of serial in vitro rechallenge of anti-ROR1 CAR-iNK cells, which lack CBLB and B2M expression but express a B2M-HLA-E fusion and an IL15 receptor fusion, with the tumor cell line Hs746t, which has high ROR1 antigen density. [Figure 48A] Figure 48 shows the results of multiple rounds of treatment of animals implanted with luciferase-expressing SKOV3 (a tumor cell line with low ROR1 antigen density) with anti-ROR1 iNK cells. Figure 48A: Bioluminescence, Figure 48B: Area under the curve (AUC) calculated from bioluminescence. [Figure 48B]Figure 48 shows the results of multiple rounds of treatment of animals implanted with luciferase-expressing SKOV3 (a tumor cell line with low ROR1 antigen density) with anti-ROR1 iNK cells. Figure 48A: Bioluminescence, Figure 48B: Area under the curve (AUC) calculated from bioluminescence. [Figure 49A] Figure 49 shows the results of multiple rounds of treatment with anti-ROR1 iNK cells in animals implanted with luciferase-expressing Hs746t (a tumor cell line with high ROR1 antigen density). Figure 49A: Bioluminescence, Figure 49B: Area under the curve (AUC) calculated from bioluminescence. [Figure 49B] Figure 49 shows the results of multiple rounds of treatment with anti-ROR1 iNK cells in animals implanted with luciferase-expressing Hs746t (a tumor cell line with high ROR1 antigen density). Figure 49A: Bioluminescence, Figure 49B: Area under the curve (AUC) calculated from bioluminescence. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition The following definitions are provided to aid in the understanding of this disclosure. Unless defined in this section, technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 4 th See Ed. Cold Spring Harbor Lab. Press (2012).

[0017] The term "activation" refers to the state of a T cell, which includes one or both of cell proliferation and cytokine secretion by the cell.

[0018] The term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. This term also includes Fv, Fab and F(ab)2, scFv, and other Fvs, as described, for example, in Antibodies: A Laboratory Manual, 2 ndEd. Greenfield, E., ed., Cold Spring Harbor Lab. Press, NY (2013), including antibody fragments including other forms.

[0019] The term "costimulatory domain" refers to a portion of a chimeric T cell receptor (CAR) that is a binding partner that specifically binds to a costimulatory ligand, thereby mediating T cell costimulatory responses, proliferation, and cytokine secretion. Examples of costimulatory ligands include 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. Examples of costimulatory domains include CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0020] The term "therapeutic benefit" refers to the effect of improving the condition of a subject with respect to a medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of the disease. For example, treating cancer can involve, for example, reducing tumor size, reducing tumor invasiveness, reducing tumor growth rate, or preventing metastasis, or extending the overall survival (OS) or progression-free survival (PFS) of a subject with cancer.

[0021] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions in patients. For example, pharmaceutically and pharmacologically acceptable preparations should meet the standards set forth by the FDA Office of Biological Standards.

[0022] The terms "pharmaceutically acceptable carrier" and "excipient" refer to aqueous solvents (e.g., water, aqueous alcoholic solutions, saline, 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, disintegrating agents, lubricants, sweeteners, flavoring agents, and dyes. The concentrations and pH of the various components in the pharmaceutical composition are adjusted according to well-known parameters for each component.

[0023] The term "domain" refers to a region in a polypeptide that folds into a particular structure independently of other regions.

[0024] The term "effector function" refers to the specialized functions of differentiated cells such as NK cells.

[0025] The term "adoptive cells" refers to cells that can be genetically modified for use in cell therapy treatments. Examples of adoptive cells include T cells, macrophages, and natural killer (NK) cells.

[0026] The term "cell therapy" refers to the treatment of a disease or disorder using genetically modified cells. The term "adoptive cell therapy (ACT)" refers to therapy using genetically modified adoptive cells. Examples of ACT include T cell therapy, CAR T cell therapy, natural killer (NK) cell therapy, and CAR NK cell therapy.

[0027] The term "lymphocyte" refers to a white blood cell that is part of the vertebrate immune system. Lymphocytes are CD4 + and / or CD8 +Lymphocytes include T cells such as cytotoxic T cells, alpha / beta T cells, gamma / delta 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).

[0028] The terms "effective amount" and "therapeutically effective amount" of a composition, e.g., a cell therapy composition, refer to the amount of the composition that is sufficient to produce a desired response in a patient to whom the composition is administered.

[0029] The terms "peptide," "polypeptide," and "protein" are interchangeable and refer to polymers of amino acids, including natural and synthetic (unnatural) amino acids, as well as amino acids not found in naturally occurring proteins, e.g., peptidomimetics and D optical isomers. Polypeptides can be branched or linear and can be interrupted by non-amino acid residues. The term also encompasses amino acid polymers modified through acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, cross-linking, or conjugation (e.g., with labels). A polypeptide need not contain the full-length amino acid sequence of a reference molecule but can include only that amount of the reference molecule necessary for the polypeptide to retain its desired activity. For example, polypeptides including full-length proteins, fragments thereof, and polypeptides with amino acid deletions, additions, and substitutions are encompassed by the terms "protein" and "polypeptide," so long as the desired activity is retained. For example, polypeptides having 95%, 90%, 80%, or less sequence identity to a reference polypeptide are included, so long as the desired activity is retained by the polypeptide.

[0030] The terms "CRISPR" (clustered regularly interspaced short palindromic repeats), "Cas" (CRISPR-associated proteins), "CRISPR-Cas," and "CRISPR system" refer to a genome editing tool derived from prokaryotes and comprising a nucleic acid guide molecule and a sequence-specific nucleic acid-guided endonuclease capable of cleaving a target nucleic acid strand at a site complementary to the sequence in the nucleic acid guide.

[0031] The term "NATNA" (nucleic acid-targeting nucleic acid) refers to the nucleic acid guide molecule of the CRISPR system. A NATNA can be composed of two nucleic acid-targeting polynucleotides ("dual guide") including a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). A NATNA can be composed of a single nucleic acid-targeting polynucleotide ("single guide") including a crRNA and a tracrRNA connected by a fusion region (linker). The crRNA can include a targeting region and an activating region. The tracrRNA can include a region capable of hybridizing to the activating region of the crRNA. The term "targeting region" refers to a region capable of hybridizing to a sequence in a target nucleic acid. The term "activating region" refers to a region that interacts with a polypeptide, e.g., a CRISPR nuclease.

[0032] Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable promise for hematological malignancies (see, e.g., U.S. Patent No. 9,464,140). However, CAR-T cells have not demonstrated such robust and reproducible antitumor activity against solid malignancies. Natural killer (NK) cells express the receptor NKG2D, promoting their innate ability to detect and kill tumors that express stress ligands.

[0033] ROR1 (also known as NTRKR1, receptor tyrosine kinase-like orphan receptor 1, neurotrophic tyrosine kinase receptor-related 1) is an orphan receptor tyrosine kinase-like antigen. ROR1 is a 937-amino acid protein, with amino acids 30–406 comprising the extracellular domain. ROR1 is a glycosylated type I membrane protein belonging to the ROR subfamily of cell surface receptors. Binding to the specific ligand WNT5A activates the downstream Rac / Rho pathway, which is involved in cell motility and invasion.

[0034] ROR1 is expressed in a wide range of tumors, including hematological malignancies (chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL)), ovarian, lung, pancreatic, and triple-negative breast cancer. ROR1 expression promotes tumor cell growth, survival, and metastasis. In particular, ROR1 is expressed on ovarian cancer stem cells and has been shown to promote 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.

[0035] Disclosed herein are engineered T cells and T cell compositions, and NK cell and NK cell compositions, in which the T cells and NK cells are engineered to express a ROR1-specific CAR (anti-ROR1-CAR). The T cells and NK cells of the present invention have enhanced specificity and function against solid tumors and inhibit ROR1. + The results show that engineered T cells and NK cells can detect tumor cells via the anti-ROR1 CAR. Furthermore, engineered NK cells can detect tumor cells via their natural NKG2D receptor.

[0036] Disclosed herein are methods and compositions for treating tumors with ROR1-targeted engineered cells, including T cells and natural killer (NK) cells.

[0037] In some embodiments, the present invention includes adoptive cells and their use in cellular immunotherapy. The adoptive cells of the present invention include T cells, CAR-T cells, natural killer (NK) cells, inducible natural killer (iNK) cells, CAR-NK cells, CAR-iNK cells, and their precursors.

[0038] In some embodiments, the cells of the invention are allogeneic cells, ie, cells isolated from a donor individual, such as a healthy human donor of either gender.

[0039] In some embodiments, cells are isolated from healthy donors using standard techniques. For example, lymphocytes can be isolated from blood, including peripheral blood and umbilical cord blood, or from lymphoid organs such as the thymus, bone marrow, lymph nodes, and mucosa-associated lymphoid tissue (MALT). Techniques for isolating lymphocytes from such tissues are well known in the art; see, e.g., Smith, JW (1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1:203-206.

[0040] In some embodiments, the isolated lymphocytes are characterized for specificity, frequency, and function, hi some embodiments, the isolated lymphocyte population is enriched for a particular cell subset, such as T cells or NK cells.

[0041] In some embodiments, the isolated lymphocyte population is CD4 + , CD8 + , CD25 + , or CD62L + In some embodiments, the isolated lymphocyte population is enriched for specific subsets of T cells, such as CD56, which is indicative of NK cells. See, e.g., Wang et al., Mol. Therapy-Oncolytics (2016) 3:16015. In some embodiments, the isolated lymphocyte population is enriched for specific subsets of T cells, such as CD56, which is indicative of NK cells. +In some embodiments, after isolation, 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.

[0042] In some embodiments, NK cells are produced by differentiating stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). For example, T. Cheng (ed.), Hematopoietic Differentiation of Human Pluripotent Stem Cells, SpringerBriefs 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. al., (2016) Human embryonic stem cells differentiate into a homogeneous population 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, PloS One, 7(1):e30264.

[0043] The differentiation process typically involves two steps, and optionally three steps: differentiation of stem cells into hematopoietic progenitor cells (HPCs), differentiation of HPCs into natural killer cells (NKs), and optional expansion of NK cells. The products of differentiation (or expansion) can also be tested for the presence of an NK phenotype, such as an NK-specific gene expression pattern, including expression of NK-specific cell surface markers, e.g., CD45 and CD56.

[0044] In some embodiments, forming hematopoietic progenitor cells (HPCs) comprises forming spheroids by allowing cells to aggregate (optionally assisted by low-speed centrifugation) and incubating the spheroids in the presence of one or more cytokines selected from 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.

[0045] In some embodiments, the step of forming NK cells comprises incubating the spheroids in the presence of one or more cytokines selected from 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, the HPC fraction is transfected with CD34 IgG before initiating the NK differentiation step. + In some embodiments, upon completion of the differentiation step, the cell fraction is tested for surface expression of NK-specific markers such as CD45 and CD56.

[0046] In some embodiments, the NK cell expansion step comprises incubating the NK cells in the presence of cytokines and antigen-presenting cells (APCs). In some embodiments, the APCs are engineered to express cytokines on their cell surface. In some embodiments, the APCs overexpress 41BBL and membrane-bound IL-21. In some embodiments, the APCs overexpress membrane-bound IL-15. In some embodiments, the APCs overexpress a cytokine receptor fusion. In some embodiments, the APCs overexpress an IL-15-IL-15R fusion. In some embodiments, the APCs overexpress an IL-21-IL-21R fusion. In some embodiments, exogenous cytokines are also added. In some embodiments, IL-2 is added. In some embodiments, the final expanded iNK product is evaluated in in vitro and in vivo functional assays.

[0047] The present invention includes 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 includes a combination of CAR-T cells and CAR-NK cells for administration to a patient. In some embodiments, the CAR-T cells and CAR-NK cells are administered to a patient simultaneously as a single formulation. In some embodiments, the CAR-T cells and CAR-NK cells are administered to a patient sequentially.

[0048] A typical chimeric antigen receptor (CAR) comprises an extracellular domain containing an antigen-binding region, a transmembrane domain, and one or more intracellular activation (costimulatory) domains. In some embodiments, the CAR also comprises a hinge domain. In some embodiments, the CAR also comprises a leader peptide that directs the CAR to the cell membrane.

[0049] The CARs disclosed herein comprise an extracellular domain comprising an antigen-binding region that targets 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 (murine) sequence. In some embodiments, the antigen-binding region comprises a human sequence. In some embodiments, the antigen-binding region comprises a humanized mouse (murine) sequence.

[0050] In some embodiments, the antigen-binding region comprises a single-chain variable fragment (scFv). H ) linked to the variable region of an antibody light chain (V L In some embodiments, the variable region of V L V via a peptide linker H is linked to.

[0051] Peptide linkers generally comprise from about 5 to about 40 amino acids. The linker may be a naturally occurring sequence or an engineered sequence. For example, in some embodiments, the linker is derived from a human protein, e.g., an immunoglobulin selected from IgG, IgA, IgD, IgE, or IgM. In some embodiments, the linker comprises 5 to 40 amino acids from the CH1, CH2, or CH3 domain of an immunoglobulin heavy chain. In some embodiments, the linker comprises the sequence (G x S y ) nExamples of 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 publication WO2014 / 087010, "Improved polypeptides directed against IgE."

[0052] In some embodiments, the sequence of the scFv is further optimized for binding to the ROR1 antigen. In some embodiments, the optimization process involves the heavy chain (V H ) and light chain (V L This includes varying the linker between the scFv and the scFv. It has been reported that the linker sequence can affect antigen-scFv binding (Navabi, et al., (2021) Designing 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, in silico design of the linker amino acid sequence within the context of a known crystal structure of the scFv-antibody complex has been shown to result in a linker sequence that confers better antigen affinity to the scFv. It has also been shown that scFv-containing chimeric antigen receptors (CARs) can be optimized for antigen binding simply by varying the linker length, 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 linker is represented by the general formula (G xS y ) n It has.

[0053] In some embodiments, the CAR comprises an scFv described in International Application No. PCT / US2023 / 067314, filed May 22, 2023, Anti-ROR1 antibody and ROR1-targeting engineered cells.

[0054] In some embodiments, the CAR comprises an scFv selected from clones 857, 858, and 862 described in PCT / US2023 / 067314. In some embodiments, the CAR comprises an 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 an 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 has been further optimized to comprise SEQ ID NO:27, which contains the linker G4S. In some embodiments, the optimized scFv consists of SEQ ID NO:27.

[0055] 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.

[0056] In some embodiments, the antigen-binding region comprises a heavy chain (V) comprising a sequence selected from SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:10. H In some embodiments, the antigen-binding region comprises a heavy chain (V) consisting of a sequence selected from SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:10. H ) is included.

[0057] In some embodiments, the antigen-binding region comprises a light chain (V) comprising a sequence selected from SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11. L In some embodiments, the antigen-binding region comprises a heavy chain (V) consisting of a sequence selected from SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11. H ) is included.

[0058] Murine antibodies and scFvs, humanized antibodies and scFvs derived therefrom, and human antibodies and scFvs are composed of heavy and light antibody chains (V H and V L ) each contain three complementarity determining regions (CDRs).

[0059] In some embodiments, CDRs are identified using a crystal structure of an antigen-antibody complex. In some embodiments, CDRs are identified using an in vitro method such as phage display. In some embodiments, CDRs are identified using in silico methods, such as IMGT (Lefranc et al., (2009) IMGT®, The international immunogenetics information system, Nucl. Acids Res. 37:D1006) and Kabat (Kabat et al., (1987) Sequences of Proteins of Immunological Interest, 4th ed., USHHS, NIH). In some embodiments, CDRs are identified using the IMGT tool. In some embodiments, CDRs are identified using the Kabat tool. In some embodiments, the minimal portion of a CDR is identified as the overlap between the sequence located by the IMGT tool and the sequence located by the Kabat tool.

[0060] In some embodiments, the scFv comprised in the CAR of the invention is V H In some embodiments, the scFv comprises SEQ ID NO: 29 in CDR1 of V HIn some embodiments, the scFv comprises SEQ ID NO: 30 in CDR2 of V H In some embodiments, the scFv comprises SEQ ID NO: 31 in the CDR3 of V L In some embodiments, the scFv comprises SEQ ID NO: 32 in CDR1 of V L In some embodiments, the scFv comprises SEQ ID NO: 33 in CDR2 of V L The CDR3 of the antibody contains SEQ ID NO: 34.

[0061] In some, the scFv included in the CAR of the present invention is V H CDR1 of SEQ ID NO: 29, V H SEQ ID NO: 30 in CDR2, and V H CDR3 of V contains SEQ ID NO: 31, L CDR1 of SEQ ID NO: 32, V L SEQ ID NO: 33 in CDR2, and V L and further comprising SEQ ID NO: 34 in CDR3.

[0062] In some embodiments, the CAR also 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.

[0063] In some embodiments, the CAR comprises a signal peptide (signal sequence) that enables transport of the CAR to the 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.

[0064] In some embodiments, the transmembrane domain of the CAR is derived from a membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain is derived from the same protein as the costimulatory domain described below. For example, the transmembrane domain of the CAR may be derived from a T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon chain, The transmembrane domain may be a transmembrane domain of 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 consists of SEQ ID NO: 21.

[0065] The cytoplasmic or intracellular signaling domain, also referred to as the costimulatory domain of a CAR, is responsible for activating one or more effector functions of an immune cell expressing the CAR. In some embodiments, the costimulatory domain of a CAR comprises a portion or the entire sequence of a TCR zeta chain, a CD3 zeta chain, a CD3 epsilon chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, ICOS / CD278, IL-2R beta / CD122, IL-2R alpha / 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 a CAR comprises a CD3 zeta costimulatory domain. In some embodiments, the cytoplasmic domain of a CAR comprises a 4-1BB costimulatory domain. In some embodiments, the cytoplasmic domain of the CAR consists of CD3 zeta and 4-1BB costimulatory domains.

[0066] 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.

[0067] In some embodiments, the 4-1BB costimulatory domain comprises SEQ ID NO: 26. In some embodiments, the 4-1BB costimulatory domain consists of SEQ ID NO: 26. In some embodiments, the 4-1BB costimulatory domain is encoded by a nucleic acid comprising SEQ ID NO: 25. In some embodiments, the 4-1BB costimulatory domain is encoded by a nucleic acid consisting of SEQ ID NO: 25.

[0068] In some embodiments, the cytoplasmic domain of an anti-ROR1 CAR comprises one or more additional elements that enhance intracellular signaling by the CAR. In some embodiments, the cytoplasmic domain comprises one or more protein association motifs that bind to and recruit one or more intracellular signaling proteins. For example, U.S. Patent No. 10,336,810 discloses increased cytotoxic activity of anti-CD19 CAR-T cells bearing CARs enhanced with JAK and STAT binding motifs in the cytoplasmic region.

[0069] In some embodiments, a CAR comprises two or more intracellular (or cytoplasmic) domains and two or more protein association motifs, each protein association motif being in a separate intracellular domain of the CAR. In some embodiments, the two or more protein association motifs are within the same intracellular domain of the CAR.

[0070] In some embodiments, the intracellular signaling 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.

[0071] In some embodiments, the intracellular signaling 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-2R β chain. In some embodiments, the STAT5 association motif is engineered into the CD3ζ intracellular signaling domain of the CAR.

[0072] In some embodiments, the intracellular signaling 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 from a JAK association motif 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, a JAK association motif is engineered into the CD3ζ intracellular signaling domain of the CAR.

[0073] In some embodiments, the chimeric antigen receptor (CAR) comprises a sequence selected from SEQ ID NOs: 4, 8, 12, 35, and 37. In some embodiments, the CAR consists of a sequence selected from 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 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 SEQ ID NOs: 14, 16, 18, 36, and 38.

[0074] In some embodiments, the CAR is fully human or humanized to reduce immunogenicity in human patients, hi some embodiments, the CAR sequence is optimized for codon usage in human cells.

[0075] Examples of anti-ROR1 CAR constructs are described in Example 1 of the present disclosure and in Figure 1. Other examples of anti-ROR1 CAR constructs are described in Example 12 of the present disclosure and in Figure 26.

[0076] A nucleic acid encoding a CAR (e.g., a nucleic acid comprising SEQ ID NO: 14, 16, 18, 36, or 38) can be introduced into a cell as a genomic DNA sequence or a cDNA sequence. The cDNA sequence comprises an open reading frame for translation of a protein (e.g., a CAR), and in some embodiments, the cDNA further comprises untranslated elements that, for example, improve the stability or rate of translation of the CAR mRNA.

[0077] In some embodiments, the CAR coding sequence is inserted into the cellular genome of a T cell or NK cell at the endogenous T cell receptor alpha chain (TRAC) locus. In some embodiments, the TRAC locus is targeted by a CRISPR endonuclease (e.g., Cas9, Cas12a, or CASCADE) and a guide polynucleotide. In some embodiments, the guide polynucleotide is a CRISPR hybrid DNA-RNA polynucleotide (chRDNA).

[0078] In some embodiments, insertion of the CAR relies on the cell's endogenous homologous recombination system. In such embodiments, the CAR coding sequence can be introduced into the cell via chemical or electrochemical means (e.g., lipid nanoparticles or electroporation). The CAR coding sequence can be introduced into the cell using a vector, such as a lentiviral vector, as discussed in detail elsewhere in this disclosure.

[0079] In some embodiments, the cells used in the present invention are engineered to express a CAR and further comprise genomic modifications that arm the cells against attack by the immune system of the recipient of allogeneic immune cells (immune cells derived from a donor). In some embodiments, the arming modifications include protection from recognition by the host's cytotoxic T cells. Cytotoxic T cells recognize MHC class I antigens. MHC class I molecules are cell surface molecules composed of beta 2 microglobulin (B2M) associated with the heavy chain of an HLA-I protein (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 binds to cytotoxic CD8 + If the allogeneic cells are recognized by T cells and HLA-I is recognized as non-self, they will be killed by the T cells. In some embodiments, the cells of the present invention contain an armed genome modification that includes disruption of the B2M gene, thus disrupting the MHC class I cell surface binding complex. This disruption eliminates MHC class I antigen recognition, which normally stimulates cytotoxic T cell attack.

[0080] In some embodiments, the armed genomic modifications include disruption of the recognition of CAR-T cells or CAR-NK cells by the host's natural killer (NK) cells. NK cells recognize cells lacking MHC-I proteins as "losing themselves" and kill such cells. NK cells are inhibited by HLA-I proteins, including HLA-E, the least polymorphic HLA-I protein. In some embodiments, the cells of the invention comprise a first armed genomic modification comprising disruption of the B2M gene, thus disrupting the MHC class I cell surface binding complex and disrupting MHC class I antigen recognition that stimulates cytotoxic T cell attack, and further comprise a second armed genomic modification comprising insertion of an HLA-E gene fused to the beta 2 microglobulin (B2M) gene, thus expressing a B2M-HLA-E construct designed to hide the cells from attack by 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.

[0081] The present inventors have successfully achieved immunohiding of iNK cells using a B2M-HLA-E fusion construct. An example of such immunohiding is shown in Example 10.

[0082] In some embodiments, the arming modification comprises transcriptionally silencing or disrupting one or more immune checkpoint 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.

[0083] In some embodiments, the silenced or disrupted immune checkpoint gene is CBLB. CBLB proteins are immune checkpoints that control the activity of NK cells. Upon phosphorylation, CBLB binds to downstream effectors of NK cell activation and downregulates them via proteasome-mediated degradation. CBLB negatively affects the cytotoxicity, cytokine production, and persistence of NK cells, and suppressing CBLB activity in NK cells enhances the anti-tumor function of NK cells. 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 stimulation cytokine,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.J ImmunoTher.of Cancer 9:e001975.

[0084] In some embodiments, disruption 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 in vitro from iPSCs). In some embodiments, disruption of the CBLB gene by the methods described herein occurs in iNK cell precursors, such as iPSCs. In some embodiments, disruption of the CBLB gene in iPSCs does not negatively affect their ability to differentiate into iNK cells.

[0085] We show herein that iNK cells lacking CBLB expression (CBLB KO) exhibit enhanced in vitro degranulation and cytotoxicity, as well as enhanced in vivo antitumor activity. We also show that iPSCs with disrupted CBLB efficiently differentiate into iNKs, and the resulting iNKs express canonical NK cell markers at levels similar to wild-type iNKs. (See Examples 8 and 9.)

[0086] 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 efficacy of NK cells against certain cancers and infections (U.S. Patent No. 11,104,375).

[0087] In some embodiments, disruption of the CISH 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 in vitro from iPSCs).

[0088] In some embodiments, disruption of the CISH gene by the methods described herein occurs in iNK cell precursors, such as iPSCs. In some embodiments, disruption of the CISH gene in iPSCs does not negatively affect their ability to differentiate into iNK cells.

[0089] We show herein that iNK cells lacking CISH expression (CISH KO) exhibit enhanced in vitro cytotoxicity. (See Example 9.)

[0090] 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 contribute to T cell exhaustion. Chronic lymphocytic leukemia (CLL) cells both express and secrete LAG3, which is thought to contribute to CLL tumor growth and escape T cell attack. Shapiro et al. (2017) Lymphocyte activation gene 3: a novel therapeutic target in chronic lymphocytic leukemia, Haematologica, 102(5):874.

[0091] In some embodiments, disruption of the LAG3 gene by the methods described herein occurs in T cells. The inventors herein show that T cells lacking LAG3 expression exhibit enhanced in vitro cytotoxicity. (See Examples 17 and 18.)

[0092] 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 mediates CD4 + T cells, CD8 + It inhibits the function of various types of T cells, including T cells, Tregs, and T helper cells. In hematological malignancies, including chronic lymphocytic leukemia (CLL), high levels of Tim3 expression upregulate inhibitory Treg cells, inhibit T helper cell function, and correlate with poor prognosis. Pang et al. (2021) Activated Galectin-9 / Tim3 promotes Tregs and suppresses Th1 effector function in chronic lymphocytic leukemia, FASEBJ.35:e21556.

[0093] In some embodiments, disruption of the Tim3 gene by the methods described herein occurs in T cells, and we show herein that T cells lacking Tim3 expression exhibit enhanced in vitro cytotoxicity (see Examples 17 and 18).

[0094] In some embodiments, the silenced or disrupted immune checkpoint gene is TIGIT. T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) mediates activation of CD8 + T and CD4 + It is expressed by T cells, natural killer (NK) cells, regulatory T cells (Treg), and follicular T helper cells. High levels of TIGIT expression in CAR-T cells are associated with a poor response 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.

[0095] In some embodiments, disruption of the Tim3 gene by the methods described herein occurs in T cells. We show herein that T cells lacking TIGIT expression exhibit enhanced in vitro cytotoxicity (see Examples 17 and 18).

[0096] 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 combinations of PD1 and Tim3, PD1 and LAG3, PD1 and TIGIT, and Tim3 and LAG3. The inventors surprisingly discovered that inactivating multiple checkpoints in CAR-T cells has the most significant synergistic effect on the duration of the cytotoxic potential of CAR-T cells (see Figures 32 and 33).

[0097] In some embodiments, immune checkpoint genes or regulatory genes are disrupted using an endonuclease that specifically cleaves nucleic acid strands within the target sequence of the disrupted gene. Strand cleavage by a sequence-specific endonuclease results in nucleic acid strand breaks that can be repaired by non-homologous end joining (NHEJ). NHEJ is an imperfect repair process that can result in direct religation, but more frequently 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 can result in a missense or nonsense mutation in the protein-coding sequence, which can eliminate production of any protein or cause the production of a non-functional protein.

[0098] In some embodiments, the immune checkpoint gene is disrupted by contacting the cell with a sequence-specific endonuclease to induce the NHEJ process in the cell, resulting in genetic mutation and elimination of protein expression of the immune checkpoint gene.

[0099] 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.

[0100] 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 that includes a CRISPR endonuclease and a CRISPR guide RNA (nucleic acid-targeting nucleic acid or NATNA). In some embodiments, the NATNA includes one or more DNA nucleotides and is a CRISPR hybrid RNA-DNA or chRDNA. In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. In some embodiments, the NATNA is selected from the embodiments described in International Application Publication No. WO 2022 / 086846, "DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same."

[0101] In some embodiments, the present invention includes a method for producing an anti-ROR1 chimeric antigen receptor (CAR). In some embodiments, a nucleic acid encoding a CAR is introduced into a target cell in which expression of the CAR is desired. In some embodiments, the introduced nucleic acid is selected from an expression vector containing a sequence encoding the CAR, an mRNA encoding the CAR, and a delivery vector containing a donor sequence encoding the CAR that is inserted into the cell genome. In some embodiments, the target cell is contacted with the nucleic acid encoding the CAR in vitro, in vivo, or ex vivo.

[0102] In some embodiments, the vector used to deliver the nucleic acid encoding a 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. Lentiviral vector packaging systems are available from several commercial vendors, such as ThermoFisher Scientific, LabCorp, etc. Such systems comprise a host cell into which one or more plasmids encoding a sequence of interest (e.g., a CAR-encoding sequence) are introduced along with the lentiviral vector-encoding gene. The transfected host cell then produces a lentiviral vector bearing the CAR sequence payload.

[0103] In some embodiments, the CAR coding sequence is introduced into cells via a viral vector. Suitable vectors are non-replicating in target cells. In some embodiments, the vector is selected from, or designed based on, SV40, EBV, HSV, or BPV. In some embodiments, the vector is a lentiviral vector or any other suitable viral vector capable of delivering an appropriately sized payload. In some embodiments, to facilitate homologous recombination, the coding sequence is linked to homology arms located 5' (upstream) and 3' (downstream) of the desired insertion site in the genome. In some embodiments, the homology arms are approximately 500 bp in length. 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 CAR coding sequence together with the homology arms is cloned into a viral vector plasmid. The plasmid is used to package the sequence into a virus.

[0104] 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 regulatory sequences, including transcriptional activator binding sequences, transcriptional repressor binding sequences, enhancers, introns, and the like. In some embodiments, the viral vector provides a constitutive or inducible promoter. In some embodiments, the promoter is selected from EF1α, PGK1, MND, Ubc, CAG, CaMKIIa, and β-actin promoters. In some embodiments, the promoter is selected from SV40 early and late promoters, cytomegalovirus (CMV) immediate early promoter, and Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, β-interferon promoter, and 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.

[0105] In some embodiments, the viral vector provides a transcription terminator.

[0106] In some embodiments, the vector comprises a nucleic acid selected from SEQ ID NOs: 14, 16, 18, 36, and 38.

[0107] In some embodiments, the nucleic acid comprises two or more coding sequences and encodes a polycistronic transcript. In some embodiments, a first coding sequence in the polycistronic transcript encodes a CAR (e.g., SEQ ID NO: 1), and a second coding sequence in the polycistronic transcript encodes a cytokine gene selected from 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 the IL36 signal peptide. In some embodiments, the cytokine is IL36 with the IL2 signal peptide. In some embodiments, the cytokine is IL36 with the IL2 mutant signal peptide described in Zhang, et al. (2005) Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo, J. Gene Medicine, 7:354. The IL-2 mutant signal peptide contains alterations in two domains of the signal peptide: the basic domain and the hydrophobic domain. In some embodiments, the coding sequences in the polycistronic transcript are separated by the coding sequence for a P2A polypeptide to allow for separation of the nascent peptides during translation.

[0108] In some embodiments, the vector is a plasmid selected from a prokaryotic plasmid, a eukaryotic plasmid, and a shuttle plasmid.

[0109] In some embodiments, the CAR is expressed in eukaryotic cells, such as mammalian, human NK cells (or precursors thereof), and the vector is a plasmid containing 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.

[0110] In some embodiments, the expression vector comprises one or more selectable markers. In some embodiments, the selectable marker is an antibiotic resistance gene or other negative selectable marker. In some embodiments, the selectable marker comprises a protein whose mRNA is co-transcribed with the fusion protein mRNA and the polycistronic transcript is cleaved before translation.

[0111] In some embodiments, the expression vector comprises a polyadenylation signal. In some embodiments, the polyadenylation site is an SV-40 polyadenylation signal.

[0112] In some embodiments, NK cells, or their precursor cells, are contacted with a viral vector, whereby 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, the transduced and transfected cells can be tested to confirm transgene expression on the cell surface using methods well known in the art, such as fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. For example, the cells can be tested by staining with an antibody or labeled antigen specific for a portion of the CAR (e.g., ROR1 in the case of engineered anti-ROR1 CAR-T and CAR-NK cells), or by flow cytometry.

[0113] The present invention involves manipulating nucleic acids, including genomic DNA and plasmid DNA, isolated or extracted from a sample. Methods for nucleic acid extraction are well known in the art. See J. Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, 2nd Ed., Cold Spring Harbor Laboratory Press: New York, NY. A variety of reagents and kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples, including products from BD Biosciences (San Jose, Calif.), Clontech (TaKaRa Bio.), Epicentre Technologies (Madison, Wisc.), Gentra Systems (Minneapolis, Minn.), Qiagen (Valencia, Calif.), Ambion (Austin, Tex.), BioRad Laboratories (Hercules, Calif.), KAPA Biosystems (Roche Sequencing Solutions, Pleasanton, Calif.), and others.

[0114] In some embodiments, the invention involves an intermediate purification or separation step for nucleic acids, for example, to remove 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 solid-phase reversible immobilization (SPRI) technology from Beckman Coulter (Brea, Calif.).

[0115] In some embodiments, an exogenous protein-encoding nucleic acid sequence (e.g., a CAR-encoding sequence) is introduced into cells such as T cells or NK cells, or NK cell precursors such as induced pluripotent stem cells (iPSCs). In some embodiments, "naked" nucleic acids are introduced into lymphocytes by electroporation as described in U.S. Patent No. 6,410,319.

[0116] 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 a tracrRNA, a crRNA, or a single guide RNA that combines elements of a tracrRNA and a crRNA in a single molecule). In some embodiments, the components of the CRISPR system are introduced into the cell (e.g., a T cell, an NK cell, or an NK cell precursor) in the form of a nucleic acid.

[0117] In some embodiments, components of the CRISPR system are introduced into cells (e.g., T cells, NK cells, or NK cell precursors) in the form of DNA encoding a nucleic acid-guided endonuclease and a NATNA guide. In some embodiments, a gene encoding the nucleic acid-guided endonuclease (e.g., a CRISPR nuclease selected from Cas9 and Cas12a) is inserted into a plasmid capable of propagating in the target cell. In some embodiments, a gene encoding the NATNA guide is inserted into a plasmid capable of propagating in the target cell.

[0118] In some embodiments, the nucleic acid-guided endonuclease and NATNA guide are introduced into a target cell (e.g., a T cell, NK cell, or NK cell precursor) in the form of RNA, e.g., mRNA, encoding the nucleic acid-guided endonuclease together with the NATNA guide.

[0119] In some embodiments, the nucleic acid-guided endonuclease and NATNA guide are introduced into the target cell (e.g., T cell, NK cell, or NK cell precursor) as a pre-assembled nucleoprotein complex. In some embodiments, the nucleic acid-guided endonuclease and NATNA guide are introduced into the target cell (e.g., T cell, NK cell, or NK cell precursor) via any combination of different means, for example, the endonuclease is introduced as DNA via a plasmid containing a gene encoding the endonuclease, and the guide is introduced in its final form as RNA (or RNA containing DNA nucleotides).

[0120] In some embodiments, the nucleic acid encoding the nucleic acid-guided endonuclease and the NATNA guide is introduced into the cell via electroporation.

[0121] In some embodiments, the nucleic acid encoding the nucleic acid-guided endonuclease is introduced into the cell via electroporation or viral pseudotransduction, e.g., in the form of mRNA as described in U.S. Pat. No. 10,584,352.

[0122] In some embodiments, one or more of the coding sequences described herein are introduced into the genome of a cell with the aid of a sequence-specific endonuclease. In some embodiments, the endonuclease is an endonuclease guided by a nucleic acid encoded by a CRISPR locus. CRISPR (clustered regularly interspaced short palindromic repeats) genomic locus is found in many prokaryotic genomes and provides resistance to the invasion of foreign nucleic acids. The structure, nomenclature, and classification of CRISPR loci are reviewed in Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology. 2011 June;9(6):467-477.

[0123] Briefly, a typical CRISPR locus contains several short repeats regularly spaced with spacers. The CRISPR locus also contains coding sequences for CRISPR-associated (Cas) genes. The spacer repeat units encode CRISPR RNA (crRNA). In vivo, mature crRNA is processed from a polycistronic transcript called pre-crRNA or pre-crRNA array. Repeats within the pre-crRNA array are recognized by Cas-encoded proteins, which bind to and cleave the repeats, liberating mature crRNA. The CRISPR system performs cleavage of the target nucleic acid, where the Cas proteins and crRNA form the CRISPR ribonucleoprotein (crRNP). The crRNA molecule guides the crRNP to the target nucleic acid (e.g., a foreign nucleic acid entering a bacterial cell), and the Cas nuclease protein cleaves the target nucleic acid.

[0124] Type I CRISPR systems contain a means for processing the pre-crRNA array, including a multiprotein complex called CASCADE (CRISPR-associated complex for antiviral defense), composed of CasA, B, C, D, and E subunits. The Cascade-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with the crRNA. A binding nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of the target nucleic acid.

[0125] The type II CRISPR system contains a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to the crRNA repeats within the pre-crRNA array and recruits endogenous RNase III to cleave the pre-crRNA array. The tracrRNA / crRNA complex can associate with a nuclease, such as Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with the crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease for target nucleic acid cleavage.

[0126] Type III CRISPR systems comprise the RAMP superfamily of endoribonucleases (e.g., Cas6), which cleave the pre-crRNA array with the aid of one or more CRISPR polymerase-like proteins.

[0127] Type VI CRISPR systems contain a different 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.

[0128] V-type CRISPR systems are 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. (Nat. Rev. Microbiol., 2020, 18:67-83) and Pausch et al. (Science, 2020, 369(6501):333-337). The VA subtype encodes the Cas12a protein (formerly known as Cpf1). Cas12a has a RuvC-like nuclease domain that is homologous to the corresponding domain in Cas9, but lacks the HNH nuclease domain present in the Cas9 protein. V-type systems can contain a single crRNA sufficient to target Cas12 to the target site, or a crRNA-tracrRNA guide pair for targeting Cas12 to the target site.

[0129] CRISPR endonucleases require nucleic acid targeting nucleic acids (NATNAs), also known as guide RNAs. The endonucleases can form ribonucleoprotein complexes (RNPs) with one or more guide RNAs. In some embodiments, the endonuclease is a type II CRISPR endonuclease, and the NATNA includes a tracrRNA and a crRNA.

[0130] In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752. Briefly, the NATNA can comprise, in 5' to 3' order, a spacer extension, a spacer, a minimal CRISPR repeat, a single guide connector, a minimal tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension. In some cases, the targeting nucleic acid can comprise, in any order, a tracrRNA extension, a 3' tracrRNA sequence, a minimal tracrRNA, a single guide connector, a minimal CRISPR repeat, a spacer, and a spacer extension.

[0131] In some embodiments, the guide nucleic acid targeting a nucleic acid can comprise a single-guide NATNA. The NATNA comprises a spacer sequence that can be engineered to hybridize to the target nucleic acid sequence. The NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to the tracrRNA sequence. Optionally, the NATNA can have a spacer extension and a tracrRNA extension. These elements can include elements that can contribute to the stability of the NATNA. The CRISPR repeat and the tracrRNA sequence can interact to form a double-stranded structure through base pairing. This structure can facilitate the binding of an endonuclease to the NATNA.

[0132] In some embodiments, the single-guide NATNA comprises a spacer sequence located 5' of the first duplex, which comprises the region of hybridization between the minimal CRISPR repeats and the minimal tracrRNA sequence. The first duplex may be interrupted by a bulge. The bulge facilitates recruitment of an endonuclease to the NATNA. Following the bulge can be a first stem comprising a linker connecting the minimal CRISPR repeats and the minimal tracrRNA sequence. The last pair of nucleotides at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to the intermediate tracrRNA. The intermediate tracrRNA can comprise one or more additional hairpins.

[0133] In some embodiments, the NATNA can comprise a dual-guide nucleic acid structure. The dual-guide NATNA comprises a spacer extension, a spacer, minimal CRISPR repeats, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension. The dual-guide NATNA does not comprise 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 via the minimal CRISPR repeats and the minimal tracrRNA sequence.

[0134] In some embodiments, the NATNA is an engineered guide RNA (CRISPR hybrid RNA-DNA or chRDNA) containing one or more DNA residues. In some embodiments, the NATNA is selected from the embodiments described in U.S. Patent No. 9,650,617. In some embodiments, the NATNA is selected from the embodiments described in International Application Publication No. 2022 / 086846, "DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same." Briefly, some chRDNAs for use with Type II CRISPR systems may be composed of two strands that form a secondary structure including an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and an activation region composed of one or more hairpins. The nucleotide sequence immediately downstream of the targeting region may contain varying proportions of DNA and RNA. Other chRDNAs can be single-guide DNAs for use with type II CRISPR systems, including a targeting region and an activation region consisting of a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins. The nucleotide sequence immediately downstream of the targeting region can contain various proportions of DNA and RNA. For example, the targeting region can contain DNA or a mixture of DNA and RNA, and the activation region can contain RNA or a mixture of DNA and RNA.

[0135] 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 CAR coding sequence) into the genome of a cell is a restriction endonuclease, e.g., a Type II restriction endonuclease.

[0136] 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 Fok I nuclease improves the specificity of genome modification, Nature biotechnology, 32(6), 577-582.)

[0137] In some embodiments, the endonuclease used to introduce one or more of the genetic modifications described herein is a zinc finger nuclease (ZFN) or a ZFN-Fok I fusion. In such embodiments, the target sequence is approximately 22-52 bases in length and comprises a pair of ZFN recognition sequences, each 9-18 nucleotides in length, separated by a spacer that is 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.)

[0138] In some embodiments, the endonuclease used to introduce one or more of the 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 approximately 48-85 nucleotides in length and comprises a pair of TALEN recognition sequences, each 18-30 bases in length, separated by a spacer that is 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.)

[0139] In some embodiments, quality control measures that evaluate one or more characteristics of the engineered anti-ROR1 CAR-T cells and CAR-NK cells are applied to the cells before they are administered to a patient.

[0140] In some embodiments, the assessed characteristic of CAR-T cells and CAR-NK cells is the presence of CAR nucleic acid in the cellular genome. The presence of CAR in the cellular genome can be assessed by a method selected from 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 cellular genome is assessed by ddPCR using amplification primers specific for one or both CAR insertion sites.

[0141] In some embodiments, the assessed characteristic of CAR-T cells and CAR-NK cells is surface expression of the CAR. Surface expression of the CAR can be assessed by fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. In some embodiments, surface expression of the CAR is assessed by flow cytometry using an anti-Fab2 antibody or labeled purified antigen. In some embodiments, surface expression of the CAR is assessed by flow cytometry using ROR1. In some embodiments, the CAR-T cell or CAR-NK cell population with the highest surface expression of the CAR is selected for administration to the patient.

[0142] In some embodiments, the fraction of cells in a cell population that harbor a CAR in their genome or the fraction of cells in a cell population that express a CAR on their cell surface is used to determine the total number of cells in the cell population that constitute a therapeutically effective dose.

[0143] In some embodiments, the properties of CAR-T cells and CAR-NK cells are assessed in vitro and are selected from antigen-dependent lysis of antigen-expressing target cells (antigen-specific lysis), proliferation in the presence of antigen-expressing target cells (antigen-dependent proliferation), and cytokine secretion, cell exhaustion, and the presence of a memory cell phenotype in the presence of antigen-expressing target cells.

[0144] In some embodiments, in vitro evaluation of 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 an antigen specific to CAR-T cells or CAR-NK cells, i.e., the tumor cells express ROR1, which is recognized by anti-ROR1 CAR-T cells and CAR-NK cells. In some embodiments, the tumor cells are derived from the tumor cell lines SKOV3, REC1, Mino, and JeKo-1. In some embodiments, the target cells are first evaluated for ROR1 expression. An example of evaluating ROR1 expression in target cells is provided as Example 3 and FIG. 4 of the present disclosure.

[0145] In some embodiments, the characteristic being evaluated is antigen-dependent lysis of antigen-bearing target cells. ROR1-dependent cytolysis can be evaluated by co-culturing a population containing engineered anti-ROR1 CAR-T cells or CAR-NK cells (effector cells or effectors) with ROR1-expressing target cells (targets). In some embodiments, the target cells are ROR1-expressing tumor cell lines. In some embodiments, the ROR1-expressing tumor cell lines are selected from JeKo1 (B-cell lymphoma, BCL), REC1 (mantle cell lymphoma, MCL), and Mino (MCL). In some embodiments, the target cells are primary cells from a patient suffering from a tumor known to express ROR1. In some embodiments, the primary cells are derived from a lymphoma patient, e.g., a B-cell lymphoma patient. Co-cultures can be established at different effector:target ratios (E:T ratios). In some embodiments, the E:T ratio ranges from about 0.1 to about 10. In some embodiments, two or more E:T ratios within a selected range are evaluated. In some embodiments, ROR1-expressing target cells are pre-labeled with a cell tracking dye (e.g., CellTrace™ Violet dye (CTV)) and co-cultured with anti-ROR1 CAR-T or CAR-NK effector cells at a range of E:T ratios. At the time of analysis, a dead cell stain (e.g., 7-aminoactinomycin (7AAD) stain) is added to the culture. The number of live target cells is then determined, e.g., by flow cytometry, based on the number of cells with no remaining tracking dye / dead stain (e.g., CTV). + 7AAD - ) cells. In some embodiments, the number of viable target cells is normalized to the number of viable target cells in control cultures lacking anti-ROR1 CAR-T or CAR-NK cells. An example of measuring in vitro target cell lysis by anti-ROR1 CAR-NK cells is provided as Example 3 and Figures 5 and 6 of the present disclosure. An example of measuring in vitro target cell lysis by anti-ROR1 CAR-T cells is provided as Example 14 and Figures 27A-27C of the present disclosure.

[0146] In some embodiments, the property assessed is repeated antigen-dependent lysis of antigen-bearing target cells in a serial rechallenge assay. Antigen-dependent cytolysis can be assessed by serially challenging the same population of engineered anti-ROR1 CAR-T cells or CAR-NK cells with two or more fresh aliquots of target cells. An example of measuring repeated in vitro target cell lysis by anti-ROR1 CAR-NK cells is provided as Example 4 and Figure 7 of the present disclosure. An example of measuring repeated in vitro target cell lysis by anti-ROR1 CAR-T cells is provided as Example 20 and Figure 32 of the present disclosure.

[0147] In some embodiments, the CAR-T or CAR-NK cell population that results in the highest percentage of ROR1-expressing target cell lysis is selected for administration to the patient. In some embodiments, a CAR-T or CAR-NK cell population that results in a high percentage of ROR1-expressing target cell lysis but has low non-specific target cell lysis is selected for administration to the patient. In some embodiments, the CAR-T or CAR-NK cell population that results in the highest percentage of repeated ROR1-expressing target cell lysis is selected for administration to the patient.

[0148] In some embodiments, the property assessed is antigen-dependent proliferation of CAR-T or CAR-NK cells. Proliferation can be assessed by co-culturing a population containing engineered anti-ROR1 CAR-T or CAR-NK cells (effector, E) with ROR1-expressing target cells (target, T). In some embodiments, the co-culture is at an E:T ratio of about 1. In some embodiments, cell proliferation is detected by labeling CAR-T or CAR-NK cells with a cell-permeable, stable fluorescent dye (e.g., CellTrace™ Violet) and measuring dye dilution within the CAR-T or CAR-NK cell population.

[0149] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the highest proliferation rate in the presence of ROR1-expressing target cells is selected for administration to the patient.

[0150] In some embodiments, the characteristic evaluated is cytokine or chemokine secretion by CAR-T or CAR-NK cells. In some embodiments, 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-ROR1 CAR-T or CAR-NK cells (effector, E) with ROR1-expressing target cells (target, T). In some embodiments, the co-culture is at an E:T ratio of about 1. In some embodiments, cytokines or chemokines in the co-culture supernatant can be detected or quantitatively detected by antibody- or antibody conjugate-based assays, such as Western blot or ELISA, and similar secondary antibody-based methods with colorimetric or fluorescent detection methods.

[0151] An example of measuring repeated in vitro target cell lysis (in a serial re-challenge assay) by anti-ROR1 CAR-NK cells is provided as Example 9 of the present disclosure, and Figures 18A-18B and 19A-19B. An example of measuring repeated in vitro target cell lysis by anti-ROR1 CAR-T cells is provided as Example 20 and Figure 32 of the present disclosure.

[0152] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the highest level of cytokine or chemokine secretion in the presence of ROR1-expressing target cells is selected for administration to the patient.

[0153] In some embodiments, the properties of the CAR-T or CAR-NK cells are evaluated in vivo and selected to affect the properties of experimental animals bearing target tumor cells. In some embodiments, the target cells are tumor cells known to express ROR1, and the experimental animals are mice implanted with the tumor cells before receiving a dose of anti-ROR1 CAR-T or CAR-NK cells. In some embodiments, the experimental animals are NGS mice implanted with JeKo-1 tumor cells. In some embodiments, evaluation of the CAR-T or CAR-NK cells includes monitoring the body weight, overall survival, and tumor burden of mice implanted with tumor cells and administered a dose of anti-ROR1 CAR-T or CAR-NK cells.

[0154] Examples of measuring in vivo reduction of tumor burden in animals implanted with JeKo-1 tumors by administration of anti-ROR1 CAR-NK cells are provided as Example 5 and Figure 8, and Example 6 and Figure 9 of the present disclosure.

[0155] An example of measuring the in vivo reduction of tumor burden in animals implanted with JeKo-1 tumors by administration of anti-ROR1 CAR-T cells is provided as Example 16 and Figures 29A-29B of the present disclosure.

[0156] In some embodiments, animals are implanted with fluorescently labeled tumor cell lines and tumor burden is assessed by measuring in vivo fluorescence (and other mouse measurements). In some embodiments, experimental animals are immunodeficient NGS mice implanted with JeKo-1-GFPluc luciferase-expressing tumor cells. In some embodiments, results are expressed as the change in tumor (or animal) fluorescence over time and assessed as the area under the curve (AUC).

[0157] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the greatest reduction in tumor burden in experimental animals implanted with ROR1-expressing tumors and injected with anti-ROR1 CAR-T or CAR-NK cells is selected for administration to the 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 the patient.

[0158] In some embodiments, the property evaluated is the persistence of anti-ROR1 CAR-T or CAR-NK cells in the circulation of experimental animals implanted with ROR1-expressing tumors 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 measuring the CD56 expression level in a volume of the animal's blood. + In some embodiments, the persistence of anti-ROR1 CAR-T cells is assessed as the presence and / or number of CD8 cells in a volume of the animal's blood. + The presence and / or number of anti-ROR1 CAR-NK cells or CAR-T cells in the blood sample is assessed. In some embodiments, qualitative and / or quantitative assessment of anti-ROR1 CAR-NK cells or CAR-T cells in the 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 antibody and anti-human CD8 antibody.

[0159] An example of measuring the in vivo persistence of anti-ROR1 CAR-NK cells in the circulation is provided as Example 7 and Figure 10 of the present disclosure.

[0160] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the highest persistence in the circulation of experimental animals implanted with ROR1-expressing tumors and injected with anti-ROR1 CAR-T or CAR-NK cells is selected for administration to patients.

[0161] In some embodiments, the characteristic evaluated is the continued expression of anti-ROR1 CAR in CAR-T or CAR-NK cells in the circulation of experimental animals implanted with ROR1-expressing tumors and injected with anti-ROR1 CAR-T or CAR-NK cells. In some embodiments, CAR expression is assessed by flow cytometry and compared to or normalized to the expression of additional NK-associated genes. In some embodiments, the additional genes are one or more genes selected from CD45 (or hCD45) and CD56.

[0162] An example of measuring the expression of anti-ROR1 CAR in CAR-NK cells is provided as Example 7 and Figure 11 of the present disclosure.

[0163] In some embodiments, the CAR-T or CAR-NK cell population that exhibits the highest level of anti-ROR1 CAR expression among cells recovered from the circulation of experimental animals implanted with ROR1-expressing tumors and injected with anti-ROR1 CAR-T or CAR-NK cells is selected for administration to the patient.

[0164] In some embodiments, CAR-NK cell clones or populations are selected for inclusion in the therapeutic compositions described herein. The inventors surprisingly discovered that anti-ROR1 CAR-NK cells with similar in vitro tumor cell killing capabilities exhibit substantial variations in in vivo anti-tumor activity. (See Figures 5-6, 9-10.)

[0165] In some embodiments, the CAR-T cells of the present invention are compared with existing anti-ROR1 CAR-T cells. One example of a cutting-edge 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 ESMO Congress 2022, Paris, France.). LYL797 CAR-T cells express a CAR containing a single-chain variable fragment (scFv) derived from the high-affinity monoclonal antibody R12, which binds to the amino-terminal Ig-like / Frizzled 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).

[0166] In some embodiments, the CAR-T cells of the invention are superior to T cells expressing a benchmark CAR (R12) in surface CAR expression, in vitro antigen-dependent cytotoxicity, in vivo tumor control, and prolonged survival in experimental animals implanted with ROR1-expressing tumors. See Example 26.

[0167] In some embodiments, the invention includes compositions comprising anti-ROR1 CAR-T or CAR-NK cells that exhibit anti-tumor properties. In some embodiments, the invention includes compositions comprising both anti-ROR1 CAR-T and CAR-NK cells that exhibit anti-tumor properties. In some embodiments, the invention includes compositions comprising anti-ROR1 CAR-T or CAR-NK cells that are evaluated for having satisfactory properties or satisfactory levels of parameters selected from one or more of the following: presence of the CAR in the cellular genome, surface expression of the CAR, antigen-dependent cytotoxicity in vitro, anti-tumor activity in vivo, antigen-dependent proliferation in vivo or in vitro, and cytokine secretion in vivo or in vitro.

[0168] Once produced and (optionally) evaluated for desired properties, the engineered cells can be formulated into a composition for delivery to a human subject to be treated. The composition comprises engineered lymphocytes and one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Suitable excipients for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates, e.g., sugars, derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and / or sugar polymers, can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, and myo-inositol. The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monosodium phosphate, disodium phosphate, and the like, and combinations thereof.

[0169] In some embodiments, the composition further comprises an antimicrobial agent to prevent or inhibit microbial growth, hi some embodiments, the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and the like, and combinations thereof.

[0170] In some embodiments, the composition further comprises an antioxidant added to prevent degradation of lymphocytes, hi some embodiments, the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium disulfite, and the like, and combinations thereof.

[0171] In some embodiments, the composition further comprises a surfactant, which in some embodiments is selected from lipids such as polysorbates, sorbitan esters, phospholipids (lecithin and other phosphatidylcholines), phosphatidylethanolamines, fatty acids and fatty acid esters, steroids such as cholesterol, and the like.

[0172] In some embodiments, the composition further comprises a cryogen, for example, 3% to 12% dimethyl sulfoxide (DMSO) or 1% to 5% human albumin.

[0173] The number of anti-ROR1 CAR-T and / or CAR-NK cells in the composition will vary depending on several factors, but will optimally contain a therapeutically effective dose per vial, which can be empirically determined by repeatedly administering increasing amounts of the anti-ROR1 CAR-T and / or CAR-NK cell-containing composition to determine which amount produces a clinically desired endpoint.

[0174] In some embodiments in which the subject is a human, the number of anti-ROR1 CAR-T cells per dose is about 2×10 7 fewer than 10 cells, and the number of anti-ROR1 CAR-NK cells per dose was approximately 2 x 10 8 In some embodiments, the dose is less than 4 x 10 cells. 7 , 8×10 7 , 1.2 × 10 8 , or 1×10 6 ~2×10 8 In some embodiments, the dose comprises a suitable number of anti-ROR1 CAR-T cells in the range of 4×10 8 , 8×10 8 , 1.2 × 10 9 , or 1×10 7 ~2×10 9 In some embodiments, the dose comprises a suitable number of anti-ROR1 CAR-NK cells in the range of 1:10. In some embodiments, the dose 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 in a ratio of approximately 1:10.

[0175] In some embodiments, the total number of cells in a dose is adjusted based on the percentage of CAR-expressing cells or cells in the total cells in the cell composition. In some embodiments, the total number of cells to be administered is multiplied by 100 / N, where N is the percentage of CAR-expressing cells in the cell composition. Expansion results in the total number of cells that need to be administered to a patient to administer the desired number of CAR-expressing cells.

[0176] In some embodiments, the present invention is a method for treating, preventing, or alleviating a disease associated with expression of ROR1, comprising administering a population of immune cells, anti-ROR1 CAR-T cells described herein, anti-ROR1 CAR-NK cells, or a combination thereof.

[0177] In some embodiments, the population of immune cells administered to the patient has been assessed for having satisfactory characteristics or satisfactory levels of a parameter selected from one or more of the following: presence of a CAR in the cellular genome, surface expression of a CAR, antigen-dependent cytotoxicity in vitro, anti-tumor activity in vivo, antigen-dependent proliferation, and cytokine secretion.

[0178] In some embodiments, diseases or conditions that can be treated by the immune cells of the present disclosure include various malignancies, including solid tumors selected from ovarian cancer, triple-negative breast cancer, colorectal cancer, non-small cell lung cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, melanoma, and endometrial cancer, and hematological tumors selected from CLL, SLL, B-ALL, B-NHL, MCL, and AML. ROR1 is an abundantly expressed tumor antigen 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.

[0179] In some embodiments, the invention is a method of inhibiting tumor growth in a patient.

[0180] In some embodiments, the invention includes methods 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 expanded prior to administration. In some embodiments, administration of immune cells according to the invention results in the treatment, prevention, or alleviation of a disease or condition in the subject or patient. In some embodiments, the disease or disorder is selected from cancer or tumor and infectious diseases that can be treated by administration of immune cells that elicit an immune response.

[0181] 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, hi some embodiments, the repeated administration is performed 1, 2, 3 or more times with 1, 2, 3 or more days between administrations.

[0182] Pharmaceutical compositions comprising T cells, NK cells, or a combination of T cells and NK cells expressing the anti-ROR1 CAR of the present disclosure can be delivered via a variety of routes and delivery methods, such as local or systemic delivery, including parenteral, intramuscular, intravenous, subcutaneous, or intradermal delivery.

[0183] In some embodiments, the methods include administering a composition of the invention to a subject who has been preconditioned with immunodepleting (e.g., lymphodepleting) therapy. In some embodiments, the preconditioning is with a lymphodepleting agent comprising a combination of cyclophosphamide and fludarabine.

[0184] In some embodiments, the composition or formulation for administration to a patient is a pharmaceutical composition or formulation that allows for the biological activity of the active ingredient and contains only non-toxic additional components, such as pharmaceutically acceptable carriers, which in some embodiments include buffers, excipients, stabilizers, and preservatives.

[0185] In some embodiments, a preservative is used. In some embodiments, the preservative includes 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 an amount of from about 0.0001% to about 2% by weight of the total composition.

[0186] 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 dextrin, 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 non-ionic surfactant such as polyethylene glycol (PEG).

[0187] In some embodiments, the carrier comprises a buffer solution. In some embodiments, the buffer solution comprises citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, the buffer solution is present at about 0.001% to about 4% by weight of the total composition.

[0188] 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.

[0189] 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 the cells in sterile water, saline, or a solution or solvent such as glucose, dextrose, etc. In some embodiments, the pharmaceutical composition further comprises dispersing or emulsifying agents, pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, dyes, etc., depending on the route of administration and the desired preparation.

[0190] In some embodiments, T cells or NK cells expressing an anti-ROR1 CAR described herein are co-administered with a cytokine. 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.

[0191] In some embodiments, T cells or NK cells expressing an anti-ROR1 CAR described herein are engineered to constitutively express a cytokine. 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 the cytokine to its receptor (cytokine-receptor fusion). In some embodiments, the membrane-bound cytokine receptor fusion is selected from an IL-15-IL-15 receptor fusion (IL-15-IL15RA fusion) and an IL-21-IL-21 receptor fusion (IL-21-IL-21RA fusion). In some embodiments, the fusion also comprises a signal peptide. In some embodiments, the leader peptide is selected from an IL-2 signal peptide and a CD2 signal peptide.

[0192] 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 generated mouse CD8 T cells expressing the fusion. + showed improved T cell survival and proliferation.

[0193] Fusions of human IL-21 with its receptor are described in International Application No. PCT / US23 / 67427, filed May 24, 2023, entitled "Cytokine-receptor fusions for immune cell stimulation."

[0194] In some embodiments, the coding sequence for the membrane-bound cytokine is introduced into the cell via chemical or electrochemical means (such as lipid nanoparticles or electroporation), hi some embodiments, the coding sequence for the membrane-bound cytokine is introduced into the cell using a vector, such as a lentiviral vector.

[0195] In some embodiments, the lentiviral vector comprises an expression construct including a promoter and coding sequence for a cytokine and its receptor. In some embodiments, the cytokine and its receptor are linked 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 virus long terminal repeat (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, β interferon promoter, and hsp70 promoter. In some embodiments, the promoter is an EF-1α promoter.

[0196] In some embodiments, the lentiviral constructs described herein are 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 in vitro from iPSCs).

[0197] In some embodiments, a lentiviral construct described herein is introduced into iNK cell precursors, such as iPSCs. In some embodiments, the introduction of a lentiviral construct described herein and the presence of a cytokine receptor fusion does not negatively affect the ability of the iPSCs to differentiate into iNK cells.

[0198] In some embodiments, T cells or NK (or iNK) cells are assessed for surface expression of membrane-bound cytokines. Surface expression of membrane-bound cytokines can be assessed by fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot. In some embodiments, surface expression of membrane-bound cytokines is assessed by flow cytometry using anti-cytokine antibodies. In some embodiments, the T cell or NK (or iNK) cell population with the highest surface expression of membrane-bound cytokines is selected for administration to the patient. In some embodiments, the iPSC cell population with the highest surface expression of membrane-bound cytokines is selected for differentiation into iNK cells.

[0199] In some embodiments, membrane-bound cytokine-expressing T cells or NK (or iNK) cells are assessed for cytotoxic properties. In some embodiments, cytotoxic properties are assessed by co-culture with human tumor cells. In some embodiments, the membrane-bound cytokine-expressing NK (or iNK) cell population with the highest cytotoxic activity is selected for administration to the patient. [Example]

[0200] Example 1. Anti-ROR1 CAR constructs The CAR construct is shown in Figure 1. The CAR contained three single-chain variable regions (scFv) described in International Application No. PCT / US2023 / 067314, filed May 22, 2023, entitled "Anti-ROR1 antibody and ROR1-targeting engineered cells." The CAR contains a CD8 hinge and transmembrane domain, and the cytoplasmic domain consists of the 4-1BB and CD3 zeta domains. The expression construct further contains a minimal CAG (mCAG) CMV-derived promoter.

[0201] Example 2. Generation of iNK cells expressing anti-ROR1 CAR In this experiment, differentiated (induced) CAR-NK cells (CAR-iNK cells) were generated according to the scheme shown in Figure 2. CAR insertion was performed before the initial differentiation step.

[0202] CAR insertion Briefly, CAR constructs (Figure 1) were introduced into induced pluripotent stem cells (iPSCs) via lentiviral transduction (HIV-1-derived virus, (SignaGen Laboratories, Frederick, Md.) MOI 30) and selection with Zeocin. Successful transduction was assessed by CAR staining and sorting (FACS) based on iNK differentiation. Co-staining with anti-ROR1 CAR and CD56 confirmed CAR expression and iNK differentiation stage.

[0203] Differentiation of iPSCs into NK cells Briefly, to initiate differentiation of iPSC clonal lines, spheroids of defined size were generated and seeded on LN-511-coated surfaces. The spheroids were grown in medium containing a specific set of cytokines (BMP4, VEGF, SCF, IL3, IL6, and TPO) to induce differentiation into hematopoietic cells (HPCs). There were two periods of HPC differentiation (each consisting of a 1-week interval) with different cytokine cocktails in the medium.

[0204] Cells were harvested from the HPC differentiation phase, enriched for CD34-positive cells, and transferred to a culture vessel containing the first irradiated feeder cell line (AFT024 - a mouse fetal liver stromal cell line) cultured on a surface (seeded 1 day prior). This transfer onto irradiated feeder line #1 in combination with a cytokine cocktail (IL3, IL15, IL7, SCT, FLT3L) initiated the iNK differentiation phase. Differentiating iNKs were replated onto fresh feeders weekly. Medium changes were performed after 3 days. The iNK differentiation phase continued for 4 weeks. Cells were characterized weekly by in-process flow cytometry to test for CD45 and CD56 positivity.

[0205] After four weeks of iNK differentiation, a two-week iNK expansion phase was performed. iNK cells were cocultured with irradiated suspensions of a second feeder cell line (41BBL and K562 cell lines engineered to overexpress membrane-bound IL-21). After seven days of expansion, cultured cells were characterized by flow cytometry. Expanded iNKs were cryopreserved at this point. For in vitro and in vivo functional assessment, cryopreserved iNKs were thawed and fresh feeders were added to begin the second week of expansion. Medium changes were performed every two to three days. After two weeks of expansion, the final expanded iNK product was evaluated in in vitro and in vivo functional assays.

[0206] Anti-ROR1 CAR expression was measured during the differentiation process by flow cytometry staining with ROR1-Avi tag (ACROBiosystems, Newark, Del.). Figure 3 shows the percentage of CAR-expressing cells during each stage of iNK differentiation in cells transduced with each of the CAR constructs shown in Figure 1.

[0207] Example 3. In vitro anti-ROR1 cytotoxicity In this experiment, the anti-ROR1 iNK cells of Example 2 were tested in vitro for cytotoxicity against the ROR1-expressing tumor cell lines SKOV3 and JeKo-1, which had been engineered to express GFPluc. ROR1 expression in SKOV3 and JeKo-1 was assessed by flow cytometry. The results are shown in Figure 4.

[0208] 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.

[0209] The results for the SKOV3 target cell line are shown in Figure 5. The percentage of remaining viable target cells after E:T = 10:1 co-culture was determined by the percentage of remaining GFP +Evaluation was performed using Incucyte® Live-Cell Analysis Systems (Sartorius, AG, Gottingen, Germany), an image analysis system capable of detecting SKOV3 cells.

[0210] JeKo-1-GFPluc target cells were pre-labeled with cell trace violet dye (CTV) and co-cultured with anti-ROR1 CAR iNK effector cells at a range of E:T ratios. At the time of analysis, 7AAD dead cell stain was added to the cultures. Assay analysis was performed using the remaining CTV normalized to target-only (no effector) wells. + 7AAD - Based on flow cytometry counting of (viable target cells), the results are shown in Figure 6.

[0211] Example 4. Repeated cytotoxic activity by anti-ROR1 iNK cells In this experiment, a repeated challenge ("serial rechallenge") assay was performed on the GFP-expressing JeKo-1 cell line. Briefly, plates seeded with target cells were co-cultured with anti-ROR1 CAR iNK cells. After 1 and 2 days, additional target cells were added to selected plates to test whether the iNK cells were still capable of mounting a cytotoxic response. The results are shown in Figure 7 as specific lysis of target cells at various E:T ratios. The three lines in each chart represent the first, second, and third encounter of target cells by iNK cells. The dashed line represents the level of cytotoxic activity of control (no CAR) iNK cells upon the first challenge with target cells.

[0212] Example 5. In vivo anti-tumor activity of anti-ROR1 CAR-iNK cells in NGS mice In this experiment, the in vivo anti-tumor activity of anti-ROR1 CAR iNK cells was evaluated in NGS mice. Assay design shown in Table 1: [Table 1]

[0213] Briefly, JeKo-1-GFPluc cells were injected intravenously (IV). Effector cells were also injected IV on day 3 post-transplant. Animals were dosed intraperitoneally (IP) with IL-2 (10,000 U / animal) on the day of iNK injection and then every 3–4 days up to day 21. Animals were also dosed IP with IL-15 (10 ng / animal) on the day of iNK injection and then daily up to day 7. Magnetic beads used for CAR enrichment were not removed prior to iNK transplant.

[0214] The results are shown in Figure 8 as the change in tumor burden measured as area under the curve (AUC) for each treatment group.

[0215] Example 6. In vivo anti-tumor activity of anti-ROR1 CAR-iNK cells in NOG mice expressing human IL-15 In this experiment, the in vivo anti-tumor activity of anti-ROR1 CAR iNK cells was evaluated in NOG mice expressing transgenic human IL-15. Assay design shown in Table 2. [Table 2]

[0216] Mice engineered to express transgenic human IL-15 (hIL-15) were used. Briefly, JeKo-1-GFPluc cells were injected intravenously (IV). Three days after transplantation, effector cells were also injected IV. Except for group 6, which did not receive IL2, animals were intraperitoneally (IP) dosed with IL2 (10,000 U / animal) on the day of iNK injection and every 3–4 days thereafter up to day 21. Unlike Example 5, no exogenous IL15 was used. Also unlike Example 5, magnetic beads used for CAR enrichment were removed prior to iNK transplantation.

[0217] Example 7. Persistence of circulating anti-ROR1 CAR-iNK cells in vivo. In this example, the characteristics of iNK cells were evaluated in treated NOG-hIL15 animals. Blood samples were collected 7 days after transplantation, and iNK cells were quantified by flow cytometry by co-staining with CD45, CD56, and using counting beads. The results are shown in Figure 10. iNK cell counts are normalized to the volume of blood collected.

[0218] The cells were further evaluated for continued expression of the anti-ROR1 CAR. Surface expression of human CD45, human CD56, GFP, and the anti-ROR1 CAR was detected by flow cytometry. The results are shown in Figure 11.

[0219] Example 8. CBLB-deficient anti-ROR1 iNK cells In this example, the CBLB gene was disrupted in an iPSC cell line using CRISPR Cas12a and DNA-containing guide RNA (chRDNA) essentially as described in International Patent Application Publication No. WO 2022 / 086846, "DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same. The chRDNA was designed to target the CBLB locus for cleavage by Cas12a and subsequent DNA repair, resulting in disruption of the gene sequence and silencing ("knockout") of the gene.

[0220] CBLB-deficient iPSCs were then differentiated into iNKs essentially as described in Example 2.

[0221] The phenotype of the resulting CBLB KO iNK cells was assessed for perforin and granzyme B secretion by commercially available immunoassays (Figure 12). The phenotype was further assessed by flow cytometry by measuring the expression levels of 11 NK cell markers relative to CD45 and CD56 (Figure 13).

[0222] CBLB KO iNK were further evaluated for cytotoxicity against SKOV3 ovarian tumor target cells in in vitro coculture essentially as described in Example 3. The cytotoxic effect of CBLB KO iNK is shown in Figure 14. CBLB KO iNK-SKOV3 cocultures were also evaluated for iNK degranulation (measured as release of CD107a). The results are shown in Figure 15.

[0223] Furthermore, CBLB KO iNKs were evaluated for their anti-tumor activity in vivo in SVOV3 tumor-implanted NSG mice essentially as described in Example 5, and the results are shown in FIG.

[0224] Example 9. CBLB-deficient anti-ROR1 iNK cells and CISH-deficient anti-ROR1 iNK cells Disruption of the CBLB gene was described in Example 8. In this example, the CISH gene was disrupted in a separate iPSC cell line using CRISPR Cas12a chRDNA essentially as described in WO2022 / 086846. The chRDNA was designed to target the CISH locus for cleavage by Cas12a and subsequent DNA repair, resulting in disruption of the gene sequence and silencing ("knockout") of the gene. CBLB-deficient and CISH-deficient iPSCs were differentiated into iNKs essentially as described in Example 2.

[0225] In vitro activity of CBLB-deficient iNK and CISH-deficient iNK 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. The number of remaining viable SKOV3 cells was determined by counting the number of GFP luc cells. + GFP from SKOV3 target cells + YO - PRO3 +Viable SKOV3 GFP at time zero was calculated via imaging subtracting labeled stained target cells + YO - PRO3 - Normalized to counts. Results are shown in Figure 17.

[0226] Effector molecule secretion by CBLB-deficient and CISH-deficient iNKs was assessed with and without coculture with SKOV3 ovarian tumor target cells. IFNγ, TNFα, perforin, and granzyme B were measured after 24 hours of culture of CISH KO or CBLB KO iNKs, or primary NK (pNK) or wild-type iNKs with or without SKOV3 target cells at a 1:1 E:T ratio as controls. Effector molecules were measured using ProcartaPlex immunoassays on a Luminex instrument (ThermoFisher Scientific, Waltham, Mass.). Results are shown in Figures 18 and 19.

[0227] In vivo antitumor activity of CBLB KO iNK and CISH KO iNK In this experiment, 3 × 10 5 SKOV3-GFPluc tumor cells were injected intraperitoneally (IP) into NSG mice on day -4. On day -1, tumor-implanted animals were analyzed by fluorescence imaging (IVIS® Spectrum in vivo Imaging System, Perkin Elmer, Waltham, Mass.). Animals were randomized across treatment groups. On day 0, 2 × 10 7 iNKs (CBLB KO iNKs and CISH KO iNKs) were injected IP. Animals were then imaged twice weekly for up to 45 days to collect bioluminescence data. Area under the curve (AUC) analysis of bioluminescence intensity is shown in Figure 20. The AUC analysis covered only the time during which all vehicle control animals survived. P-value annotation *>0.05. The survival probability for vehicle and iNK-treated groups is shown in Figure 21.

[0228] Example 10. Immunomasculination of anti-ROR1 iNK cells 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 disrupted by CRISPR Cas12a cleavage, but no construct was inserted ("B2M KO iNK").

[0229] The design and insertion of a B2M-HLA-E fusion construct into the B2M locus was performed essentially as described in WO 2022 / 086846. Briefly, the fusion nucleic acid construct encoded, in N-direction, an N-terminal B2M secretion signal, an HLA-G-derived peptide sequence, a first linker sequence, a B2M sequence, a second linker sequence, and an HLA-E sequence. The nucleic acid construct further contained an EF1α mammalian promoter sequence and a C-terminal BGH polyadenylation signal sequence.

[0230] The resulting edited iNK cells were tested for cytotoxic properties and survival in co-culture with allogeneic T cells. + T cells were obtained by enrichment with negative selection from donor-derived PBMCs. T cells were co-cultured with wild-type iNK, B2M KO 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 with PMA / ionomycin. On day 6, T cells in all cultures were counted by CD8 staining and counting beads. The results are shown in Figure 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 Figure 22 ("NK cell count"). Also, the CD56 of iNK cells was counted. + CD8 in the population + Population proportions were also determined (Figure 23).

[0231] Example 11. Cytokine-expressing anti-ROR1 iNK This example describes iNKs engineered to constitutively express the membrane-bound cytokine IL-15 (mbIL-15).

[0232] a. Production of mbIL-15-expressing iNK Lentiviral constructs were designed to transduce iPSCs and differentiate the engineered iPSCs into iNK cells. The design of the lentiviral construct for expressing membrane-bound IL-15 (mbIL-15) is shown in Figure 24. The expression construct inserted into the lentiviral vector contains the EF-1α promoter and the coding sequences for IL-15 and IL-15 receptor (IL-15RA) connected by a serine-glycine linker. The fusion also contains a CD2 signal sequence. Transduction of iPSCs with the lentiviral construct was performed as described in Example 2. The iPSCs were then selected for IL-15-positive cells using a cell sorter (SH800, Sony Biotechnology, San Jose, Calif.) to sort IL-15-positive cells using an anti-IL-15 primary antibody and an anti-mouse Alexa Fluor 488-conjugated secondary antibody.

[0233] Once selected, mbIL-15-expressing iPSC clones were differentiated in three stages: a 2-week stage to differentiate iPSCs into HPCs; a 2-week stage to differentiate HPCs into CD34 + iNK cells were differentiated according to a differentiation protocol involving a 4-week step to differentiate into iNK positively selected for IL-1 and a 2-week expansion step.

[0234] b. Cytotoxic properties of iNK expressing mbIL-15 The engineered iNKs were tested for cytotoxic properties in coculture with SKOV-3 ovarian tumor cells expressing a firefly luciferase-GFP fusion protein as a target. Control cocultures included wild-type iNKs. Cytotoxicity assays were performed using Incucyte® Live-Cell Analysis Systems (Sartorius, AG, Goettingen, Germany). Cocultures had E:T ratios of 10:1 and 3:1 and were imaged every 2 hours. YO-PRO-3 viability dye (ThermoFisher Scientific, Waltham, Massachusetts) was included in the cocultures. The percentage of viable SKOV-3 cells was calculated as an overlay of the percent of total SKOV-3 cells from the start of the assay. The results are shown in Figure 25.

[0235] Example 12. Optimization of anti-ROR1 scFv by varying linker length In this experiment, a previously disclosed humanized single-chain variable fragment (scFv) (International Application No. PCT / US2023 / 067314, filed May 22, 2023, Anti-ROR1 antibody and ROR1-targeting engineered cells) was optimized by varying the length of the linker between the light and heavy chains of the scFv. The amino acid sequence of scFv862, disclosed herein as SEQ ID NO: 12, contains a linker consisting of four repeats of the glycine-serine linker G4S (G4S)4. A variant of this sequence contained a single copy of the linker sequence (G4S)1. A CAR containing two scFvs was designed as shown in Figure 26. The CAR contained a signal peptide (sp), hinge (h), and transmembrane domain (tm) from CD8A, and the cytoplasmic domain of the CAR consisted of the 4-1BB and CD3 zeta domains. The CAR expression construct included an EF1α promoter. The original design CAR was present in the plasmid construct pCB7306, and the short linker CAR was present in the plasmid pCB7339.

[0236] Example 13. Insertion of anti-ROR1 CAR into T cells In this experiment, the CAR design shown in Figure 26 was introduced into T cells for insertion into the TRAC locus. The TRAC locus was targeted using CRISPR Cas12a and CRISPR hybrid RNA-DNA guides (chRDNA) essentially as described in International Patent Publication No. 2022 / 086846, "DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same." To target the TRAC locus, exons were analyzed for the presence of a suitable PAM sequence for Cas12a in Type V Acidaminococcus spp. The target site was selected at the TRAC locus on human chromosome 14 between nucleotides 22547529 and 22547552. A stretch of approximately 20 nucleotides 3' of the PAM sequence was used to design the targeting region for the Cas12a guide.

[0237] To design the donor sequence for insertion, 500 bp long homology arms 5' and 3' of the cleavage site were identified. The 5' and 3' homology arms were added to the ends of a DNA donor containing a CAR (Example 12) in the reverse orientation (i.e., 3' to 5') relative to the homology arms. The nucleotide sequences of the DNA donor polynucleotides were provided to commercial manufacturers for synthesis into suitable recombinant AAV (rAAV) plasmids and packaging into AAV6 viruses.

[0238] Primary T cell transduction with rAAV Primary activated T cells were obtained from PBMCs of healthy donors and transfected with rAAV6 essentially as described in WO2022 / 086846. Briefly, T cells were electroporated with a Cas12a chRDNA-guided nucleoprotein complex targeting TRAC, and 1 minute to 4 hours after nucleofection, cells were transfected at a concentration of 4 × 10 5T cells were infected with rAAV6 virus packaged with a CAR donor sequence at an MOI of 100. T cells were cultured in ImmunoCult-XF complete medium (STEMCELL Technologies, Cambridge, Mass.) supplemented with IL-2 (100 units / mL) for 24 hours after transduction. The following day, transduced T cells were transferred to a 50 mL conical tube and centrifuged at 300 × g for approximately 7–10 minutes to pellet the cells. The supernatant was discarded, the pellet gently resuspended, and the T cells were pooled in an appropriate volume of ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL). The counted T cells were diluted to 1 × 10 in ImmunoCult-XF complete medium supplemented with IL-2 (100 units / mL). 6 The cells were resuspended at 1000 cells / mL and seeded into the required number of T-175 suspension flasks (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) (ACROBiosystems, Newark, Del.).

[0239] Example 14. In vitro anti-ROR1 cytotoxicity of anti-ROR1 CAR-T cells In this experiment, the anti-ROR1 CAR-T cells of Example 13 were tested in vitro for cytotoxicity against the ROR1-expressing target tumor cell lines JeKo-1, REC1, and Mino, and primary B-cell chronic lymphocytic leukemia (B-CLL) cells (donor PBMC fraction). The control effector T cell population had a disrupted TRAC locus but no CAR insertion ("TRAC KO").

[0240] 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 hours. At the time of analysis, propidium iodide (PI) dead cell stain was added to the cultures. Assay analysis was performed using the remaining CTV normalized to target-only (E:T = 0:1) wells. + PI - Based on flow cytometry counting of (viable target cells), the results are shown as percentage of specific cell lysis in Figure 27A (Jeko-1 and REC1), Figure 27B (Mino and B-CLL, donor 1), and Figure 27C (B-CLL, donor 2).

[0241] Example 15. In vitro cytokine secretion by anti-ROR1 CAR-T cells In this experiment, cocultures of CAR-T cells and ROR1-expressing targets described in Example 14 were evaluated for cytokine secretion. 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 from cocultures (1:1 E:T ratio) at 24 hours. Secreted cytokine levels were quantified using a Luminex-based multiplex assay. Results are shown as cytokine concentrations in the coculture supernatants in Figure 28A (IFNγ and TNFα), Figure 28B (granzyme A and granzyme B), Figure 28C (IL-2 and perforin), and Figure 28D (IL-4 and IL-6).

[0242] Example 16. In vivo antitumor activity of anti-ROR1 CAR-T cells in NGS mice In this experiment, the in vivo anti-tumor activity of anti-ROR1 CAR-T cells (Example 13) was evaluated in NSG mice. Assay design shown in Table 1. [Table 3]

[0243] Briefly, JeKo-1-GFPluc cells were injected intravenously (IV). Three days after transplantation, effector cells were also injected IV.

[0244] The in vivo antitumor activity of anti-ROR1 CAR-T cells was measured for each treatment group as changes in body weight, survival, and tumor burden measured as bioluminescence intensity and the area under the curve (AUC) of bioluminescence intensity. The results are shown in Figure 29A (changes in body weight and survival probability) and Figure 29B (tumor burden measured as bioluminescence and the AUC of bioluminescence (BLI) measurements). Figure 29A: Comparison of Kaplan-Meier survival curves was analyzed with 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 Dunn's post hoc test for multiple comparisons. Vehicle control vs. pCB7306 p=0.0218 (***), vehicle control vs. pCB7339 p=0.0006 (**). Figure 29B: To compare BLI from day 0 to day 28 for each treatment group, a Kruskal-Wallis test followed by Dunn's post-hoc test for multiple comparisons was applied to the AUC. Vehicle control vs. pCB7306 p=0.0004, vehicle control vs. pCB7339 p=0.0042.

[0245] Example 17. Checkpoint-deficient anti-ROR1 CAR-T cells In this experiment, one of the immune checkpoint genes PDCD1, TIGIT, TIM3, and LAG3 was disrupted in the CAR-T cells of Example 13. Gene inactivation using CRISPR Cas12a and CRISPR hybrid RNA-DNA guides (chRDNA) was performed essentially as described in International Patent Publication No. 2022 / 086846, "DNA-containing polynucleotides and guides for CRSIPR Type V systems, and methods of making and using the same." Briefly, CRISPR hybrid RNA-DNA guides (chRDNA) were designed to target gene loci for cleavage by Cas12a and subsequent DNA repair, resulting in disruption of the gene sequence and gene silencing ("knockout"). To target each gene, exons were analyzed for the presence of a PAM sequence suitable for Cas12a in Type V Acidaminococcus spp. A stretch of approximately 20 nucleotides 3' of the PAM sequence was used to design the targeting region of the Cas12a guide for each gene.

[0246] To target the PDCD1 gene, a target site was selected at the PDCD1 locus on human chromosome 2 between nucleotides 241852860 and 241852883. To target the TIGIT gene, a target site was selected at the TIGIT locus on human chromosome 3 between nucleotides 114299581 and 114299604. To target the TIM3 gene, a target site was selected at the TIM3 locus on human chromosome 5 between nucleotides 157104679 and 157104702. To target the LAG3 gene, a target site was selected at the LAG3 locus on human chromosome 12 between nucleotides 6773330 and 6773353.

[0247] A gene-targeting Cas12a chRDNA-guided nucleoprotein complex was formed and introduced into anti-ROR1 CAR-T cells (Example 13) to generate a gene knockout, essentially as described in WO2022 / 086846. The resulting cell populations were designated anti-ROR1 CAR+PD1 KO, anti-ROR1 CAR+TIM3 KO, anti-ROR1 CAR+LAG3 KO, and anti-ROR1 CAR+TIGIT KO, respectively. The cell populations with multiple checkpoint inactivation are anti-ROR1 CAR+PD1 KO+TIM3 KO, anti-ROR1 CAR+PD1 KO+LAG3 KO, anti-ROR1 CAR+PD1 KO+TIGIT KO, and anti-ROR1 CAR+TIM3 KO+LAG3 KO.

[0248] Example 18. In vitro cytotoxicity of checkpoint-deficient anti-ROR1 CAR-T cells In this experiment, the checkpoint-deficient anti-ROR1 CAR-T cells of Example 17 were tested for in vitro cytotoxicity against the ROR1-expressing target JeKo-1 cell line and primary B-CLL donor cells essentially as described in Example 14. For single checkpoint inactivation, the results are shown in Figure 30A (JeKo-1 cells) and Figure 30B (primary B-CLL cells). For dual checkpoint inactivation, the results are shown in Figure 33 (JeKo-1 cells and primary B-CLL cells).

[0249] Example 19. In vitro cytokine secretion by checkpoint-deficient anti-ROR1 CAR-T cells In this experiment, cocultures of checkpoint-deficient anti-ROR1 CAR-T cells and ROR1-expressing targets, as described in Example 18, were evaluated for cytokine secretion. 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 from the cocultures at 24 hours. Secreted cytokine levels were quantified using a Luminex-based multiplex assay. Results are shown in Figure 31A (IL-2 and IL-4), Figure 31B (IL-6 and IFNγ), Figure 31C (TNFα and perforin), and Figure 31D (granzyme A and granzyme B).

[0250] Example 20. Serial rechallenge of checkpoint-deficient anti-ROR1 CAR-T cells with ROR1-expressing tumor cells In this experiment, the in vitro cytotoxicity of the checkpoint-deficient anti-ROR1 CAR-T cells from Example 17 was evaluated by serial rechallenge with the ROR1-expressing target cell line JeKo-1. 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 (for a maximum of six rechallenges), and viable cell counts were assessed by luminescence readout. Cytotoxicity (cell lysis) was measured after challenges 1–6 with ROR1-expressing target cells and used to calculate the area under the curve AUC. The results are shown in Figure 32.

[0251] Example 21. Alternative anti-ROR1CAR designs [Table 4] [Table 5]

[0252] Example 22. Antigen-dependent cytotoxicity of surrogate anti-ROR1 CAR-designed CAR-T cells against ROR1-expressing targets In this example, the CAR-T cells of Example 21 were tested for ROR1-specific cytotoxicity in coculture with the ROR1-positive cell line JeKo1 and ROR1-positive primary B cells from a patient with B-CLL. ROR1 positivity was determined by staining the cells with a phycoerythrin (PE)-conjugated anti-ROR1 antibody (BioLegend, San Diego, Calif., clone 2A2) and quantifying the number of anti-ROR1 antibody bound per cell, and therefore the number of ROR1 molecules per cell, using BD PE Quantibrite™ beads according to the manufacturer's instructions.

[0253] Target cells were labeled with Cell Trace Violet™ (CTV) dye. Anti-ROR1 CAR-Ts were co-cultured with CTV-labeled target cells at various effector:target (E:T) ratios in triplicate for 48 hours. After 48 hours, propidium iodide (PI) dead cell stain was added to each well, and PI uptake was assessed via flow cytometry to quantify target cell death. Viable target cell counts were extracted, and specific lysis was calculated relative to the 0:1 E:T control using the following formula:

number

[0254] The area under the curve (AUC) was calculated for each effector to represent the overall specific lysis against each ROR1-expressing target. The results are shown in Figure 34 (pCB7432, pCB7437, or pCB7438 with JeKo-1 or B-CLL), Figure 35A (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, or pCB7436 with K562 or B-CLL), and Figure 35B (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, or pCB7436 with JeKo-1).

[0255] Example 23. Cytokine secretion by surrogate anti-ROR1 CAR-designed CAR-T cells in the presence of ROR1-expressing targets In this example, the CAR-T cells of Example 21 were evaluated for cytokine secretion in the presence of ROR1-expressing target cells. 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.

[0256] Effector and target cells were co-cultured at a 1:1 ratio for 24 hours. 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 ProcartaPlex™ immunoassay kits and the manufacturer's protocol (Life Technologies, Waltham, Mass.). The results are shown in Figure 36A (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, or pCB7436 secreting IL-2, IL-4, TNFα, or perforin), Figure 36B (pCB7429, pCB7430, pCB7431, pCB7433, pCB7434, or pCB7436 secreting IL-6, IFNγ, granzyme A, or granzyme B), Figure 37A (pCB7432, pCB7437, or pCB7438 secreting IL-2, IL-4, TNFα, or perforin), and Figure 37B (pCB7432, pCB7437, or pCB7438 secreting IL-6, IFNγ, granzyme A, or granzyme B).

[0257] Example 24. IL-36γ secretion by alternative anti-ROR1 CAR-designed CAR-T cells In this example, IL-36γ secretion by anti-ROR1 CAR-T cells (pCB7306 (Figure 26), pCB7436, pCB7437, and pCB7438, Example 21, Table 3) and negative T cell controls was assessed using an IL-36γ ELISA kit from R&D Systems (catalog no. DY2320-05) (Minneapolis, Minn.) according to the manufacturer's instructions. Target JeKo-1 cells, which were 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 assessing cytotoxicity (see Example 22). Supernatants from round 1 and round 5 were assayed for IL-36γ concentration. The results are shown in Figure 38.

[0258] Example 25. In vivo anti-tumor activity of alternative anti-ROR1 CAR-designed CAR-T cells 5×10 5 JeKo-1-GFPluc tumor cells were injected intravenously (IV) into NGS female mice. Seven days after implantation, CAR-T cells (Example 21) were also injected intravenously at 1 x 10 7 Cars + Mice were injected IV with T cells. Body weight and IVIS measurements were taken twice weekly, and mice were assessed for anti-tumor activity by tracking weight change, survival, and changes in tumor burden measured as bioluminescence intensity. Results are shown in Figure 39A (body weight and survival probability) and Figure 39B (bioluminescence as photons and AUC).

[0259] Example 26. Comparison of new anti-ROR1 CAR-T cells with benchmark anti-ROR1 CAR-T cells In this example, the CAR construct (pCB7306) shown in Figure 26 was compared to the same CAR except that the scFv was replaced with the R12 scFv from the 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.

[0260] The resulting CAR-T cells expressing scFv PMC862 (invention) or scFv R12 (Hudecek et al., supra) were tested in parallel to compare their in vitro and in vivo anti-tumor properties.

[0261] In vitro serial re-challenge with JeKo1 tumor cells CAR-T cells "PMC862" and "R12," along with TRAC KO control T cells, were thawed and harvested in IL-2-containing medium 24 hours prior to the first stimulation. PE-conjugated human recombinant ROR1 protein was used to assess pre-stimulation (round zero, R0) CAR expression. Comparable surface CAR expression was observed between PMC862 and R12 CAR-T cells.

[0262] T cells were then seeded at different effector:target (E:T) ratios of Jeko1-GFP:ffLuc target cells. Every 72–96 h, specific lysis of target cells was quantified via luciferase-based luminescence readings. CAR-T cell phenotyping via flow cytometry was also performed. +T cell counts were assessed using BD Countbright™ Absolute Counting Beads. After each round of analyte measurement, remaining CAR-T cells were restimulated with fresh JeKo1-GFP:ffLuc target cells. Serial rechallenges were continued until loss of specific lysis was observed for one or both anti-ROR1 CAR-T cells. The results are shown in Figures 40 and 41.

[0263] PMC862 CAR-T cells retained high CAR expression through five rounds of re-challenge, while benchmark R12 CAR-T cells began to lose CAR expression at round 3, with expression becoming undetectable at round 5. This loss in R12 CAR-T cells correlates with a complete loss of specific lysis at round 5 (Figure 40). Loss of CAR expression in R12 CAR-T cells correlated with a loss of antigen-specific target cell lysis (Figure 41).

[0264] In vivo antitumor activity in NGS mice 5 × 10 JeKo-1-GFP:ffLuc target cells in PBS 5 Seven days after transplantation, PMC862 and R12 anti-ROR1 CAR-T cells in X-Vivo serum-free medium were intravenously transplanted at 10 cells / animal. 7 Cars + T cells / animal were dosed intravenously. Vehicle-only (X-Vivo medium) injection was used as a negative control. The CAR-positive rates of PMC862 and R12 CAR-T cells were 89.6% and 88.4%, respectively.

[0265] Starting on day 5 after CAR-T infusion, animals were subjected to bioluminescence imaging (BLI) twice weekly using an IVIS® Spectrum in vivo imaging system. Individual BLI measurements (n=8 mice / group) were used to calculate the mean BLI and area under the curve (AUC) from days 0 to 22 (Figure 42). A Mann-Whitney test comparing the AUC of PMC862 (CB-013CAR) and the AUC of R12 CAR indicates superior tumor control of PMC862 CAR-T cells (*** indicates p<0.001).

[0266] Animals were euthanized after losing more than 20% of their body weight compared to day 0 or developing paralysis. Kaplan-Meier survival curves (Mantel-Cox log-rank test) at any time point were developed (Figure 43). Median survival for the vehicle-treated group was 24 days, for the R12 CAR-T-treated group was 28 days, and for the PMC862 CAR-T-treated group was 39.5 days.

[0267] Example 27. In vivo anti-tumor activity of anti-ROR1 CAR-iNK cells with multiple genomic modifications In vivo experiments were designed essentially as in Example 25. 5 SKOV3-GFPluc tumor cells were injected intraperitoneally (IP) into NGS female mice. On day 7 post-implantation, 2 × 10 7 CAR-iNK cells were also injected IP.

[0268] CAR-iNK cells contained the CAR from pCB7306 (Figure 26), a disrupted CBLB gene (Example 8), and a B2M-HLA-E fusion peptide at the B2M locus (Example 10). CAR-iNK cells also expressed an IL15 receptor fusion (Example 11). Unmodified iNK cells were used as a control.

[0269] IVIS measurements were performed on mice from days 2 to 13 after treatment, and changes in tumor burden were assessed by bioluminescence intensity. The results are shown in Figure 44 (mean bioluminescence, BLI) and Figure 45 (area under the curve, AUC; ** indicates p<0.01).

[0270] Example 28. Serial rechallenge of anti-ROR1 iNK cells with tumor cells The experiment was designed essentially as in Example 20. iNK cells were subjected to six consecutive challenges with tumor cells. Two CAR-iNK cells were tested: pCB7306, which expressed the CAR shown in Figure 26, and pCB7447, which expressed the same CAR except for an additional CD27 costimulatory domain inserted between the CD3 zeta and 4-1BB domains (see Figure 26). Both types of CAR-iNK cells contained a disrupted CBLB gene, a B2M-HLA-E fusion peptide at the B2M locus, and expression of an IL15 receptor fusion (Example 27). Control iNK cells contained a disrupted TRAC gene and, like the CAR-iNK cells, also contained a disrupted CBLB gene, a B2M-HLA-E fusion peptide at the B2M locus, and expression of an IL15 receptor fusion as described in Example 27, except that the control iNK cells lacked the CAR. Unmodified iNK cells and iNK cells expressing an IL15 receptor fusion were used as two additional controls.

[0271] Two types of target cells were used: SKOV3, an ovarian tumor cell line with low ROR1 antigen density, and Hs764t, a gastric cancer cell line with high ROR1 antigen density.

[0272] Target cells engineered to express luciferase were added to the effector cells at an E:T ratio of 1:1. Every 42 hours, 5,000 fresh target cells were added to each culture five more times (a total of six challenges). The percentage of remaining viable tumor cells was assessed by luminescence readout. The results are shown in Figure 46 (SKOV3 target) and Figure 47 (Hs746t target).

[0273] Example 29. Multiple doses of anti-ROR1 CAR-iNK cells with multiple genomic modifications This in vivo experiment was designed essentially as in Example 27. Two CAR-iNK designs were the same as in Example 28, with the first CAR-iNK having CAR design pCB7306 and the second CAR-iNK having CAR design pCB7447, with both sets of iNKs having a disrupted CBLB gene, a B2M-HLA-E fusion peptide at the B2M locus, and expressing an IL15 receptor fusion (see Example 28). Unedited ("WT") iNKs were used as a control.

[0274] Two types of target tumor cell lines were used: SKOV3, an ovarian tumor cell line with low ROR1 antigen density, and Hs764t, a gastric cancer cell line with high ROR1 antigen density.

[0275] 3×10 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 and 6 mice in the Hs746t pCB7306 treatment groups). On day 7 post-implantation, 2 × 10 7 CAR-iNK cells were also injected IP. 2 × 10 7 A second injection of 2 × 10 CAR-iNKs was administered on day 8 after tumor cell implantation. 7 A third injection of CAR-iNK was administered 11 days after tumor cell implantation.

[0276] IVIS measurements of mice were performed twice weekly after the initial iNK treatment, and changes in tumor burden were assessed from bioluminescence intensity. The results are shown in Figure 48A (SKOV3 mean bioluminescence, BLI), Figure 48B (SKOV3 area under the curve, AUC calculated from bioluminescence), Figure 49A (Hs746t mean bioluminescence, BLI), Figure 49B (Hs746t area under the curve, AUC), and Figure 45 (area under the curve, AUC calculated from bioluminescence).

[0277] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Accordingly, the scope of the invention should be limited not by the embodiments described herein, but by the claims set forth below.

[0278] Unofficial sequence listing [Table 6-1] [Table 6-2] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9]

Claims

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

2. The chimeric antigen receptor (CAR) of claim 1, wherein the cytoplasmic domain comprises a CD3 zeta domain and a 4-1BB domain.

3. The anti-ROR1 scFv comprises a light chain (V Ｌ ) and heavy chain (V Ｈ ) and V Ｌ comprises a sequence selected from SEQ ID NOs: 3, 7, and 11, Ｈ The chimeric antigen receptor (CAR) of claim 1, wherein said chimeric antigen receptor (CAR) comprises a sequence selected from SEQ ID NOs: 2, 6, and 10.

4. The anti-ROR1 scFv comprises a light chain (V Ｌ ) and heavy chain (V Ｈ ) and V Ｌ consists essentially of a sequence selected from SEQ ID NOs: 3, 7, and 11, Ｈ The chimeric antigen receptor (CAR) of claim 3, wherein the chimeric antigen receptor (CAR) essentially consists of a sequence selected from SEQ ID NOs: 2, 6, and 10.

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

6. The linker is represented by the formula (G ｘ S ｙ ) ｎ wherein G is glycine and S is serine.

7. G ４ The chimeric antigen receptor (CAR) of claim 6, comprising S.

8. G ４ The chimeric antigen receptor (CAR) of claim 7, comprising S.

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

34.

10. In the anti-ROR1 scFv, the V Ｈ wherein the CDR1 of the V Ｈ wherein the CDR2 of the V Ｈ wherein the CDR3 of the V Ｌ wherein the CDR1 of the V Ｌ wherein the CDR2 of the V Ｌ The chimeric antigen receptor (CAR) of claim 9, wherein the CDR3 of

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

22.

14. 13. 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. 16. The chimeric antigen receptor (CAR) of claim 15, wherein the CD8 hinge domain consists essentially of SEQ ID NO:

20.

17. 16. 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. 2. The chimeric antigen receptor (CAR) of claim 1, wherein the CD3 zeta domain consists essentially of SEQ ID NO:

24.

19. 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 CD3 zeta domain.

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

27. 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 to 42.

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

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

50.

31. 30. 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. 2. The chimeric antigen receptor (CAR) of claim 1, comprising a sequence selected from SEQ ID NOs: 4, 8, 12, and 27.

33. The chimeric antigen receptor (CAR) of claim 1, consisting essentially of a sequence selected from 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 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. 10. An isolated nucleic acid comprising a vector sequence and a sequence encoding the chimeric antigen receptor (CAR) of claim 1.

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

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

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

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

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

42. 37. 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. 37. The isolated nucleic acid of claim 36, further comprising a coding sequence for a cytokine.

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

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

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

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

48. 48. The immune cell of claim 47, selected from a T cell, a natural killer (NK) cell, and an inducible natural killer (iNK) cell.

49. 48. The immune cell of claim 47, wherein the chimeric antigen receptor (CAR) comprises a sequence selected from SEQ ID NOs: 4, 8, 12, and 27.

50. 48. The immune cell of claim 47, further comprising an armoring genomic modification.

51. 51. The immune cell of claim 50, wherein the armed genome modification comprises inactivation of an immune checkpoint 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 armed genome modification comprises inactivation of CISH.

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

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

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

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

57. The immune cell of claim 50, wherein the armed genome modification comprises inactivation of TIGIT.

58. 51. The immune cell of claim 50, wherein the armed 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 armed genome modification comprises inactivation of B2M.

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

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

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

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

66. 48. A method of producing the immune cell of claim 47, the method comprising introducing into the cell a nucleic acid comprising a sequence selected from SEQ ID NOs: 14, 16, 18, 36, and 38.

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

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

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

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

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

72. 71. The method of Claim 70, wherein said endonuclease comprises a catalytically inactive CRISPR endonuclease conjugated to the cleavage domain of the restriction endonuclease Fok I.

73. 70. The method of claim 69, wherein the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a ZFN-Fok I fusion, a transcription activator-like effector nuclease (TALEN), and a TALEN-Fok I fusion.

74. 70. 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. 67. The method of claim 66, wherein the nucleic acid comprising a sequence selected from SEQ ID NOs: 14, 16, 18, 36, and 38 comprises a vector.

76. 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. 48. A composition comprising the immune cells of claim 47 and a pharmaceutically acceptable excipient.

78. The immune cells are 1×10 ６ ~2 x 10 ８ The composition of claim 77, wherein the CAR-T cells are in an amount of cells.

79. The immune cells are 1×10 ７ ~2 x 10 ９ The composition of claim 77, wherein the CAR-NK cells are in an amount of cells.

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

10.

81. 78. 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, glycerin, vegetable oils, phospholipids, surfactants, sugars, derivatized sugars, alditol, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, myo-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.

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

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

84. 78. The composition of claim 77, further comprising a surfactant selected from polysorbates, sorbitan esters, lecithin, phosphatidylcholines, phosphatidylethanolamines, fatty acids, fatty acid esters, and cholesterol.

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

86. 78. 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. 78. A method of inhibiting tumor growth in a patient, comprising administering to a patient having said tumor the composition of claim 77.

88. 88. The method of claim 87, wherein the tumor is a solid tumor selected from 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 MCL, CLL, SLL, B-ALL, B-NHL, and AML.

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

90. 88. The method of claim 87, wherein the composition further comprises a delivery timing component that allows for time-release, delayed release, or sustained release of the composition.

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

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

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

94. 88. The method of claim 87, further comprising applying a quality control measure to the immune cells prior to administration to the patient, comprising assessing one or more properties selected from the presence of an anti-ROR1 CAR in a cellular genome, surface expression of the anti-ROR1 CAR, ROR1-dependent lysis of ROR1-expressing target cells, proliferation in the presence of ROR1-expressing target cells, cytokine or chemokine secretion in the presence of the ROR1-expressing target cells, reduced tumor burden in an experimental animal bearing a ROR1-expressing tumor, and persistence in the circulation of an experimental animal bearing a ROR1-expressing tumor upon administration of the immune cells to the animal.

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

96. 95. 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. 95. The method of claim 94, wherein the surface expression of the anti-ROR1 CAR is assessed by flow cytometry.

98. 95. 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. 95. The method of claim 94, wherein the ROR1-dependent lysis of ROR1-bearing target cells is assessed by co-culturing the immune cells of claim 26 with ROR1-expressing target cells at an effector:target ratio of about 0.1 to about 10 and assessing target cell lysis.

100. 95. The method of claim 94, wherein the immune cell population having the highest rate of lysis of ROR1-bearing target cells is selected for administration to the patient.

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

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

103. 95. 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. 95. The method of claim 94, wherein the cytokine secretion is assessed by co-culturing the immune cells with ROR1-expressing target cells and measuring the amount of cytokine in the co-culture supernatant.

105. 95. The method of claim 94, wherein the immune cell population with the highest cytokine secretion is selected for administration to the patient.

106. 95. The method of claim 94, wherein reducing the tumor burden in an experimental animal bearing a ROR1-expressing tumor is measured as a change in bioluminescence of bioluminescent tumor cells in a period after the animal is injected with the immune cells.

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

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

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

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

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

112. 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. 95. The method of claim 94, wherein the immune cells are engineered to express a cytokine.

114. 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, comprising evaluating one or more properties selected from surface expression of the cytokine, ROR1-dependent lysis of ROR1-expressing target cells, and reduced tumor burden in experimental animals bearing ROR1-expressing tumors.

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

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

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

119. The composition is 1×10 ７ ~2 x 10 ８ CAR-T cells in the amount of 1 x 10 cells, or 1 x 10 ８ ~2 x 10 ９ 95. The method of claim 94, comprising an amount of CAR-NK cells in an amount of CAR-T cells or a mixture of CAR-T cells and CAR-NK cells present in a ratio of approximately 1:10 CAR-T to CAR-NK.

120. 1. A method of producing anti-ROR1 immune cells, the method comprising introducing into a cell population a nucleic acid encoding a chimeric antigen receptor (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 NOs: 4, 8, 12, and 27.

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

123. 123. The method of claim 122, wherein said introducing is into an iPSC, thereby forming a CAR-iPSC.

124. The method of claim 123, further comprising inducing differentiation of the CAR-iPSC into an inducible CAR-NK (CAR-iNK).

125. Inducing differentiation (i) contacting the CAR-iPSCs with one or more cytokines selected from BMP4, VEGF, SCF, IL3, IL6, and TPO to produce hematopoietic progenitor cells (HPCs); (ii) CD34 ＋ enriching said HPCs by selecting cells; 125. The method of claim 124, comprising: (iii) contacting the HPCs with one or more cytokines selected from IL3, IL15, IL7, SCT, FLT3L in the presence of feeder cells to produce inducible natural killer cells (iNK).

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

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

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

129. 121. The method of claim 120, wherein the nucleic acid is introduced into the progenitor cell population via chemical or electrochemical means.

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

131. 121. 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. 121. The method of Claim 120, wherein the introducing step comprises introducing into the cell a CRISPR system comprising a nucleic acid-guided endonuclease and a nucleic acid-targeting nucleic acid (NATNA) guide.

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

134. 121. The method of Claim 120, wherein the endonuclease comprises a catalytically inactive CRISPR endonuclease conjugated to the cleavage domain of the restriction endonuclease Fok I.

135. 121. The method of claim 120, wherein the endonuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a ZFN-Fok I fusion, a transcription activator-like effector nuclease (TALEN), and a TALEN-Fok I fusion.

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