COMPOSITIONS AND USES OF CD45 TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS

Abstract

Immune cells, including T cells, expressing a chimeric antigen receptor targeted to CD45 are described. In some cases, the immune cells lack a functional CD45 gene. In some cases, the immune cells also include a modification (a suicide sequence) that allows the cells to be killed in vivo. The immune cells are useful for treating a variety of cancers.

Claims

1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a polypeptide, wherein the chimeric antigen receptor or polypeptide comprises: an scFv targeting CD45, a spacer, a transmembrane domain, a co-stimulatory domain, and a CD3 ζ signaling domain.

2. The nucleic acid molecule of claim 1, wherein the scFv comprises the amino acid sequence of SEQ ID NO:1 or variant thereof having 1-5 amino acid modifications.

3. The nucleic acid molecule of claim 1, wherein the scFv comprises the amino acid sequence of SEQ ID NO:32 or variant thereof having 1-5 amino acid modifications and the amino acid sequence of SEQ ID NO: 33 or variant thereof having 1-5 amino acid modifications.

4. The nucleic acid molecule of claim 1, wherein the transmembrane domain is selected from the group consisting of: a CD4 transmembrane domain or variant thereof having 1-5 amino acid modifications, a CD8 transmembrane domain or variant thereof having 1-5 amino acid modifications, a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications, and a NKG2D transmembrane domain or a variant thereof having 1-5 amino acid modifications.

5. The nucleic acid molecule of claim 1, wherein the transmembrane domain is a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain or a NKG2D transmembrane domain.

6. The nucleic acid molecule of claim 1, wherein the costimulatory domain is a CD28, 4-1BB, or a 2B4 costimulatory domain.

7. The nucleic acid molecule of claim 1, wherein the costimulatory domain comprises the amino acid sequence of any of SEQ ID NOs:22-25 and 54 or a variant thereof having 1-5 amino acid modifications.

8. The nucleic acid molecule of claim 1, wherein the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO:21.

9. The nucleic acid molecule of claim 1, wherein a linker of 3 to 15 amino acids is located between the costimulatory domain and the CD3 ζ signaling domain or variant thereof.

10. The nucleic acid molecule of claim 1, wherein the spacer comprises any one of SEQ ID NOs:2-12 or a variant thereof having 1-5 amino acid modifications.

11. The nucleic acid molecule of claim 1, wherein the CAR or the polypeptide comprises the amino acid sequence of SEQ ID NO:29 or 30, or a variant thereof having 1-5 amino acid modifications.

12. The nucleic acid molecule of claim 1, which is an mRNA molecule.

13. An expression vector comprising the nucleic acid molecule of claim 1.

14. A population of human T cells harboring the mRNA molecule of claim 12.

15. A population of human T cells transduced by a vector comprising the nucleic acid molecule of claim 1.

16. The population of human T cells of claim 15, wherein the population of human T cells comprise central memory T cells, naive memory T cells, CD4+ cells and CD8+ cells enriched from PBMC cells, T cells isolated via negative depletion, or PBMC substantially depleted for CD25+ cells and CD14+ cells.

17. The population of T cells of claim 15, wherein CD45 (PTPRC) is knocked out, knocked down, or mutated.

17a. The population of T cells of claim 17, wherein the mutation is a deletion that eliminates CD45 expression.

18. The population of T cells of claim 17, wherein CD45 (PTPRC) is knocked out, knocked down, or mutated by CRISPR-Cas9 or TALEN system.

19. A population of human NK cells transduced by a vector comprising the nucleic acid molecule of claim 1 or harboring the mRNA molecule of claim 12.

20. The population of NK cells of claim 19, wherein CD45 (PTPRC) is knocked out, knocked down, or mutated.

20a. The population of NK cells of claim 20, wherein the mutation is a deletion that eliminates CD45 expression,

21. The population of NK cells of claim 19, wherein CD45 (PTPRC) is knocked out, knocked down, or mutated by CRISPR-Cas9 or TALEN system.

22. The population of T cells of claim 18 or the population of NK cells of claim 21, wherein the CRISPR/CAS9 system comprises a gRNA targeted to a CD45 exon.

23. The population of T cells of claim 18 or the population of NK cells of claim 21, wherein the CRISPR/CAS9 system comprises a gRNA targeted to CD45 exon #3, CD45 exon #8, CD45 exon #12, or CD45 exon #25.

24. The population of T cells of claim 18 or the population of NK cells of claim 21, wherein the CRISPR/CAS9 system comprises a gRNA comprising SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, or 45, or a variant thereof having 1-5 nucleotide changes.

25. A method of treating a hematopoietic malignancy or hematopoietic disorder in a patient comprising administering a population of autologous or allogeneic human T or NK cells transduced by a vector comprising the nucleic acid molecule of claim 1, wherein the hematopoietic malignancy or hematopoietic disorder comprises cells expressing CD45, where in the CD45 (PTPRC) is knocked out, knocked down, or mutated in the human T or NK cells.

26. The method of claim 25, wherein the hematopoietic malignancy or hematopoietic disorder is any one or more of a leukemia, a lymphoma, a myeloma, a myeloid leukemia, a T cell leukemia, a T cell lymphoma, a B cell leukemia, a B cell lymphoma, AML, CML, ALL, multiple myeloma, sickle cell anemia, aplastic anemia, severe combined immunodeficiency, myelodysplastic syndromes, myeloproliferative neoplasms, histiocytic and dendritic cell neoplasms.

27. The method of claim 25, wherein the population of T cells or NK cells expressing the chimeric antigen receptor or the polypeptide is administered locally or systemically.

28. The method of claim 25, wherein the CD45-expressing cells are cancerous cells.

29. The method of claim 25, wherein the population of human T cells expressing the chimeric antigen receptor or the polypeptide is administered by single or repeat dosing.

30. A method of reducing or eliminating CD45-positive cells in a subject comprising administering a population of autologous or allogeneic human T or NK cells transduced by a vector comprising the nucleic acid molecule of claim 1, wherein the PTPRC is knocked out, knocked down, or mutated in the T or NK cells.

31. The method of claim 30, wherein the population of T cells or NK cells expressing the chimeric antigen receptor or the polypeptide is administered locally or systemically.

32. The method of claim 30, wherein the CD45-positive cells are cancerous cells or noncancerous cells.

33. The method of claim 30, wherein the population of human T cells expressing the chimeric antigen receptor or the polypeptide is administered by single or repeat dosing.

34. A method of hematological cell transplantation conditioning in a patient comprising administering a population of autologous or allogeneic human T or NK cells transduced by a vector comprising the nucleic acid molecule of claim 1, wherein the PTPRC is knocked out, knocked down, or mutated in the T or NK cells.

35. The method of claim 34, wherein the population of T cells or NK cells expressing the chimeric antigen receptor or the polypeptide is administered locally or systemically.

36. The method of claim 34, wherein CD45-expressing cells are reduced or eliminated.

37. The method of claim 34, wherein the population of human T cells expressing the chimeric antigen receptor or the polypeptide is administered by single or repeat dosing.

38. The method of claim 34, wherein the hematological cell transplantation conditioning precedes a hematopoietic cell transplantation.

39. The method of claim 38, wherein the hematopoietic cell transplantation is a bone marrow transplant.

40. A method of preparing CD45 CAR T cells comprising: providing a population of autologous or allogeneic human T cells or NK cells modifying the T cells or NK cells to reduce expression of CD45, and introducing into the T cells or NK cells the nucleic acid molecule of claim 1.

41. The method of claim 40, wherein the T cells are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% CD14 negative and at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% CD25 negative.

42. The method of claim 40, wherein the T cells comprise CD4+ T cells or CD8+ T cells or both.

43. A method of enhancing T cell proliferation in T cells expressing a CAR comprising knocking out, knocking down, or mutating the PTPRC gene in the T cells or NK cells thereby creating CD45− CAR T cells.

44. The method of claim 43, wherein there is less than 25%, 20%, 15%, 10%, 9% 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% decrease in antigen-specific toxicity of the CD45-CAR T or NK cells cells compared the CD45+ CAR T cells or NK cells expressing the same CAR.

45. The population of T cells of claim 18 or the population of NK cells of claim 21, wherein the fratricide effect is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

Description

DESCRIPTION OF DRAWINGS

[0057] FIGS. 1A-1B show a schematic depicting CD45 CAR constructs (A) and the annotated amino acid sequences of a CD45 CAR (SEQ ID NO.29; B).

[0058] FIGS. 2A-2D show generation of CD45 knockout CD45CAR T cells. FIG. 2A shows a timeline schematic to generate CD45 knockout CD45CAR T cells. To prevent the strong fratricide effect of WT CD45CAR T cells, CD45 (PTPRC) gene was knocked out on T cells before CD45CAR lentivirus transduction. CRISPR-cas9 RNP system were used to edit the CD45 genes with guide RNAs (gRNA) targeting different exons (E3, 8, 12, 25) of CD45. FIG. 2B shows the growth curves of CD45CAR T cells. FIG. 2C shows the expression profiles of CAR was evaluated by truncated EGFR staining. FIG. 2D shows the presence of CD45, CD4 and CD8 of the CAR+ T cells.

[0059] FIG. 3 shows CD45 expression profiles of hematopoietic malignant cells. Myeloid leukemia cell lines (KG1A, MV4-11, K562), T cell leukemia and lymphoma cell lines (Jurkat, CEM and Hut78), B cell leukemia and blast cell lines (TM-LCL, Raji and NALM6), multiple myeloma cell line MM.1S were stained with anti-CD45 antibody (BD55482, clone HI30) and analyzed by flow cytometry.

[0060] FIGS. 4A-4E show Cytotoxicity of CD45CAR T cells against different hematopoietic malignant cell lines. Indicated tumor cells were co-cultured with CAR T/T cells in series of Effector: Target (E:T) ratios for 4h (FIGS. 4A-4D) or 48h (FIG. 4E) and cytotoxic effect was measured by flow cytometry. (FIG. 4A) Cytotoxicity of CD45KO CD45CAR T cells generated by different gRNAs. 4h cytotoxicity of gRNA #3 generated CD45KO CD45 CAR T cells against myeloid leukemia cell line KG1A (FIG. 4B), B-ALL line Raji (FIG. 4C), and blast crisis CML cell line KCL22M with T315I BCR-ABL mutation (FIG. 4D). 48h cytotoxicity effect against AML cell line MV4-11, multiple myeloma cell line MM.1S, and B lymphoblast cell line TM-LCL (FIG. 4E).

[0061] FIGS. 5A-5C show myeloid and lymphoid depletion effect of CD45KO CD45CAR T cells against CD45+ healthy hematopoietic cells. PBMCs from heathy donor were co-cultured with CFSE labeled CAR T (FIGS. 5A and 5B) or T cells (FIG. 5C) in series of Effector: Target (E:T) ratios for 4h (FIG. 5A) or 24h (FIGS. 5B and 5C) and cytotoxic effect was analyzed by flow cytometry in combination with myeloid cells (CD11b+), T (CD3+) and B (CD19+) cell markers.

[0062] FIG. 6 shows degranulation activity and IFNγ production of CD45CAR T cells against CD45+ AML KG1A cells. CD45KO CD45 CAR T or mock T cells were cocultured with KG1A cells with Effector: Target (E:T) ratios 1:1 for 5h and the degranulation activity was indicated by CD107a staining together with IFNγ intracellular staining.

[0063] FIGS. 7A-7D shows myeloid depletion effect of CD45CAR T cells in an AML mouse model. (FIG. 7A) NSG mice were engrafted with 1 million MV4-11.eGFP.ffluc cells by i.v. injection. 5 days later, tumor cells were measured by noninvasive optical bio-photonic imaging using a Xenogen IVIS system and the mice were treated with 2 million mock T cells or CD45 CAR T cells (CD45KO). The tumor growth was monitored every week by imaging (FIGS. 7B-7C). (FIG. 7D) The health status and survival was tracked, and survival curve was monitored.

[0064] FIGS. 8A-8E shows CD45 Knockout enhance CD19CAR T cells proliferation and preserve the functionality. (FIG. 8A) Surface marker profiles of wild type (WT) and CD45KO CD19-CAR T cells. (FIG. 8B) Degranulation activity and IFNγ production of WT and CD45KO CD19-CAR T cells with 4h-coculture with CD19 negative KG1A cells and CD19 positive cells (TM-LCL, NALM6 and Raji). The labeled number is the percentage of positive populations. (FIG. 8C) Proliferation of FACS sorted T cells and CD19-CART cells with and without CD45 knockout. The cells were cultured for 26 days. (FIG. 8D) Cytotoxicity of WT and CD45KO CD19-CAR T cells against CD19 negative KG1A cells and CD19 positive NALM6 cells with effector: target (E:T) ratio of 5:1 for 4h. (FIG. 8E) Cytotoxicity of WT and CD45KO CD19-CAR T cells against CD19 positive NALM6 cells and CD19KO NALM6 cells with series of effector: target (E:T) ratios for 4h.

[0065] FIG. 9 shows antigen specific anti-tumor and myeloid-/lymphoid-ablation effect of CD45-CAR T cells. Luciferase-based Cytotoxicity Assay (LCA) of CD45-CAR T cells against different target cells with 48-hour co-culture in different Effector (E): Target (T) ratios. All the target cells were engineered with firefly luciferase (ffLuc+).

[0066] FIGS. 10A-10C show induced cell depletion effect of rimiducid on iCasp9-CD45-CAR lentivirus transduced cells. FIG. 10A is a schematic showing an iCasp9-CD45-CAR construct containing iCaspase9 as safety switch. The order of iCaspase 9 and CD45CAR components can be switched (e.g., promoter-CD45CAR-T2A-iCaspase9-etc). FIG. 10B shows flow cytometry results of HT1080 cells transduced by iCasp9-CD45-CAR lentivirus.

[0067] FIG. 10C shows a bar graph quantifying relative survival cell number in an experiment where the same number of transduced HT1080 cells as in FIG. 10B were seeded in 24 well plate and treated with 100 μM ramiducid for 24h normalized and compared to non-treated group.

[0068] FIGS. 11A-11B show CD45 protein expression profile after PTPRC (CD45) gene knockout by CRISPR/Cas9 gene editing. Data show flow cytometry profiles (FIG. 11A) and quantified MFI (FIG. 11B) of CD45 expression on wild type and CD45 knock out T cells. hCD45gRNA #3_E12, CUUCUACAAAAAAUAAUCUG (SEQ ID NO: 39) was used for this experiment.

[0069] FIGS. 12A-12B show a schematic (FIG. 12A) and sequence (FIG. 12B) of the cDNA of a CD45 mRNA CAR construct with a T7 promoter and stabilization sequences (SEQ ID NO:58).

[0070] FIGS. 13A-13B show a schematic (FIG. 13A) and amino acid sequence (FIG. 13B) of the iCaspase 9 mRNA as safety switch construct (SEQ ID NO:59).

[0071] FIGS. 14A-14C show characteristics of CD45KO CD45CAR T cells generated by mRNA transduction. FIG. 14A shows the CD45 expression profile on wild type T cells, CD45KO T cells, and CD45KO CD45CAR T cells. FIG. 14B shows CAR expression profile of CD45KO CD45CAR T cells 24h after mRNA electroporation. FIG. 14C shows cytotoxicity of CD45KO CD45CAR T cells generated by mRNA transduction against tumor cells. Raji cells were co-cultured with CAR T cells for 24h.

[0072] FIG. 15 shows mRNA electroporated T cells preserve expression for 2 weeks. T cells cultured for 7 days were transduced via electroporation with GFP mRNA in a dose of 2.5 ug/million and the expression level of GFP expression was tracked by flow cytometry.

[0073] FIGS. 16A-16G. Amino acid sequences of CD45 scFv CAR. (FIG. 16A) CD45 scFv CAR with IgG4(HL-CH3) spacer, CD28 transmembrane domain, CD28 co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:62), without signal sequence (SEQ ID NO:63), and with tEGFR (SEQ ID NO:64). (FIG. 16B) CD45 scFv CAR with IgG4(S228P, L235E,N297Q) spacer, NKG2D transmembrane domain, 2B4 co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:65), without signal sequence (SEQ ID NO:66), and with tEGFR (SEQ ID NO:67). (FIG. 16C) CD45 scFv CAR with IgG4(HL-CH3) spacer, NKG2D transmembrane domain, 2B4 co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:68), without signal sequence (SEQ ID NO:69) and with tEGFR (SEQ ID NO:70). (FIG. 16D) CD45 scFv CAR with CD8h spacer, NKG2D transmembrane domain, 2B4 co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:71), without signal sequence (SEQ ID NO:72), and with tEGFR (SEQ ID NO:73). (FIG. 16E) CD45 scFv CAR with CD8h spacer, CD8 transmembrane domain, 41BB co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:74), without signal sequence (SEQ ID NO:75), and with tEGFR (SEQ ID NO:76). (FIG. 16F) CD45 scFv CAR with IgG4(HL-CH3) spacer, CD4 transmembrane domain, 41BB co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:77), without signal sequence (SEQ ID NO:78), and with tEGFR (SEQ ID NO:79). (FIG. 16G) CD45 scFv CAR IgG4(S228P, L235E,N297Q) spacer, CD4 transmembrane domain, 41BB co-stimulatory domain, and CD3 zeta domain (SEQ ID NO:80), without signal sequence (SEQ ID NO:81), and with tEGFR (SEQ ID NO:82).

DETAILED DESCRIPTION

[0074] In this disclosure, the generation and anti-tumor efficacy of CAR with an anti-CD45 scFv antigen-binding domain are described, inter alia.

[0075] Hematopoietic transplantation has been proven effective to treat a wide array of malignant and non-malignant hematological diseases. The preparative regimen, however, routinely entails aggressive and genotoxic treatment with whole body irradiation and/or chemotherapy, which can introduce severe and even life-threatening complications. Ablation of recipient bone marrow cells, including myeloid cells and HSCs, is a requirement of these conditioning regimens in order allow successful engraftment of the composite donor HSCs. Alternative conditioning approaches for bone marrow transplantation (BMT) with less toxic side-effects are desirable. CD45 is a hematopoietic lineage specific marker. Precise hematopoietic cells targeting may be achieved by the application of CD45 targeting chimeric antigen receptor (CAR) T or CAR NK cells for hematological cell transplantation conditioning. CD45 is also a widely expressed surface marker of different types of hematological malignancies including AML, B-ALL, T-ALL, and CML. CD45CAR T cells or CAR NK cells can also be used to treat CD45 positive hematological malignancies. To prevent the fratricide effect of CD45 CAR T or CAR NK cells, the CD45 (PTPRC) gene can be knockout out or mutated by gene editing technologies.

[0076] The present disclosure relates to novel chimeric antigen receptors (CARs) and applications thereof. CARs are able to redirect immune cell specificity and reactivity toward a selected target through exploiting the ligand-binding domain properties. In particular, the present disclosure relates to a Chimeric Antigen Receptor with extracellular scFv domain of a CD45 monoclonal antibody (e.g., BC8 clone). The present disclosure also relates to polynucleotides, vectors encoding said CAR and genetically modified immune cells expressing said CAR at their surface. The present disclosure also relates to methods to gene edit immune cells by knockout, knockdown or mutant CD45 gene along with co-expressing CD45CAR to produce fratricide resistant CD45CAR T cells or CD45CAR NK cells. The present disclosure is particularly useful for myeloid ablation, hematological cell transplantation conditioning and for the treatment of CD45 positive hematopoietic malignancies such as myeloid leukemia, T cell leukemia/lymphomas, and the like.

Examples

[0077] The CD45 CAR and their use is further described in the following examples, which do not limit the scope the claims.

[0078] Materials and Methods

[0079] The following materials and methods were used in the Examples set forth herein.

[0080] Cell Lines Myeloid leukemia cell lines (KG1A, MV4-11, K562), T cell leukemia and lymphoma cell lines (Jurkat, CEM, and Hut78), B cell leukemia and blast cell lines (TM-LCL, Raji and NALM6), multiple myeloma cell line (MM.1S) were cultured in RPMI-1640 (Lonza) containing 10% fetal bovine serum (FBS, Hyclone) (complete RPMI). The 293T and HT1080 cell lines were cultured in Dulbecco's Modified Eagles Medium (DMEM, Life Technologies) containing 10% FBS, 1×AA, 25 mM HEPES (Irvine Scientific), and 2 mM L-Glutamine (Fisher Scientific) (complete DMEM). All cells were cultured at 37° C. with 5% CO.sub.2. HUT78 cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium; Fisher Scientific) with 20% FBS.

[0081] DNA Constructs and Lentivirus Production

[0082] Tumor cells were engineered to express enhanced green fluorescent protein and firefly luciferase (eGFP/ffluc) by transduction with epHIV7 lentivirus carrying the eGFP/ffluc fusion under the control of the EF1α promoter as described previously (Lenalidomide Enhances the Function of CS1 Chimeric Antigen Receptor-Redirected T Cells Against Multiple Myeloma (Wang et al). Clinical Cancer Research 2018).

[0083] Research grade lentivirus was generated using a modified polyethylenimine (PEI) mediated transfection method (Optimization of lentiviral vector production using polyethylenimine-mediated transfection. Yong Tang, et al. Oncology Letters. 2015). Briefly, 293T cells were transfected with packaging plasmid and CAR lentiviral backbone plasmid using a modified PEI method. Viral supernatants were collected after 3 to 4 days. Supernatants were concentrated via high-speed centrifugation and lentiviral pellets were resuspended in phosphate-buffered saline (PBS)-lactose solution (4 g lactose per 100 mL PBS), aliquoted and stored at −80° C. Lentiviral titers were quantified using HT1080 cells based on EGFRt expression.

TABLE-US-00010 (CD45 CAR with signal sequence) SEQ ID NO: 29 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (CD45 CAR) SEQ ID NO: 30 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKYGPPCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTV AFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD45 CAR with signal sequence and EGFRt) SEQ ID NO: 31 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHP AFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLD PQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVC HALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECL PQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCH LCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMT (CD45 scFv-IgG4(HL-CH3)-CD28 TM-CD28GG-CD3ζ with signal) SEQ ID NO: 62 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-IgG4(HL-CH3)-CD28 TM-CD28GG-CD39) SEQ ID NO: 63 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKYGPPCPPCPGGGSSGGGSGGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (CD45 scFv-IgG4(HL-CH3)-CD28 TM-CD28GG-CD3ζ with signal and EGFRt) SEQ ID NO: 64 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLL VTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAF RGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNR GENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFV ENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLV WKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALG IGLFMT (CD45 scFv-IgG4(S228P, L235E, N297Q)-NKG2D TM-2B4-CD3ζ with signal) SEQ ID NO: 65 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKPFF FCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFP GGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQP KAQNPARLSRKELENFDVYSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-IgG4(S228P, L235E, N297Q)-NKG2D TM-2B4-CD3ζ) SEQ ID NO: 66 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKYGPPCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKPFFFCCFIAVAMGIRFIIMVTWR RKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQE PAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS GGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (CD45 scFv-IgG4(S228P, L235E, N297Q)-NKG2D TM-2B4-CD3ζ with signal and EGFRt) SEQ ID NO: 67 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKPFF FCCFIA VAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFP GGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQP KAQNPARLSRKELENFDVYSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVT SLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRG DSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFS LAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGE NSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVEN SECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVW KYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIG LFMT (CD45 scFv-IgG4(HL-CH3)-NKG2D TM-2B4-CD3ζ with signal) SEQ ID NO: 68 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDL KTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPS FNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSGGGRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-IgG4(HL-CH3)-NKG2D TM-2B4-CD3ζ) SEQ ID NO: 69 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKYGPPCPPCPGGGSSGGGSGGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKPFFFCCFIA VAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTI YSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNP ARLSRKELENFDVYSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR (CD45 scFv-IgG4(HL-CH3)-NKG2D TM-2B4-CD3ζ with signal and EGFRt) SEQ ID NO: 70 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIYEDVKDL KTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPS FNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSGGGRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVE ENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSI SGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEI IRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTS GQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCN LLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAG VMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGA LLLLLVVALGIGLFMT (CD45 scFv-CD8h-NKG2D TM-2B4-CD3ζ with signal) SEQ ID NO: 71 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIMVT WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPT SQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFD VYSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (CD45 scFv-CD8h-NKG2D TM-2B4-CD3ζ) SEQ ID NO: 72 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIIMVTWRRKRKEKQSETSPKEFLTIY EDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRK RNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYSGGGRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-CD8h-NKG2D TM-2B4-CD3ζ with signal and EGFRt) SEQ ID NO: 73 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFFFCCFIAVAMGIRFIIMVT WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPT SQEPA YTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFD VYSGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVC NGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKT VKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEIS DGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEG CWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITC TGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTY GCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMT (CD45 scFv-CD8h-CD8 TM-41BB-CD3ζ with signal) SEQ ID NO: 74 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-CD8h-CD8 TM-41BB-CD3ζ) SEQ ID NO: 75 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-CD8h-CD8 TM-41BB-CD3ζ with signal and EGFRt) SEQ ID NO: 76 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRG SLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNI KHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRT DLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTI NWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGP HCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIP SIATGMVGALLLLLVVALGIGLFMT (CD45 scFv-IgG4(HL-CH3)-CD4 TM-41BB-CD3ζ with signal) SEQ ID NO: 77 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (CD45 scFv-IgG4(HL-CH3)-CD4 TM-41BB-CD3ζ) SEQ ID NO: 78 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKYGPPCPPCPGGGSSGGGSGGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMALIVLGG VAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGG GRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR (CD45 scFv-IgG4(HL-CH3)-CD4 TM-41BB-CD3ζ with signal and EGFRt) SEQ ID NO: 79 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLL CELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFT HTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQ CHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYAD AGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMT (CD45 scFv-IgG4(S228P, L235E, N297Q)-CD4 TM-41BB-CD3ζ with signal) SEQ ID NO: 80 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMA LIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (CD45 scFv-IgG4(S228P, L235E, N297Q)-CD4 TM-41BB-CD3ζ) SEQ ID NO: 81 QVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPTSS TINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTALYYCARGNYYRYGDAMDYW GQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRA TISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTL NIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKYGPPCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (CD45 scFv-gG4(S228P, L235E, N297Q)- CD4 TM-41BB-CD3ζ with signal and EGFRt) SEQ ID NO: 82 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLKLSCAASGFDFSRYWMS WVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSEDTAL YYCARGNYYRYGDAMDYWGQGTSVTVSKISGGGGSGGGGSGGGGSGGGGSGGGG SSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYLHWYQQKPGQPPKLLIYLAS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKESKY GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMA LIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVC NGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKT VKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEIS DGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEG CWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITC TGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTY GCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMT

[0084] Cell Isolation, CD45 gene editing, CD45-CAR Lentiviral Transduction, and Ex Vivo Expansion_Leukapheresis products were obtained from consented research participants (healthy donors) under protocols approved by the City of Hope Internal Review Board (IRB). On the day of leukapheresis, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) followed by multiple washes in PBS/EDTA (Miltenyi Biotec). Cells were rested overnight at room temperature (RT) on a rotator, and subsequently washed and resuspended in X-VIVO T cell medium (Lonza) containing 10% FBS (complete X-VIVO). Up to 5.0×10.sup.9 PBMC were incubated with anti-CD14 and anti-CD25 microbeads (Miltenyi Biotec) for 30 min at RT and magnetically depleted using the CliniMACS® system (Miltenyi Biotec) according to the manufacturer's protocol and these were termed depleted PBMCs (dPBMC). dPBMC were frozen in CryoStor® CS5 (StemCell Technologies) until further processing. Tn/mem cells were prepared from dPBMC by staining with anti-CD62L microbeads(Miltenyi Biotec) and enriching CD62L+ cells using AutoMACS system..dPBMC or Tn/mem were stimulated with CD3/CD28 Dyna-beads (Thermal Fisher Scientific, Ratio of Cell to Beads is 1 to 2) in X-vivo15 medium with 10 U/mL IL2 and 0.5 ng/mL IL5. After one day, the cells were harvested and PTPRC (CD45) gene was knocked out by CRISPR-Cas9 ribonucleoprotein (RNP) system. For small scale experiment, the RNP was prepared by mixing 60 pmol Truecut Cas9 V2 protein (Thermo Fisher) and 180 pmol gRNA targeting PTPRC in 50 uL electroporation P3 buffer (Lonza) and incubate for 15 min at room temperature. The RNP solution was then mixed with 50 uL T cell suspension of 2 million cells and delivered by electroporation using 4D Nucleofector system (Lonza). After electroporation, T cells were incubated with 0.5 mL culture medium for 15 min then transferred to wells with 2 mL medium and fresh CD3/CD28 beads (Ratio of Cells to Beads is 1 to 1.)

[0085] Lentiviral transduction was performed at 2-5 days after gene editing. Briefly gene modified T cells were cultured with CD3/CD28 Dynabeads® (Life Technologies), protamine sulfate (APP Pharmaceuticals), cytokine mixture (as stated above) and desired lentivirus at a multiplicity of infection (MOI) of 1-3. Cells were then cultured in and replenished with fresh complete X-VIVO containing cytokines every 2-3 days. After 7 days, beads were magnetically removed, and cells were further expanded in complete X-VIVO containing cytokines to achieve desired cell yield. Following further expansion, cells were frozen in CryoStor® CS5 prior to in vitro functional assays and in vivo tumor models. Purity and phenotype of CAR T cells were verified by flow cytometry. We designed multiple gRNAs targeting different exons of CD45 (PTPRC) gene to knock out it. Examples used are as follows: hCD45gRNA #1_E3, AUAUUAAUUCUUACCAGUGG (SEQ ID NO: 37); hCD45gRNA #2_E8, ACUCCAUCUAAGCCAACAUG (SEQ ID NO: 38); hCD45gRNA #3_E12, CUUCUACAAAAAAUAAUCUG (SEQ ID NO: 39); hCD45gRNA #4_E25, GUGCUGGUGUUGGGCGCAC (SEQ ID NO: 45).

[0086] Flow Cytometry

[0087] T cells were harvested and stained as described previously (Jonnalagadda, M., et al., Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. Mol Ther, 2015. 23(4): p. 757-68.). T cell phenotype was examined using fluorochrome-conjugated antibodies against CD3, CD4, CD8α, CD45 (clone HI30, BC-8 or 94.1). Transgenic CAR expression was determined by staining of the truncated EGFR tag. Data were acquired on MacsQuant Analyzer 10 (Miltenyi Biotec) flow cytometers and analyzed with FlowJo (v10.6.1).

[0088] In Vitro T Cell Assays

[0089] For tumor killing assays, CAR T cells and tumor targets were co-cultured at indicated effector:tumor (E:T) ratios. To test cytotoxicity effect of CD45CAR T cells, GFP expressing tumor cells were plated in 96-well U-bottom plates at the indicated density. Effector cells (CD45KO CD45CAR T or Mock T cells) were washed, resuspended in fresh medium without cytokines and co-cultured with the indicated tumor cells for 4 hours (short term) or 48 hours (long term). Cytotoxicity was routinely evaluated by flow cytometry with enumeration of GFP+DAPI-tumor cells for viable GFP-expressing tumor cells. For primary PBMC, viable T cells (CD3+), B cells (CD19+) and myeloid cells (CD11b+) were analyzed by staining with lineage specific markers.

[0090] To test for degranulation activity, CAR T or control T cells were incubated with tumor cells for five hours in the presence of CD107a antibody and GolgiStop protein transport inhibitor (BD Biosciences). After the co-culture, cells were harvested, fixed, permeabilized, and stained for intracellular cytokines. Degranulation (CD107a staining) and intracellular cytokine staining (e.g. IFNγ) were examined by flow cytometry.

[0091] In Vivo Tumor Studies

[0092] All animal experiments were performed under protocols approved by the City of Hope Institutional Animal Care and Use Committee. Tumor xenograft models were generated using 6 to 8 week-old NOD/SCID/IL2R−/− (NSG) mice as previously described (Jackson Laboratory) [Urak, R., et al., Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells foradoptive immunotherapy. J Immunother Cancer, 2017. 5:26]. Briefly, on day 0, ffLuc+MV4-11 cells (1×10.sup.6) were injected intravenously (i.v.) into the NSG mice. After 5 days, mice were then treated with CAR T cells or mock T cells as described for each experiment. Tumor growth was determined by in vivo bio-photonic imaging using a Xenogen IVIS 100. Mice were also monitored for survival, with euthanasia applied according to the American Veterinary Medical Association Guidelines.

Example 1: Generation of CD45KO CD45 CAR T cells

[0093] We utilized the single chain variable fragment (scFv) sequence of an anti-CD45 antibody clone BC-8, which already shown good profiles of safety and specificity [Mawad, R., et al., Radiolabeled anti-CD45 antibody with reduced-intensity conditioning and allogeneic transplantation for younger patients with advanced acute myeloid leukemia or myelodysplastic syndrome. Biol Blood Marrow Transplant, 2014. 20(9):1363-8; Lin, Y., et al., A genetically engineered anti-CD45 single-chain antibody-streptavidin fusion protein for pretargeted radioimmunotherapy of hematologic malignancies. Cancer Res, 2006. 66(7):3884-92; Orozco, J. J., J. Zeller, and J. M. Pagel, Radiolabeled antibodies directed at CD45 for conditioning prior to allogeneic transplantation in acute myeloid leukemia and myelodysplastic syndrome. Ther Adv Hematol, 2012. 3(1):5-16]. The CD45 CAR construct for T cells is composed with anti-CD45 scfv domain, an IgG4 spacer with two point-mutations (L235E and N297Q) within the CH2 region, a CD28GG costimulatory domain, CD3ζ, and a truncated human epidermal growth factor receptor (huEGFRt) as a marker (FIG. 1A-1B). The CAR construct can also use other costimulatory domains such as 41BB, other spacer domains and transmembrane domains such as CD8 hinge and CD4 transmembrane domain. For CAR constructs for NK cells, the transmembrane domain can be the NKG2D transmembrane domain and the costimulatory domain can be the 2B4 costimulatory domain.

[0094] CD45KO CD45CAR T cells can be prepare from, for example, PBMC, dPBMC (PBMC with depletion of CD14+ and CD25+ cells), Tn/mem (naïve and memory T cells, CD62L+ enriched from dPBMC), or Tcm (central memory T cells). In this case, CD45KO CD45CAR T cells were generated from Tn/mem cells (FIG. 2A). A number of different gRNAs targeting different exons of PTPRC gene were used knock out PTPRC gene. The generated CD45CAR T cells with PTPRC gene knock out demonstrated better proliferation capacity and generated more cells compared to WT CD45CAR T cells (FIG. 2B), in which CD45CAR can cause a fratricidal effect. There was a CAR positive percentage range from 35.5% to 48.3% with MOI of 2 for lenti virus transduction (FIG. 2C). Surface marker analysis by flow cytometry showed that CD45KO CD45CAR T cells had no detectable cell surface CD45 expression (FIG. 2D). Moreover, the relative CD4+ and CD8+ population was not significantly altered (FIG. 2D).

Example 2: Validation that CD45 CAR T Cells Selectively Target CD45-Positive Cells In Vitro

[0095] To determine whether CD45 CAR T cells demonstrate selective activity against CD45-positive cancer and noncancerous cells, the CD45 CAR T cells were grown in presence of either CD45-positive cells.

[0096] As shown in FIG. 3, CD45 is widely expressed on different hematopoietic malignant cells, including acute myeloid leukemia (KG1A, MV4-11, K562), T cell leukemia and lymphoma (Jurkat, CEM, Hut78), B cell leukemia and lymphoma (TM-LCL, Raji, NALM6) and Multiple Myeloma (MM.1S). It was also well established that CD45 is a hematopoietic specific marker in healthy physiological condition [Rheinlander, A., B. Schraven, and U. Bommhardt, CD45 in human physiology and clinical medicine. Immunol Lett, 2018. 196:22-32]. By coculturing with CD45+KG1A and Raji cells, all CD45KO CD45CAR T cells demonstrated potent antigen specific cytotoxicity while mock T cells did not (FIG. 4A).

[0097] The gRNA #3 (target PTPRC exon 12) CD45 knock out cells were used for further functional characterization. The CD45KO CD45CAR T cells demonstrated potent cytotoxicity against AML (KG1a, MV4-11) (FIGS. 4B and 4E), B-ALL (Raji, TM-LCL) (FIG. 4C), blast crisis CML (KCL22M with T315I mutation) (FIG. 4D) and multiple myeloma (MM.1 S) (FIG. 4E).

[0098] By co-culturing with PBMC from healthy donor, CD45KO CD45CAR T cells were shown to eliminate healthy myeloid cells (CD11b+), B cells (CD19+) and T cells (CD3+) (FIGS. 5A and 5B), while mock T cells did not (FIG. 5C). This functional phenotype indicated potential application of CD45KO CD45 CAR T cells for HCT conditioning.

[0099] The CD45KO CD45CAR T cells also demonstrated potent antigen specific degranulation activity and IFNγ secretion (FIG. 6).

Example 3: Validation that CD45 CAR T Cells Delivered In Vivo in a Mouse Model Exhibit Potent Anti-Tumor Activity and Confer Extended Lifespan to the Mice

[0100] To evaluate in vivo efficacy of CD45 CAR T cells to selectively target CD45-positive cells in the AML model, CD45 CAR T cells were delivered and tumor size and survival was evaluated over time.

[0101] To further evaluate the in vivo activity, we tested in a tumor xenograft mouse model with MV4-11 AML cells (FIG. 7A). CD45KO CD45 CAR T cells treatment significantly eliminated tumor engrafts and prolonged the survival of the mice (FIGS. 7B-7D).

Example 4: Validation that CD45KO CAR T Cells Targeted to Other Antigens Benefit from Enhanced Features

[0102] CD45 is reported to play key roles in T cell development and function regulation in both negative and positive way [Alexander, D. R., The CD45 tyrosine phosphatase: a positive and negative regulator of immune cell function. Semin Immunol, 2000. 12(4):349-59; Cho, J. H., et al., CD45-mediated control of TCR tuning in naive and memory CD8(+) T cells. Nat Commun, 2016. 7:13373; Virts, E. L., O. Diago, and W. C. Raschke, A CD45 minigene restores regulated isoform expression and immune function in CD45-deficient mice: therapeutic implications for human CD45-null severe combined immunodeficiency. Blood, 2003. 101(3):849-55]. However, the role of CD45 in CAR T cells is not well studied. We explored the functions of CD45 on CD19-CAR T cells by knocking out CD45. As shown in FIG. 8A, knocking out CD45 on CD19-CAR T cells or mock T cells increased CD4+ population with expense of CD8+ population. CD45KO CD19-CAR T cells demonstrated potent degranulation, IFNγ secretion activity (FIG. 8B) and comparable antigen specific cytotoxicity (FIG. 8D). As shown in FIG. 8E, which depicts the results of a 4 hr cytotoxicity assay using wt or CD45KO CD19-CAR T cells against WT NALM6 or CD19KO NALM6, knockout of CD45 did not impair antigen specific cytotoxicity of CD19-CAR T cells. The CD45 knockout enhanced the proliferation of T cells and CD19CAR T cells (FIG. 8C). This phenotype suggests various CAR T cells, e.g., CAR T cells targeted to CD19 may benefit from knockout or knockdown of CD45.

Example 5: Myeloid/Lymphoid Ablation Effect of CD45 CAR T Cells in Humanized Mouse Model with Human PBMC or HSC Engraftments

[0103] In some circumstances it is desirable to reduce or eliminate myeloid and/or lymphoid cells in vivo. Experiments will be conducted in a humanized mouse model to measure depletion of myeloid and lymphoid cells as a function of treatment with CD45KO CD45 CAR T cells and CD45KO CD45 CAR NK cells. Results will show a reduction of myeloid and/or lymphoid cells and in increase of success of PBMC and/or HCS (hematopoietic stem cells) engraftment with CD45 CAR T cells and/or CD45KO CD45 CAR NK cells treatment.

Example 6: CD45KO CD45CAR T Cells have Antigen-Specific Anti-Tumor and Myeloid-Ablation and Lymphoid-Ablation Activity

[0104] Killing assays demonstrated that CD45KO CD45CAR T cells have antigen specific anti-tumor and myeloid-ablation and lymphoid-ablation activity. Luciferase-based Cytotoxicity Assay (LCA) of CD45KO CD45-CAR T cells against different CD45+ target cells (MOLM14, MV4-11, Jurkat, and Hut78) with 48-hour co-culture in different Effector (E): Target (T) ratios showed effective killing of the CD45+ target cells (FIG. 9).

Example 7: Validation of iCasp9-CD45-CAR Expression and Function

[0105] To test the effects of a suicide switch as a safety precaution, experiments were conducted to investigate the induced cell depletion effect of rimiducid on iCasp9-CD45-CAR lentivirus transduced cells. iCasp9 can be activated by a specific chemical inducer of dimerization (CID) such as rimiducid, leading to efficient elimination of iCasp9 engineered cell.

[0106] The design of an iCasp9-CD45-CAR construct containing iCaspase9 is shown in FIG. 10A. The order of iCaspase 9 and CD45 scFv components can be switched (e.g., promoter-CD45CAR-T2A-iCaspase9-etc). HT1080 cells were transduced by lentivirus. Flow cytometry showed 22% of the transduced T cells expressed CD45 CAR (FIG. 10B). Transduced HT1080 cells were seeded in 24 well plate and treated with 100 μM ramiducid for 24h normalized and compared to non-treated group, and the relative survival cell number was quantified (FIG. 10C). Treating the iCasp9-CD45-CAR cells with ramiducid reduced cell survival to 36% (FIG. 10C).

Example 8: Validation of CD45 Depletion on T Cells Following CRISPR/Cas9 Gene Editing

[0107] Experiments were then conducted to quantify the CD45 protein expression on wild type and CD45 knock out T cells after PTPRC (CD45) gene knockout by CRISPR/Cas9 gene editing. hCD45gRNA #3_E12, CUUCUACAAAAAAUAAUCUG (SEQ ID NO: 39) was used for this experiment. This experiment demonstrated the decreasing expression of CD45 via flow cytometry (FIG. 11A) and quantified MFI (FIG. 11B) after CRISPR/cas9 mediated CD45KO, which provided evidence that transduction CD45-CAR after 5 days following CRISPR can avoid fratricide effect.

Example 9: CD45KO CD45CAR T Cells Generated by mRNA Transduction/iCaspase9 for Generating mRNA by In Vitro Transcription Method/mRNA Electroporated T Cells can Preserve Transitory Expression for 2 Weeks

[0108] The design of a CD45 mRNA CAR is shown in FIG. 12A. The sequence of the CD45 mRNA CAR is shown in FIG. 12B. CD45KO CD45CAR T cells are used here but it works the same for a CD45KO CD45CAR NK cells (data not shown). The design of an iCaspase 9 mRNA is shown in FIG. 13A. The sequence of the iCaspase 9 mRNA is shown in FIG. 13B. The iCaspase 9 mRNA is used as a safety switch construct.

[0109] Experiments were then conducted to investigate the characteristics of CD45KO CD45CAR T cells generated by mRNA transduction. Flow cytometry probed the CD45 expression profile on wild type T cells, CD45KO T cells, and CD45KO CD45CAR T cells and showed CD45 was effectively knocked out (FIG. 14A). The CAR expression profile of CD45KO CD45CAR T cells 24h after mRNA electroporation shows 20% of the cells expressed the CAR (FIG. 14B).

[0110] T cells cultured for 7 days were transduced via electroporation with GFP mRNA in a dose of 2.5 ug/million and the expression level of GFP expression was tracked by flow cytometry. This data demonstrated that GFP mRNA can express GFP protein for about 2 weeks. Importantly, mRNA electroporated T cells preserved expression for 2 weeks indicates the feasibility of mRNA CD45CAR T cells with transitional expression as a strategy to make these CAR T and NK cells safer for patients.

Other Embodiments

[0111] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention.