Compositions and methods for inhibition of lineage-specific antigens

Abstract

To provide a method for administering a material targeting a lineage-specific cell surface antigen such as CD33 and a hematopoietic cell population lacking the lineage-specific cell surface antigen such as CD33 for immunotherapy of hematopoietic malignancies.SOLUTION: Provided is an engineered hematopoietic stem or progenitor cell comprising a genetic mutation in an exon 3 of an endogenous CD33 gene, wherein the genetic mutation is at a site targeted by a gRNA comprising the nucleotide sequence of CCUCAACUGAUCAAGCAAGC, wherein the genetic mutation results in a reduced expression level of CD33 relative to a wild-type counterpart cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 62 / 793,210, filed January 16, 2019, and 62 / 852,573, filed May 24, 2019, the entire contents of each of which are incorporated herein by reference. Sequence Listing This application contains an electronically submitted Sequence Listing in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on January 16, 2020, is named 01001-004965-WO1_SL.txt and is 83 kilobytes in size. [Background technology]

[0002] Despite decades of attempts, curative immunotherapy for cancer has been extremely difficult to achieve. The fundamental principle is antigen recognition by either antibodies or T cells (via T cell receptors) (Cousin-Frankel, Science (2013) 342:1432). Antibody-based immunotherapy has been widely used for cancer when the target antigen is upregulated in tumor cells compared to normal cells (e.g., Her-2 in Her-2-proliferating breast cancer) or when tumor cells express an antigen that can be recognized by an antibody or antibody-toxin conjugate (e.g., rituximab against CD20) (Baselga et al., Annals Oncology (2001) 12:S35). Clinical trials using antibody-based immunotherapy have shown improved patient survival in a limited number of cancer types (usually when combined with standard chemotherapy), but these effects are often accompanied by significant safety and efficacy concerns (Cousin-Frankel Cancer, Science (2013) 342:1432).

[0003] Effective T cell therapy for cancer is even more difficult to achieve clinically (Schmitt et al., Hum. Gene Ther. (2009) 20(11):1240). Effective T cell therapy for cancer relies on T cells with high affinity binding to antigens on cancer cells. Chimeric antigen receptor T cells (CAR T cells) are widely used and recognize antigens on cells with both high affinity and specificity, without the need for auxiliary recognition molecules such as HLA antigens to "present" peptides. The T cell receptor of the CAR T cell is "swapped" with antigen-binding heavy and light chains, thus eliminating the need for HLA auxiliary molecules. The recombinant CAR T receptor is fused to a signaling domain that results in T cell activation upon binding of the CAR T receptor to the target antigen.

[0004] The clinical use of CAR T cells has been limited to targeting a narrow range of cell surface antigens, further supporting the need for improved and novel approaches in the treatment of cancer, particularly for diseases such as acute myeloid leukemia (AML), where outcomes in elderly patients who cannot undergo the current standard of care, intensive chemotherapy, remain extremely poor, with a median survival of only 5–10 months (Dohner et al., NEJM (2015) 373:1136).

[0005] Described herein is a novel approach to cancer immunotherapy that targets specific types of lineage-specific cell surface antigens on tumor cells. CAR T cell therapy is then combined with the replacement of non-tumor cells by injecting or reinjecting a modified cell population lacking the lineage-specific cell surface antigen. Tumor recurrence is prevented or reduced by maintaining in vivo patient surveillance with CAR T cells. Summary of the Invention

[0006] The present disclosure is based, at least in part, on the discovery that an agent comprising an antigen-binding fragment that binds a lineage-specific cell surface antigen (e.g., immune cells expressing a chimeric receptor that targets CD33) selectively causes cell death of cells that express the lineage-specific cell surface antigen, while cells lacking that antigen (e.g., genetically engineered hematopoietic cells) escape the resulting cell death. Based on this discovery, it is expected that immunotherapy comprising a combination of an agent that targets a lineage-specific cell surface antigen, e.g., CAR-T cells that target CD33, with hematopoietic cells lacking the lineage-specific cell antigen (e.g., CD33) will provide an effective method for treating hematopoietic malignancies.

[0007] In some aspects, the present disclosure provides genetically engineered hematopoietic stem or progenitor cells that comprise a genetic mutation in exon 3 of the endogenous CD33 gene, where the genetic mutation is at a position described herein. One aspect of the present disclosure provides genetically engineered hematopoietic stem and / or progenitor cells that comprise a genetic mutation in exon 3 of the endogenous CD33 gene, where the genetic mutation is at a site targeted by a gRNA, which comprises the nucleotide sequence AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67), GGCCGGGUUCUAGAGUGCCA (SEQ ID NO: 68), or CCUCACUAGACUUGACCCAC (SEQ ID NO: 70), and the genetically engineered hematopoietic stem and / or progenitor cells have reduced expression levels of CD33 compared to their wild-type counterparts. In some embodiments, the genetically engineered hematopoietic stem and / or progenitor cells express less than 10% of the CD33 expressed by their wild-type counterparts. In some embodiments, the genetically engineered hematopoietic stem and / or progenitor cells do not express CD33. In some embodiments, the genetically engineered hematopoietic stem and / or progenitor cells do not express CD34. + In some embodiments, the genetically engineered hematopoietic stem and / or progenitor cells are derived from bone marrow cells or peripheral blood mononuclear cells of a subject (e.g., a human patient with a hematopoietic malignancy or a healthy donor).

[0008] In some embodiments, the present disclosure also provides cell populations comprising a plurality of genetically engineered hematopoietic stem and / or progenitor cells described herein.

[0009] In another aspect, the disclosure provides a method for producing genetically engineered hematopoietic stem and / or progenitor cells, the method comprising: (i) providing hematopoietic stem and / or progenitor cells; and (ii) introducing into the cells (a) a guide RNA (gRNA) comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 67, SEQ ID NO: 68, and / or SEQ ID NO: 70, and (b) a Cas9 endonuclease, thereby producing genetically engineered hematopoietic stem and / or progenitor cells having reduced expression levels of CD33. In some embodiments, the gRNA and Cas9 endonuclease are encoded on a vector, which is introduced into the cells. In some embodiments, the vector is a viral vector. In some embodiments, the gRNA and Cas9 endonuclease are introduced into the cells as a preformed ribonucleoprotein complex. In some embodiments, the ribonucleoprotein complex is introduced into the cells by electroporation.

[0010] The present disclosure also provides, in some embodiments, the use of gRNAs described herein to reduce the expression of CD33 in a hematopoietic stem or progenitor cell sample using the CRISPR / Cas9 system.

[0011] The present disclosure also provides, in some embodiments, the use of a CRISPR / Cas9 system to reduce the expression of CD33 in a hematopoietic stem or progenitor cell sample, wherein the gRNA of the CRISPR / Cas9 system is a gRNA described herein.

[0012] In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the gRNA is a modified sgRNA. In some embodiments, the hematopoietic stem and / or progenitor cells are CD34 +In some embodiments, the hematopoietic stem and / or progenitor cells are derived from bone marrow cells or peripheral blood mononuclear cells (PBMCs). In some embodiments, the subject has a hematopoietic disorder. In some embodiments, the subject is a healthy HLA-matched donor.

[0013] In some embodiments, the present disclosure provides genetically engineered hematopoietic stem and / or progenitor cells, which are produced by the methods described herein.

[0014] In another aspect, the disclosure provides methods of treating a hematopoietic disorder, the method comprising administering to a subject in need thereof an effective amount of genetically engineered hematopoietic stem and / or progenitor cells or cell populations described herein. In some embodiments, the hematopoietic disorder is a hematopoietic malignancy.

[0015] In some embodiments, the method includes administering to the subject an effective amount of an agent that targets CD33, wherein the agent comprises an antigen-binding fragment that binds CD33. In some embodiments, the agent that targets CD33 is an immune cell that expresses a chimeric antigen receptor (CAR), which comprises an antigen-binding fragment that binds CD33.

[0016] In some aspects, the present disclosure provides genetically engineered hematopoietic stem and / or progenitor cells or cell populations described herein for use in treating a hematopoietic disorder, wherein the treatment comprises administering to a subject in need thereof an effective amount of the genetically engineered hematopoietic stem or progenitor cells or cell population, and further comprises administering to the subject an effective amount of an agent that targets CD33, wherein the agent comprises an antigen-binding fragment that binds CD33.

[0017] In some aspects, the present disclosure provides an agent that targets CD33 for use in treating a hematopoietic disorder, wherein the agent comprises an antigen-binding fragment that binds CD33, and wherein the treatment comprises administering to a subject in need thereof an effective amount of the agent that targets CD33, and further comprises administering to the subject an effective amount of a genetically engineered hematopoietic stem or progenitor cell described herein or a cell population described herein.

[0018] The combination of the genetically engineered hematopoietic stem or progenitor cells described herein or the cell population described herein with an agent that targets CD33 is for use in the treatment of a hematopoietic disorder, wherein the agent comprises an antigen-binding fragment that binds CD33, and the treatment comprises administering to a subject in need thereof an effective amount of the genetically engineered hematopoietic stem or progenitor cells or cell population and the agent that binds CD33.

[0019] In some embodiments, the genetically engineered hematopoietic stem or progenitor cells or cell populations are administered simultaneously with the agent that targets CD33. In some embodiments, the genetically engineered hematopoietic stem or progenitor cells or cell populations are administered before the agent that targets CD33. In some embodiments, the agent that targets CD33 is administered before the genetically engineered hematopoietic stem or progenitor cells or cell populations.

[0020] In some embodiments, the immune cells are T cells. In some embodiments, the immune cells, the genetically engineered hematopoietic stem and / or progenitor cells, or both are allogeneic. In some embodiments, the immune cells, the genetically engineered hematopoietic stem and / or progenitor cells, or both are autologous. In some embodiments, the antigen-binding fragment in the chimeric receptor is a single-chain antibody fragment (scFv) that specifically binds human CD33.

[0021] In some embodiments, the subject is a human patient with Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma, hi some embodiments, the subject is a human patient with leukemia that is acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.

[0022] In another aspect, the present disclosure provides a guide ribonucleic acid (gRNA) comprising a spacer sequence that is at least about 90% identical to SEQ ID NO:67 or SEQ ID NO:68, or SEQ ID NO:70. In some embodiments, the gRNA is a single-molecule gRNA (sgRNA). In some embodiments, the gRNA is modified. In particular examples, the spacer sequence is SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:70.

[0023] Features of the compositions and methods herein are also described in the following enumerated embodiments. 1. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having a sequence selected from the following: ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50), ATCCCTGGCACTCTAGAACCCGG (SEQ ID NO: 49), GGCCGGGTTCTAGAGTGCCA (SEQ ID NO: 51), GGCCGGGTTCTAGAGTGCCAGGG (SEQ ID NO: 29), CCTCACTAGACTTGACCCAC (SEQ ID NO: 58); or CCTCACTAGACTTGACCCACAGG (SEQ ID NO: 48). 2. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50). 3. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50), which genetic mutation results in reduced expression levels of CD33 compared to wild-type counterpart cells. 4. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50), which genetic mutation results in a reduced expression level of CD33 that is less than 20% of the level of CD33 in a wild-type counterpart cell. 5. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACCCGG (SEQ ID NO: 49). 6. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58). 7. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58), which genetic mutation results in reduced expression levels of CD33 compared to wild-type counterpart cells. 8. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58), which genetic mutation results in a reduced expression level of CD33 that is less than 20% of the level of CD33 in a wild-type counterpart cell. 9. A genetically engineered hematopoietic stem or progenitor cell containing a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCACAGG (SEQ ID NO: 48). 10. A genetically engineered hematopoietic stem or progenitor cell comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site targeted by a gRNA, wherein the gRNA comprises the nucleotide sequence AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67) or CCUCACUAGACUUGACCCAC (SEQ ID NO: 70), and wherein the genetically engineered hematopoietic stem and / or progenitor cell has a reduced expression level of CD33 compared to its wild-type counterpart. 11. A genetically engineered hematopoietic stem or progenitor cell comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site targeted by a gRNA comprising the nucleotide sequence CCUCACUAGACUUGACCCAC (SEQ ID NO: 70). 12. A genetically engineered hematopoietic stem or progenitor cell comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site targeted by a gRNA comprising the nucleotide sequence AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67). 13. The genetically engineered hematopoietic stem or progenitor cell of any of embodiments 1-12, wherein the genetically engineered hematopoietic stem or progenitor cell does not comprise a mutation in any predicted off-target site, e.g., at any site listed in Figure 30, e.g., at any site in SEQ ID NOs: 99-112. 14. The genetically engineered hematopoietic stem or progenitor cell of any of embodiments 1-13, which does not contain a mutation in any of the predicted off-target sites listed in Figure 29, for example, in any of SEQ ID NOs: 79-98. 15. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 14, wherein the genetic mutation is a substitution (e.g., a single-base variant), an insertion, or a deletion, or a combination thereof. 16. The genetically engineered hematopoietic stem or progenitor cell of embodiment 15, wherein the deletion is entirely within SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 58. 17. The genetically engineered hematopoietic stem or progenitor cell of embodiment 15 or 16, wherein the deletion is 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, or 17 nucleotides in length. 18. The genetically engineered hematopoietic stem or progenitor cell of embodiment 15, wherein the deletion extends outside of SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 58. 19. The genetically engineered hematopoietic stem or progenitor cell of any of embodiments 1 to 18, wherein the genetic mutation results in a frameshift. 20. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 19, wherein the genetic mutation results in a reduced expression level of wild-type CD33 compared to a wild-type counterpart cell (e.g., less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% of the level of a wild-type counterpart cell). 21. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 20, wherein the genetically engineered hematopoietic stem or progenitor cells produce reduced levels of wild-type CD33 compared to wild-type counterpart cells (e.g., less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% of the levels of wild-type counterpart cells). 22. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 21, wherein the genetic mutation results in a reduced expression level of CD33 compared to a wild-type counterpart cell (e.g., less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% of the level of a wild-type counterpart cell). 23. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 22, wherein the genetically engineered hematopoietic stem or progenitor cells result in reduced levels of CD33 compared to wild-type counterparts (e.g., less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% of the levels of wild-type counterparts). 24. The genetically engineered hematopoietic stem or progenitor cell of any of embodiments 1 to 23, wherein the genetic mutation results in a lack of expression of CD33. 25. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 24, which express CD33 that is less than 20% of the CD33 expressed by their wild-type counterparts. 26. The genetically engineered hematopoietic stem or progenitor cell of any of embodiments 1-25, wherein the reduced expression level of CD33 is in a cell differentiated (e.g., terminally differentiated) from a hematopoietic stem or progenitor cell, and the wild-type counterpart is a cell differentiated (e.g., terminally differentiated) from a wild-type hematopoietic stem or progenitor cell. 27. The genetically engineered hematopoietic stem or progenitor cells of embodiment 26, wherein the cells differentiated from hematopoietic stem or progenitor cells are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 28. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 27, which do not express CD33. 29. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 28, which are CD34+. 30. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 29, which are bone marrow or peripheral blood mononuclear cells of a subject. 31. The genetically engineered hematopoietic stem or progenitor cells of embodiment 30, wherein the subject is a human patient with a hematopoietic malignancy. 32. The genetically engineered hematopoietic stem or progenitor cells of embodiment 30, wherein the subject is a healthy human donor (e.g., an HLA-matched donor). 33. The genetically engineered hematopoietic stem or progenitor cell of any of embodiments 1 to 32, produced by a process comprising contacting the endogenous CD33 gene with a nuclease selected from a CRISPR endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a meganuclease (e.g., by contacting the cell with a nuclease or a nucleic acid encoding the nuclease). 34. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells according to any one of embodiments 1 to 33 (e.g., comprising hematopoietic stem cells, hematopoietic progenitor cells, or a combination thereof). 35. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50). 36. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50), and the genetic mutation resulting in a reduced expression level of CD33 compared to a wild-type counterpart cell population. 37. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50), and the genetic mutation resulting in a reduced expression level of CD33 that is less than 20% of the level of CD33 in a wild-type counterpart cell. 38. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58). 39. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58), and the genetic mutation resulting in reduced expression levels of CD33 compared to a wild-type counterpart cell population. 40. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells comprising a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58), and the genetic mutation resulting in a reduced expression level of CD33 that is less than 20% of the level of CD33 in a wild-type counterpart cell. 41. The cell population of any of embodiments 34-40, further comprising one or more cells comprising one or more non-genetically engineered CD33 genes. 42. The cell population of any of embodiments 34-41, further comprising one or more cells that are homozygous wild type for CD33. 43. The cell population of any of embodiments 34-42, further comprising one or more cells that are heterozygous wild type for CD33. 44. A cell population described in any of embodiments 34 to 43, wherein at least 50%, 60%, 70%, 80%, 85%, 90% or 95% of the cells in the population contain a genetic mutation in both copies of CD33. 45. A cell population according to any of embodiments 34 to 44, which expresses less than 20% of the CD33 expressed by a wild-type counterpart cell population. 46. ​​A cell population according to any of embodiments 34-45, wherein the reduced expression level of CD33 is in cells differentiated (e.g., terminally differentiated) from hematopoietic stem or progenitor cells, and the wild-type counterpart cells are cells differentiated (e.g., terminally differentiated) from wild-type hematopoietic stem or progenitor cells. 47. The cell population of embodiment 46, wherein the cells differentiated from hematopoietic stem or progenitor cells are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 48. A cell population according to any of embodiments 34 to 47, comprising hematopoietic stem cells and hematopoietic progenitor cells. 49. A pharmaceutical composition comprising genetically engineered hematopoietic stem or progenitor cells according to any of embodiments 1 to 33. 50. A pharmaceutical composition comprising a cell population according to any one of embodiments 34 to 48. 51. A method for producing genetically engineered hematopoietic stem or progenitor cells according to any one of embodiments 1 to 33, or a cell population according to any one of embodiments 34 to 48, comprising: (i) providing hematopoietic stem or progenitor cells (e.g., wild-type hematopoietic stem or progenitor cells); and (ii) introducing into the cell a nuclease (e.g., an endonuclease) that cleaves at that site; thereby producing genetically engineered hematopoietic stem or progenitor cells. 52. The method of embodiment 51, wherein (ii) comprises introducing into the cell a gRNA that binds the site and an endonuclease that binds the gRNA. 53. The method of embodiment 51, wherein the endonuclease is a ZFN, TALEN, or meganuclease. 54. AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67), GGCCGGGUUCUAGAGUGCCA (SEQ ID NO: 68), or CCUCACUAGACUUGACCCAC (SEQ ID NO: 70), or its reverse complement, or a sequence having at least 90% or 95% identity to any of the foregoing, or a sequence having no more than 1, 2, or 3 mutations relative to any of the foregoing. 55. A guide ribonucleic acid (gRNA) comprising a spacer sequence that is at least about 90% identical to SEQ ID NO: 70. 56. The gRNA of embodiment 55, wherein the spacer sequence comprises SEQ ID NO: 70. 57. A guide ribonucleic acid (gRNA) comprising a spacer sequence that is at least about 90% identical to SEQ ID NO: 67. 58. The gRNA of embodiment 57, wherein the spacer sequence comprises SEQ ID NO: 67. 59. CCUCAUCCCUGGCACUCUAGAACCCGGC (SEQ ID NO: 71), GAGUGGCCGGGUUCUAGAGUGCCAGGGA (SEQ ID NO: 72), or UUCUCCUCACUAGACUUGACCCACAGGC (SEQ ID NO: 73), or its reverse complement, or a sequence having at least 90% or 95% identity to any of the foregoing, or a sequence having no more than 1, 2, or 3 mutations relative to any of the foregoing. 60. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides start at position 1 of the sequence. 61. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides begin at position 2 of the sequence. 62. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides begin at position 3 of the sequence. 63. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides begin at position 4 of the sequence. 64. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides begin at position 5 of the sequence. 65. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides begin at position 6 of the sequence. 66. The gRNA of embodiment 59, wherein at least 18, 19, 20, 21, or 22 consecutive nucleotides begin at position 7 of the sequence. 67. The gRNA of embodiment 59, wherein at least 18, 19, 20, or 21 consecutive nucleotides begin at position 8 of the sequence. 68. The gRNA of embodiment 59, wherein at least 18, 19, or 20 consecutive nucleotides start at position 9 of the sequence. 69. A gRNA according to any one of embodiments 54 or 59 to 68, wherein the two mutations are not adjacent to each other. 70. A gRNA described in any of embodiments 54, or 59-68, wherein none of the three mutations are adjacent to one another. 71. A gRNA according to any one of embodiments 54, or 59 to 70, wherein one, two, or three mutations are substitutions. 72. The gRNA of any of embodiments 54, or 59 to 70, wherein the one or more mutations are insertions or deletions. 73. A gRNA according to any one of embodiments 54 to 72, wherein the spacer sequence is about 18 to 23, e.g., 20, nucleotides in length. 74. A gRNA described in any one of embodiments 54 to 73, wherein the spacer sequence has a GC content of 45% to 65% or 50 to 60%, for example, 55%. 75. The gRNA of any of embodiments 54-74, comprising one or more chemical modifications (e.g., chemical modifications to the nucleobase, sugar, or backbone moieties). 76. A gRNA described in any of embodiments 54 to 75, comprising one or more 2'O-methyl nucleotides, for example at the positions described herein. 77. A gRNA described in any of embodiments 54 to 76, comprising one or more phosphorothioate or thioPACE linkages, for example, at positions described herein. 78. A gRNA described in any of embodiments 54 to 77, which is a single-molecule gRNA (sgRNA). 79. A gRNA according to any one of embodiments 54 to 78, which binds Cas9. 80. A gRNA according to any of embodiments 54 to 79, which binds to tracrRNA. 81. A gRNA according to any one of embodiments 54 to 79, comprising a scaffold sequence. 82. a) a gRNA according to any of embodiments 54 to 81, or a nucleic acid encoding the gRNA, and b) a second gRNA, or a nucleic acid encoding a second gRNA; A kit or composition comprising: 83. The kit or composition of embodiment 82, wherein the gRNA of (a) comprises a spacer sequence of CCUCACUAGACUUGACCCAC (sequence number 70). 84. The kit or composition of embodiment 82, wherein the gRNA of (a) comprises a spacer sequence of AUCCCUGGCACUCUAGAACC (sequence number 67). 85. A kit or composition according to any of embodiments 82 to 84, wherein the second gRNA targets a lineage-specific cell surface antigen. 86. The kit or composition of any of embodiments 82-85, wherein the second gRNA targets a lineage-specific cell surface antigen other than CD33. 87. A kit or composition described in any of embodiments 82 to 86, wherein the second gRNA targets CLL-1. 88. A kit or composition described in any of embodiments 82 to 87, wherein the second gRNA comprises a spacer sequence of GUUGUAGAGAAAUAUUUCUC (SEQ ID NO: 115), GGAGAGGUUCCUGAUCUUGU (SEQ ID NO: 116), or UGAAUAUCUCCAACAAGAUC (SEQ ID NO: 119). 89. A kit or composition described in any of embodiments 82 to 88, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 70 and the second gRNA comprises a spacer sequence according to SEQ ID NO: 115. 90. A kit or composition described in any of embodiments 82 to 88, wherein the gRNA of (a) comprises a spacer sequence according to SEQ ID NO: 70 and the second gRNA comprises a spacer sequence according to SEQ ID NO: 116. 91. A kit or composition described in any of embodiments 82 to 88, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 67 and the second gRNA comprises a spacer sequence according to SEQ ID NO: 115. 92. A kit or composition described in any of embodiments 82 to 88, wherein the gRNA of (a) comprises a spacer sequence according to SEQ ID NO: 67 and the second gRNA comprises a spacer sequence according to SEQ ID NO: 116. 93. A kit or composition described in any of embodiments 82 to 88, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 67 and the second gRNA comprises a spacer sequence according to SEQ ID NO: 70. 94. A kit or composition described in any of embodiments 82 to 88, wherein the gRNA of (a) comprises a spacer sequence according to SEQ ID NO: 115 and the second gRNA comprises a spacer sequence according to SEQ ID NO: 116. 95. The kit or composition of any of embodiments 82 to 94, further comprising a third gRNA, or a nucleic acid encoding a third gRNA. 96. The kit or composition of embodiment 95, wherein the third gRNA targets a lineage-specific cell surface antigen. 97. The kit or composition of embodiment 95, wherein the third gRNA targets CD33 or CLL-1. 98. A kit or composition described in any of embodiments 95 to 97, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 70, the second gRNA comprises a spacer sequence according to SEQ ID NO: 115, and the third gRNA comprises a spacer sequence according to SEQ ID NO: 116. 99. A kit or composition described in any of embodiments 95 to 97, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 67, the second gRNA comprises a spacer sequence according to SEQ ID NO: 115, and the third gRNA comprises a spacer sequence according to SEQ ID NO: 116. 100. A kit or composition described in any of embodiments 95 to 97, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 67, the second gRNA comprises a spacer sequence according to SEQ ID NO: 70, and the third gRNA comprises a spacer sequence according to SEQ ID NO: 116. 101. A kit or composition described in any of embodiments 95 to 97, wherein the gRNA (a) comprises a spacer sequence according to SEQ ID NO: 67, the second gRNA comprises a spacer sequence according to SEQ ID NO: 70, and the third gRNA comprises a spacer sequence according to SEQ ID NO: 115. 102. The kit or composition of any of embodiments 95-101, further comprising a fourth gRNA, or a nucleic acid encoding a fourth gRNA. 103. The kit or composition of embodiment 102, wherein the fourth gRNA targets a lineage-specific cell surface antigen. 104. The kit or composition of embodiment 102, wherein the fourth gRNA targets CD33 or CLL-1. 105. The kit or composition of embodiment 102, wherein the gRNA of (a) comprises a spacer sequence according to SEQ ID NO: 67, the second gRNA comprises a spacer sequence according to SEQ ID NO: 70, the third gRNA comprises a spacer sequence according to SEQ ID NO: 115, and the fourth gRNA comprises a spacer sequence according to SEQ ID NO: 116. 106. A kit or composition described in any of embodiments 102 to 105, wherein the gRNA (a), the second gRNA, the third gRNA, and the fourth gRNA are mixed. 107. A kit or composition described in any of embodiments 102 to 105, wherein the gRNA (a), the second gRNA, the third gRNA, and the fourth gRNA are in separate containers. 108. A kit or composition according to any of embodiments 82-105, wherein (a) and (b) are mixed. 109. A kit or composition according to any of embodiments 82-105, wherein (a) and (b) are in separate containers. 110. The kit or composition according to any of embodiments 82 to 109, wherein the nucleic acid of (a) and the nucleic acid of (b) are part of the same nucleic acid. 111. The kit or composition according to any of embodiments 82-109, wherein the nucleic acid of (a) and the nucleic acid of (b) are separate nucleic acids. 112. (a) A genetic mutation in exon 3 of the endogenous CD33 gene, located at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50); (b) a genetic mutation in exon 3 of the endogenous CD33 gene, the genetic mutation being at a site having the sequence CCTCACTAGACTTGACCCAC (SEQ ID NO: 58); (c) a genetic mutation in CLL-1 at a site having the sequence GTTGTAGAGAAATATTTCTC (SEQ ID NO: 117); (d) a genetic mutation in CLL-1 at a site having the sequence GGAGAGGTTCCTGATCTTGT (SEQ ID NO: 118); Genetically engineered hematopoietic cells (e.g., hematopoietic stem or progenitor cells) comprising one or more (e.g., two, three, or all) of the following: 113. The hematopoietic cell of embodiment 112, comprising (a) and (b). 114. The hematopoietic cell of embodiment 112, comprising (a) and (c). 115. The hematopoietic cell of embodiment 112, comprising (a) and (d). 116. The hematopoietic cell of embodiment 112, comprising (b) and (c). 117. The hematopoietic cell of embodiment 112, comprising (b) and (d). 118. The hematopoietic cell of embodiment 112, comprising (c) and (d). 119. The hematopoietic cell of embodiment 112, comprising (a), (b) and (c). 120. The hematopoietic cell of embodiment 112, comprising (a), (b) and (d). 121. A hematopoietic cell according to embodiment 112, comprising (a), (c) and (d). 122. The hematopoietic cell of embodiment 112, comprising (b), (c) and (d). 123. The hematopoietic cell of embodiment 112, comprising (a), (b), (c) and (d). 124. A hematopoietic cell according to any of embodiments 112-123, comprising a deletion spanning from a position within SEQ ID NO: 50 to a position within SEQ ID NO: 58. 125. A hematopoietic cell according to any of embodiments 112-124, comprising a deletion spanning from a position within SEQ ID NO: 117 to a position within SEQ ID NO: 118. 126. Use of a gRNA according to any one of embodiments 54 to 81 or a composition or kit according to any one of embodiments 82 to 111 for reducing the expression of CD33 in a hematopoietic stem or progenitor cell sample using the CRISPR / Cas9 system. 127. Use of a CRISPR / Cas9 system for reducing the expression of CD33 in a hematopoietic stem or progenitor cell sample, wherein the gRNA of the CRISPR / Cas9 system is a gRNA described in any one of embodiments 54 to 81 or a gRNA of a composition or kit described in any one of embodiments 82 to 111. 128. A method for producing genetically engineered hematopoietic stem or progenitor cells, comprising: (i) providing hematopoietic stem or progenitor cells (e.g., wild-type hematopoietic stem or progenitor cells); and (ii) introducing into the cell: (a) a guide RNA (gRNA) according to any one of embodiments 22 to 39, or a gRNA of a composition or kit according to any one of embodiments 82 to 111; and (b) a nuclease (e.g., an endonuclease) that binds to the gRNA (e.g., Cas9 endonuclease); thereby producing genetically engineered hematopoietic stem or progenitor cells. 129. A method for producing genetically engineered hematopoietic stem or progenitor cells, comprising: (i) providing genetically engineered hematopoietic stem and / or progenitor cells; and (ii) introducing into the cells (a) a guide RNA (gRNA) comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 67 or SEQ ID NO: 70, and (b) a Cas9 endonuclease, thereby producing engineered hematopoietic stem and / or progenitor cells having reduced expression levels of CD33; A method comprising: 130. A method for producing genetically engineered hematopoietic stem or progenitor cells, comprising: (i) providing hematopoietic stem or progenitor cells; and (ii) introducing (a) a guide RNA (gRNA) comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 67 or SEQ ID NO: 70, and (b) a Cas9 endonuclease into the cell, thereby producing a genetically engineered hematopoietic stem or progenitor cell; A method comprising: 131. A method for producing genetically engineered hematopoietic stem or progenitor cells with reduced expression levels of CD33, comprising: (i) providing hematopoietic stem or progenitor cells; and (ii) introducing into the cell (a) a guide RNA (gRNA) comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 67 or SEQ ID NO: 70, and (b) a Cas9 endonuclease, thereby producing a genetically engineered hematopoietic stem or progenitor cell having a reduced expression level of CD33; A method comprising: 132. A method for producing genetically engineered hematopoietic stem or progenitor cells, comprising: (i) providing hematopoietic stem or progenitor cells; and (ii) introducing (a) a guide RNA (gRNA) comprising a nucleotide sequence according to SEQ ID NO: 67, and (b) a Cas9 endonuclease into the cell, thereby producing a genetically engineered hematopoietic stem or progenitor cell; A method comprising: 133. A method for producing genetically engineered hematopoietic stem or progenitor cells, comprising: (i) providing hematopoietic stem or progenitor cells; and (ii) introducing (a) a guide RNA (gRNA) comprising a nucleotide sequence according to SEQ ID NO: 70, and (b) a Cas9 endonuclease into the cell, thereby producing a genetically engineered hematopoietic stem or progenitor cell; A method comprising: 134. A method or use according to any of embodiments 51-53 or 126-133, which results in genetically engineered hematopoietic stem or progenitor cells having a reduced expression level of CD33 compared to the wild-type counterpart. 135. A method or use according to any of embodiments 51-53 or 126-134, which results in genetically engineered hematopoietic stem or progenitor cells having an expression level of CD33 that is less than 20% of the level of CD33 in the wild-type counterpart. 136. The method or use according to any of embodiments 51-53 or 126-135, which is carried out on a plurality of hematopoietic stem or progenitor cells. 137. The method or use according to any of embodiments 51-53 or 126-136, which is carried out on a cell population comprising a plurality of hematopoietic stem cells and a plurality of hematopoietic progenitor cells. 138. The method or use according to any one of embodiments 51 to 53 or 126 to 137, for producing a cell population according to any one of embodiments 34 to 48. 139. The method of any of embodiments 128 to 138, wherein the nucleic acids of (a) and (b) are encoded in a single vector that is introduced into the cell. 140. The method of embodiment 139, wherein the vector is a viral vector. 141. The method of embodiment 139, wherein (a) and (b) are introduced into the cell as a preformed ribonucleoprotein complex. 142. The method of embodiment 141, wherein the ribonucleoprotein complex is introduced into the cell by electroporation. 143. The method of any of embodiments 128-142, wherein the endonuclease (e.g., Cas9 endonuclease) is introduced into the cell by delivering a nucleic acid molecule (e.g., mRNA molecule) encoding the Cas9 endonuclease into the cell. 144. The method of any of embodiments 128-143, wherein the gRNA is a single-molecule guide RNA (sgRNA). 145. The method of any of embodiments 128 to 144, wherein the gRNA is a modified sgRNA. 146. The method of any of embodiments 128 to 145, wherein the gRNA is a chemically modified sgRNA. 147. The method of any of embodiments 128-146, wherein the hematopoietic stem or progenitor cells are CD34+. 148. The method of any of embodiments 128-147, wherein the hematopoietic stem or progenitor cells are derived from the subject's bone marrow cells or peripheral blood mononuclear cells (PBMCs). 149. The method of embodiment 148, wherein the subject has a hematopoietic disorder. 150. The method of embodiment 148, wherein the subject is a healthy donor. 151. The method or use according to any of embodiments 51 to 53 or 126 to 150, which results in a mutation that confers a reduced expression level of CD33 compared to a wild-type counterpart cell. 152. A method or use according to any of embodiments 51-53 or 126-151, which results in a mutation that confers a reduced expression level of wild-type CD33 compared to a wild-type counterpart cell. 153. The method or use according to any of embodiments 51-53 or 126-152, for producing genetically engineered hematopoietic stem or progenitor cells having reduced expression levels of CD33 compared to wild-type counterparts. 154. A method or use according to any of embodiments 51-53 or 126-153, for producing genetically engineered hematopoietic stem or progenitor cells having reduced expression levels of wild-type CD33 compared to wild-type counterpart cells. 155. Genetically engineered hematopoietic stem or progenitor cells produced by the method or use of any of embodiments 51-53 or 126-154. 156. A method for treating a hematopoietic disorder, comprising administering to a subject in need thereof an effective amount of genetically engineered hematopoietic stem or progenitor cells according to any of embodiments 1-33, or a cell population according to any of embodiments 34-48, or a hematopoietic cell according to any of embodiments 112-125. 157. The genetically engineered hematopoietic stem or progenitor cells of any of embodiments 1 to 33, the cell population of any of embodiments 34 to 38, or the hematopoietic cells of any of embodiments 112 to 125, for use in the treatment of a hematopoietic disorder, wherein the treatment comprises administering to a subject in need thereof an effective amount of the genetically engineered hematopoietic stem or progenitor cells or cell population, and further comprises administering to the subject an effective amount of an agent that targets CD33, wherein the agent comprises an antigen-binding fragment that binds CD33. 158. An agent targeting CD33 for use in the treatment of a hematopoietic disorder, wherein the agent comprises an antigen-binding fragment that binds to CD33, and wherein treatment comprises administering to a patient in need thereof an effective amount of an agent that targets CD33, and further comprises administering to the patient an effective amount of a genetically engineered hematopoietic stem or progenitor cell described in any of embodiments 1-33, a cell population described in any of embodiments 34-38, or a hematopoietic cell described in any of embodiments 112-125. 159. A combination of genetically engineered hematopoietic stem or progenitor cells according to any of embodiments 1 to 33, a cell population according to any of embodiments 34 to 38, or hematopoietic cells according to any of embodiments 112 to 125, and an agent that targets CD33, for use in the treatment of a hematopoietic disorder, wherein the agent comprises an antigen-binding fragment that binds to CD33, and the treatment comprises administering to a patient in need thereof an effective amount of the genetically engineered hematopoietic stem or progenitor cells or cell population, and an agent that binds CD33. 160. A genetically engineered hematopoietic stem or progenitor cell according to any one of embodiments 1 to 33, or a cell population according to any one of embodiments 34 to 38, or a hematopoietic cell according to any one of embodiments 112 to 125, for use in cancer immunotherapy. 161. A genetically engineered hematopoietic stem or progenitor cell according to any one of embodiments 1 to 33, or a cell population according to any one of embodiments 34 to 38, or a hematopoietic cell according to any one of embodiments 112 to 125, for use in cancer immunotherapy, in which the patient has a hematopoietic disorder. 162. Genetically engineered hematopoietic stem or progenitor cells according to any of embodiments 1 to 33, or a cell population according to any of embodiments 34 to 38, or hematopoietic cells according to any of embodiments 112 to 125, for use in hematopoietic repopulation in patients with hematopoietic disorders. 163. A genetically engineered hematopoietic stem or progenitor cell according to any of embodiments 1 to 33, or a cell population according to any of embodiments 34 to 38, or a hematopoietic cell according to any of embodiments 112 to 125, for use in a method for treating a hematopoietic disorder, whereby the genetically engineered hematopoietic stem or progenitor cell described herein or the cell population described herein repopulates the patient. 164. A genetically engineered hematopoietic stem or progenitor cell according to any one of embodiments 1 to 33, or a cell population according to any one of embodiments 34 to 38, or a hematopoietic cell according to any one of embodiments 112 to 125, for use in reducing the cytotoxicity of substances targeting CD33 in immunotherapy. 165. A genetically engineered hematopoietic stem or progenitor cell according to any one of embodiments 1 to 33, or a cell population according to any one of embodiments 34 to 38, or a hematopoietic cell according to any one of embodiments 112 to 125, for use in immunotherapy with an agent that targets CD33, whereby the genetically engineered hematopoietic stem or progenitor cell described herein or the cell population described herein reduces the cytotoxic effect of the agent that targets CD33. 166. The method, cell, substance, or combination according to any of embodiments 156-165, wherein the genetically engineered hematopoietic stem or progenitor cells or cell population are administered simultaneously with a substance that targets CD33. 167. The method, cell, substance, or combination according to any of embodiments 156 to 165, wherein the genetically engineered hematopoietic stem or progenitor cells or cell population are administered before the substance targeting CD33. 168. The method, cell, substance or combination according to any of embodiments 156-165, wherein the substance targeting CD33 is administered before the genetically engineered hematopoietic stem or progenitor cells or cell population. 169. The method, cell, substance, use, or combination according to any of embodiments 156-168, wherein the hematopoietic disorder is a hematopoietic malignancy. 170. The method, cell, substance, use, or combination of any of embodiments 156-169, further comprising administering to the subject an effective amount of a substance that targets CD33, wherein the substance comprises an antigen-binding fragment that binds CD33. 171. The method, cell, substance, use, or combination of embodiment 170, wherein the substance that targets CD33 is an immune cell expressing a chimeric antigen receptor (CAR), which comprises an antigen-binding fragment that binds CD33. 172. The method, cell, substance, use, or combination according to embodiment 171, wherein the immune cells are T cells. 173. The method, cell, substance, use, or combination according to any of embodiments 170 to 172, wherein the immune cells, the genetically engineered hematopoietic stem and / or progenitor cells, or both, are allogeneic. 174. The method, cell, substance, use, or combination according to any of embodiments 170 to 173, wherein the immune cells, genetically engineered hematopoietic stem and / or progenitor cells, or both, are autologous. 175. The method, cell, substance, use, or combination according to any of embodiments 170 to 174, wherein the antigen-binding fragment in the chimeric receptor is a single-chain antibody fragment (scFv) that specifically binds human CD33. 176. The method, cell, substance, use, or combination according to any of embodiments 170 to 175, wherein the subject is a human patient with Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. 177. The method, cell, substance, use, or combination according to embodiment 176, wherein the subject is a human patient with leukemia, which is acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.

[0024] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the detailed description of some embodiments and the appended claims. The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these figures in conjunction with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is an exemplary illustration of type 0, type 1, type 2, and type 3 lineage-specific antigens. [Figure 2] Schematic representation of immune cells expressing chimeric receptors targeting the type 0 lineage-specific cell surface antigen, CD307. CD307-expressing multiple myeloma (MM) cells, as well as other cells expressing CD307, such as plasma cells, are targeted by immune cells expressing anti-CD307 chimeric receptors. [Figure 3] Schematic representation of immune cells expressing a chimeric receptor targeting the type 2 lineage-specific cell surface antigen, CD33. Acute myeloid leukemia (AML) cells express CD33. Human hematopoietic stem cells (HSCs) are genetically engineered to lack CD33 and are therefore not recognized by immune cells expressing the anti-CD33 chimeric receptor. HSCs can give rise to myeloid cells. [Figure 4] Schematic diagram of genome editing using the CRISPR / Cas system. The sgRNA hybridizes to a portion of the exon of a lineage-specific cell surface antigen, and the Cas9 endonuclease cleaves upstream of the protospacer adjacent motif (PAM) sequence (5'-NGG-3'). The sequence corresponds to SEQ ID NOs: 45 and 46, from upstream to downstream. [Figure 5]This diagram shows a schematic of a genome editing method using the CRISPR / Cas9 system to disrupt CD33. The PX458 vector encoding the Cas9 protein and a guide RNA targeting CD33 were transfected into K-562 cells, a human leukemia cell line, by nucleofection. Flow cytometry was performed on the cell population using an anti-CD33 antibody before (top panel) and after (bottom panel) delivery of Cas9 and the guide RNA into the cells. Genome editing resulted in deletion of the gene's coding region and a significant reduction of CD33 expression from the cell surface. [Figure 6] A schematic diagram of a genome editing method using the CRISPR / Cas9 system to disrupt CD45RA. The PX458 vector encoding the Cas9 protein and a guide RNA targeting CD45RA were transfected into TIB-67 reticulum cell sarcoma mouse macrophage-like cells by nucleofection. Flow cytometry was performed on the cell population using an anti-CD45RA antibody before (top panel) and after (bottom panel) delivery of Cas9 and the guide RNA into the cells. Genome editing resulted in deletion of the coding region of the gene and a significant reduction of CD45RA from the cell surface. [Figure 7A-D] Schematics of exemplary chimeric receptors containing antigen-binding fragments targeting CD33 are shown. Figure 7A: A typical chimeric receptor targeting CD33 containing an anti-CD33 scFv, a hinge region, a transmembrane region, a costimulatory region, and a signaling region. Figure 7B: A chimeric receptor targeting CD33 containing an anti-CD33 scFv, a CD8-derived hinge region, a CD8-derived transmembrane region, and an intracellular region derived from CD28 and CD3ζ. Figure 7C: A chimeric receptor targeting CD33 containing an anti-CD33 scFv, a CD8-derived hinge region, a CD8-derived transmembrane region, and an intracellular region derived from ICOS (or CD27, 4-1BB, or OX-40) and CD3ζ. Figure 7D: A chimeric receptor targeting CD33 containing an anti-CD33 scFv, a CD8-derived hinge region, a CD8-derived transmembrane region, and an intracellular region derived from OX40, CD28, and CD3ζ. [Figure 8] FIG. 1 is a schematic diagram of an immunotoxin. [Figure 9A-B] Figure 9 shows the expression of anti-CD33 chimeric receptors in K562 cells transduced with an empty vector or a vector encoding an anti-CD33 chimeric receptor. Figure 9A: Western blot using a primary antibody that recognizes CD3ζ. The table shows the estimated molecular weight of each chimeric receptor tested. Figure 9B: Flow cytometry analysis showing an increased population of cells staining positively with the anti-CD33 chimeric receptor. [Figure 10A-C] Figure 10A: Ponceau-stained protein gel. Lanes 1, 3, and 5: CD33 molecules. Lanes 2, 4, and 6: CD33 molecule + APC complex. Figure 10B: Western blot using a primary antibody that recognizes CD3ζ. Lanes 1, 3, and 5 contain chimeric receptors co-incubated with CD33 molecules, and lanes 2, 4, and 6 contain chimeric receptors co-incubated with CD33-APC complexes. Figure 10C: Flow cytometry analysis showing an increase in the cell population that expresses anti-CD33 chimeric receptors and binds CD33. [Figure 11A-B] Cytotoxicity of K562 cells by NK92 cells expressing the indicated chimeric receptors. Figure 11A: CART1 and CART2 compared to empty HIVzsG vector. Figure 11B: CART3 compared to empty HIVzsG vector. [Figure 12A-B] Cytotoxicity of CD33-deficient K562 cells by NK92 cells expressing the indicated chimeric receptors (shown as percent cytotoxicity on the y-axis) is shown. Figure 12A: Unsorted population of K562 cells pretreated with CD33-targeting CRISPR / Cas reagents. Figure 12B: Single clone of CD33-deficient K562 cells. Bars, from left to right, represent empty HIVzsG vector, CART1, CART2, and CART3. [Figure 13A-B] Flow cytometry analysis of primary T cell populations is shown. Figure 13A: Sorting of cells based on expression of T cell markers CD4+, CD8+, or both CD4+CD8+. Figure 13B: Relative expression of CD33 in the indicated populations of primary T cells. [Figure 14A-B]Cytotoxicity of K562 cells by primary T cells expressing the indicated chimeric receptors is shown. Figure 14A: CD4+ T cells. Figure 14B: CD4+ / CD8+ (CD4 / 8) and CD8+ (CD8). [Figure 15] Flow cytometry analysis of CD33 editing in K652 cells using the CRISPR / Cas9 system and two different gRNAs (Crispr3, top right panel, and Crispr5, bottom right panel). [Figures 16A-C] Figure 16 shows that K562 cells lacking CD33 exhibit normal cell proliferation and erythropoietic differentiation. Figure 16A: Flow cytometry analysis of the indicated cell populations at day 1 + 50 μM hemin. Figure 16B: Flow cytometry analysis of the indicated cell populations at day 9. Figure 16C: MTT cell proliferation assay. [Figures 17A-C] Flow cytometry analysis of CD33 editing in human CD34+ cells using the CRISPR / Cas9 system and two different gRNAs (crispr3, lower left panel, and crispr5, lower right panel). Figure 17A: Flow cytometry analysis of CD33 editing in human CD34+ cells using the CRISPR / Cas9 system. Figure 17B: crispr3. Figure 17C: crispr5. [Figure 18] Colony formation of human CD34+ / CD33- cells compared to human CD34+ / CD33+ cells is shown. Bars (left to right) represent uninfected lentivirus, empty vector control, crispr1, crispr3, and crispr5. [Figures 19A-F]CRISPR / Cas9-mediated gene ablation of the CD33 antigen is shown. Figure 19A illustrates the approach: stem cells, mobilized blood, or umbilical cord blood from a donor are genetically engineered to ablate CD33 expression using gene editing techniques such as CRISPR / Cas9 and transplanted into HSCT-eligible relapsed patients. After transplantation, T cells from the allogeneic donor are genetically engineered to express a chimeric antigen receptor targeting CD33 using a viral delivery system and infused into the recipient. Alternatively, patients may receive an ADC (GO) alone or in combination with a CAR-T. Figures 19B-19F illustrate CD33 expression and its ablation in human cells. Figure 19B: Expression of CD33 in the human AML cell line HL-60, in bone marrow (BM)- and umbilical cord blood-derived human primary CD34+CD33WT cells, and in human primary CD34+CD33Del cells after CRISPR / Cas9-mediated ablation. Figure 19C: Schematic representation of the CD33 genome and locus showing exons 2-4, and the location and sequence of the sgRNA targeting CD33 (bold). Sequences correspond, from upstream to downstream, to SEQ ID NOs: 52-53. Figure 19D: Surface expression of CD33 by flow cytometry in CD34+CD33WT cells and CD34+CD33Del cells after electroporation. All cells maintain their stem cell phenotype, as assessed by CD90 expression. Figure 19E: Sanger sequencing chromatograms showing the region around the DNA double-strand break site; top panel: CD34+CD33WT cells and bottom panel: CD34+CD33Del cells. Sequences correspond, from upstream to downstream, to SEQ ID NOs: 54-55. Figure 19F: Five to seven days after electroporation, CD34+ cultured cells show consistent loss of CD33 compared to controls. [Figures 20A-E]Figure 20A shows that CD33 deletion does not impair engraftment and hematopoietic repopulation in NSG-SGM3 mice. Figure 20A: Schematic of the experimental design. Figures 20B-20C show bone marrow-derived CD34+ cell engraftment and repopulation: Post-transplant peripheral blood (7 weeks; Figure 20B) and whole bone marrow (21 weeks; Figure 20C) were analyzed for cells of various lineages, as indicated. CD34+CD33Del cells show the same engraftment (CD45+) as control cells and comparable percentages of mature myeloid and lymphoid cells. Bone marrow CD34+CD33Del cells show comparable percentages of myeloid (progenitor cells CD123+, mature CD14+) and lymphoid (progenitor cells CD10+, mature CD19+), T cells (CD3+), and stem cells CD34+38-. Figures 20D-20E show bone marrow-derived CD34+ cell engraftment and repopulation: Post-transplant peripheral blood (week 9; Figure 20D) and whole bone marrow (week 21; Figure 20E) were analyzed for cells of various lineages, as indicated. CD34+CD33Del cells show the same engraftment (CD45+) as control cells and comparable percentages of mature myeloid and lymphoid cells. Bone marrow CD34+CD33Del cells show comparable percentages of myeloid (progenitor CD123+, mature CD14+) and lymphoid (progenitor CD10+, mature CD19+), T cells (CD3+), and stem cells CD34+38-. Data were analyzed using an unpaired t-test, and no significant differences were found among all groups examined (p>0.05). All data are presented as mean ± SEM. [Figure 21A-D]Integrated Genomic Viewer (IGV) screenshots of the genomic regions of the CD33 (Figure 21A) and Siglec-9 (Figure 21B) genes surrounding the guides in cells with Cas9 + sgRNA (top panel) and Cas9 alone (bottom panel) are shown on the left. The gray bars in the coverage track (shown on the right) indicate the depth of the reads represented at each locus. Generally, coverage should be uniform, so the bar height should be the same, but deletions will result in a decrease in height. The read track shows all reads (gray boxes) mapped in this region. Deletions are represented by solid black lines, and insertions are represented by diagonal hatched boxes. Reads with dark borders have no mapping partners. One read in each group had no mapping partners. Mismatched bases are filled with diamonds, open circles, solid circles, and white for nucleotides A, C, G, and T, respectively. The Siglec-9 genomic region was selected as an exemplary region showing the absence of off-target indels because: 1) it belongs to the Siglec family, which shares homology with CD33, and 2) it has the highest homology within 10 bp of the predicted cleavage site compared to any other guide. Chromosomal coordinates at the bottom are based on hg38. Figure 21C: Scatter plot showing the correlation between log10 mean normalized counts, normalized using the DESeq2 method, between CD33 and control cells. Figure 21D: Volcano plot showing the log2 (fold change) and -log10 (p-value) of genes analyzed using the edgeR method. Significantly differentially expressed (p<0.05) genes are indicated as red open circles, and the CD33 gene is indicated by a left arrow. [Figure 22A-F]Figure 22A shows that CD33 deletion protects CD34+ cells from CART33 cytotoxicity in vitro. Figure 22A: Schematic of CART33 constructs. Figure 22B: Contour plots showing CAR expression in human primary T cells after lentiviral transduction with control (black) or CART33 (green or blue) virus. The percentage transduction of each group is shown next to the contour plots. CD4+ and CD8+ cells were transduced independently and mixed 1:1 prior to the experiment. Figures 22C-22F: Cytotoxicity assays. Figure 22C: CART33 cells or control T cells were incubated with HL-60 or CD34+CD33WT or CD34+CD33Del and assessed by flow cytometry. Figures 22D-22F: Triple culture cytotoxicity assays. CART33 cells or control T cells were co-incubated with HL-60 and CD34+CD33WT (Figure 22D) cells, or with HL-60 and CD34+CD33Del (Figure 22E) cells, or with CD34+CD33WT and CD34+CD33Del (Figure 22F) cells. [Figure 23A-F]Treatment model shown: CD34+CD33Del cells tolerate CD33-targeted immunotherapy. Figure 23A: Schematic of experimental design: 5*105 HL-60 and 5*105 CD34+CD33Del were injected into NSGM3 mice on day 0. One week later, mice were treated with PBS, allogeneic CART33, or control T cells. Three days later, a new group received GO only, while allogeneic CART33 and control T cell-injected mice received GO or PBS. Treatment was repeated at week 3. Leukemia progression and CD34+CD33Del engraftment were then monitored by serial bone marrow aspirations. Figure 23B: Monitoring leukemia burden in bone marrow aspirates. Leukemia cells were gated on Ter119-dtomato+. Figure 23C: Leukemia burden measurement by epifluorescence quantification of images shown at 3.5 weeks (Figure 23D) and 8 weeks (Figure 23E). Background was removed using untreated mice (*imaging control). Figure 23F: CART33 or GO leukemia purging does not impair engraftment of CD34+CD33Del cells (%hCD45+ cells), as shown by flow cytometry of bone marrow aspirates. CD34+ infusion-derived human cells were gated on Ter119-dtomato-, Ly5- / H2kd-human CD45+CART-. [Figure 24A-D]Figure 24A shows that CD34+CD33Del HSPCs exhibit multilineage engraftment and differentiation in a therapeutic model. Figure 24A: CD34+CD33Del cells tolerate CD33-targeted immunotherapy and contribute to myelopoiesis and lymphopoiesis. The left two panels for each condition are time-course monitoring of myeloid progenitor cell repopulation, and the right two panels are lymphoid progenitors and mature cells in BM aspirates. No significant differences were observed between the different treatment groups at any time point analyzed. Figures 24B-24D show that CD34+CD33WT cells are sensitive to CD33-targeted immunotherapy. Figure 24B: Schematic of the experimental design: 5 x 105 CD34+CD33WT cells alone or in combination with 5 x 105 HL-60 cells were injected into NSG-SGM3 mice on day 0. One week later, mice were treated with PBS or allogeneic CART33 cells. Leukemia progression and CD34+CD33WT engraftment were subsequently monitored by bone marrow aspirate at week 3 for CART33. On the same day, a group of mice was injected with GO and analyzed 4 days later. Figures 24C-24D: BM aspirates show complete elimination of CD33WT leukemia cells (Figure 24C) and CD33WT primary cells (Figure 24D) in mice treated with CART33 or GO compared to PBS alone. Significant differences were observed between CART33 and GO compared to PBS. CD34+ injection-derived human cells were gated on Ter119-dtomato-, Ly5- / H2kd- human CD45+CART-. All data are presented as mean ± SEM. [Figure 25A-B] Figure 25A-25B: Schematic representation of the CD33 genome and locus showing exons 2-4, as well as the location and sequence of sgRNAs targeting two additional CD33 loci. The chromatograms at the bottom are screenshots of Sanger sequencing showing the region around the DNA double-strand break site; left panel: CD34+CD33WT cells and right panel: CD34+CD33Del. The guide sequence is highlighted in blue on the chromatogram, and the occurrence of indels is indicated by a red down arrow. The sequences correspond to SEQ ID NOs: 56-57, 75-76, 113, 59, and 77-78, respectively, in order of appearance. [Figure 26A-D] Figures 26A-26B show engraftment and repopulation of bone marrow-derived CD34+ cells. Figure 26A: Bone marrow aspirates (15 weeks) after transplantation were analyzed for cells of various lineages, as indicated. CD34+CD33Del cells show the same engraftment (CD45+) as control cells and a similar percentage of engraftment with mature myeloid and lymphoid cells. Bar graphs represent summaries of individual panels. Figure 26B: Summary of data from the main section of Figure 20C. Figures 26C-26D show engraftment and repopulation of cord blood-derived CD34+ cells. Figure 26C: Bone marrow aspirates (16 weeks) after transplantation were analyzed for cells of various lineages, as indicated. CD34+CD33Del cells show the same engraftment (CD45+) as control cells and a similar percentage of engraftment with mature myeloid and lymphoid cells. Bar graphs represent summaries of individual panels. Figure 26D: Summary of data from Figure 20E. No significant differences were observed between both groups in all cell types analyzed (p>0.05), unpaired t-test. All data are expressed as mean ± SEM. [Figure 27] Figure 27 shows the read coverage of RNA sequencing data. An Integrated Genome Viewer (IGV) screenshot of the coverage track shows the CD33 genomic region surrounding the guide in cells with Cas9+sgRNA (n=5; bottom panel) and Cas9 alone (n=5; top panel), as shown on the left. The gray bars in the coverage track (shown on the right) indicate the depth of the reads shown at each locus. Generally, coverage should be uniform, and therefore the height of the bars should be the same, although deletions, marked by dotted rectangles in the bottom panel, result in reduced height. Figure 27 discloses SEQ ID NOs: 114 and 114, respectively, in order of appearance. [Figure 28] A summary of the reads and variants found in whole-genome sequencing is shown. [Figure 29]Off-target sites for sgRNA846 are shown (sgRNA:AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67); PAM:CGG). No indels were observed within 100 bp of the predicted off-target cleavage site in whole genome sequence analysis. Mismatches with the guide sequence are shown in bold. Figure 29 discloses SEQ ID NOs: 79-98, respectively, in order of appearance. [Figure 30] Off-target sites for sgRNA811 are shown (sgRNA:CCUCACUAGACUUGACCCAC (SEQ ID NO: 70); PAM:AGG). No indels were observed within 100 bp of the predicted off-target cleavage site in whole genome sequence analysis. Mismatches with the guide sequence are represented in bold. Figure 30 discloses SEQ ID NOs: 99-107, 106, 107, 107, 107, 107-112, respectively, in order of appearance. [Figure 31] A list of genes differentially expressed in CD33-depleted cells is shown. Genes whose expression is less in CD33-depleted cells compared to CD33 wild-type cells are indicated by a minus sign. [Figure 32A-C]These results demonstrate that GO-targeted immunotherapy eliminates primary AML and rescues CD33Del cells. Figure 32A: Schematic of the experimental design: 0.5 million primary AML cells were injected into NSGS mice on day 1. Once minimal residual disease was estimated, the treatment group received chronic GO administration (1 μg every 10 days). Ten days after the first GO injection in the treatment group, the mice were transplanted with 0.5 million CD34+CD33Del cells. Figure 32B, left panel: AML (minimal residual disease) burden assessed by flow cytometry of bone marrow aspirates before treatment. Leukemia cells were gated on Ter119-hCD45+hCD33+. Right panel: AML burden and hCD45+hCD33Del engraftment in BM aspirates after chronic GO treatment. Figure 32B (continued): Hematopoietic repopulation (myeloid / lymphoid progenitors and mature cells) of Ter119-, Ly5- / H2kd-, hCD45+, hCD33-gated CD34+CD33Del cells. Figure 32C: Survival curves and analysis of control whole bone marrow (BM), spleen, and peripheral blood (PB) at time of death (solid line, untreated; dotted line, treated). [Figure 33A-D] Figure 33A shows a series of flow cytometry images demonstrating that CD33 and CLL-1 levels can be reduced individually or in combination. Figure 33B shows a series of flow cytometry images demonstrating that CD33 and CLL-1 levels in cells allow for engraftment in mice. Figures 33C and 33D show the frequency of the indicated cell types within the hCD45+ cell population in whole bone marrow samples (Figure 33C) or spleen samples (Figure 34D). From left to right, the four bars in each set represent the following cell types: CD34+WT (circles); CD34+CD33Del (squares), CD34+CLL1Del (triangles), and CD34+CD33DelCLL1Del (inverted triangles). DETAILED DESCRIPTION OF THE INVENTION

[0026] Cancer immunotherapy that targets antigens present on the cell surface of cancer cells is particularly difficult when the target antigen is also present on the surface of normal, non-cancerous cells that are necessary for or significantly involved in the development and / or survival of the subject. Targeting these antigens may result in adverse effects in the subject due to the cytotoxic effects of immunotherapy on such cells in addition to the cancer cells.

[0027] The methods, nucleic acids, and cells described herein allow for targeting antigens (e.g., type 1 or type 2 antigens) that are present not only on cancer cells but also on cells essential for the development and / or survival of a subject. The methods involve (1) reducing the number of cells bearing the target lineage-specific cell surface antigen using an agent that targets the antigen, and (2) replacing normal cells (e.g., non-cancerous cells) that present the antigen and, as a result, may be killed by administration of the agent, with hematopoietic cells that lack the lineage-specific cell surface antigen. The methods described herein can maintain surveillance of target cells, including cancer cells that express the lineage-specific cell surface antigen of interest, and can also maintain a population of non-cancerous cells that express the lineage-specific antigen that may be important for the development and / or survival of a subject.

[0028] Thus, described herein is the co-use of immune cells expressing chimeric receptors containing antigen-binding fragments targeting lineage-specific cell surface antigens (e.g., CD33) and hematopoietic cells, such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs), lacking the lineage-specific cell surface antigen to treat hematopoietic malignancies. Also provided herein are chimeric receptors, nucleic acids encoding the same, vectors containing the same, and immune cells (e.g., T cells) expressing such chimeric receptors. The present disclosure also provides genetically engineered hematopoietic cells lacking lineage-specific antigens as described herein, as well as methods for producing them (e.g., genome editing methods).

[0029] definition The terms "subject," "individual," and "patient" are used interchangeably and refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, human primates, non-human primates, or murine, bovine, equine, canine, or feline species. For the purposes of this disclosure, the term "subject" also encompasses tissues or cells that can be cultured in vitro or ex vivo, or manipulated in vivo. The term "subject" may be used interchangeably with the term "organism."

[0030] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Examples of polynucleotides include, but are not limited to, coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. One or more nucleotides within a polynucleotide may be further modified. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may also be modified after polymerization, such as by conjugation with a labeling agent.

[0031] The term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. The hydrogen bonds can occur through Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can contain two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence that can hybridize to a given sequence is called the "complement" of the given sequence.

[0032] The term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows for expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed in the cell. The vectors of the present disclosure are not generally of natural origin. Portions of the vector may be of natural origin. A non-naturally occurring recombinant expression vector of the present disclosure may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or derived in part from natural sources, and which may contain natural, non-natural, or modified nucleotides.

[0033] As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by physical or chemical methods.

[0034] The terms "antibody," "antibody fragment," "antibody fragment," "functional fragment of an antibody," or "antigen-binding portion" are used interchangeably and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to a particular antigen (Holliger et al., Nat. Biotech. (2005) 23(9):1126). The antibodies of the present invention can be antibodies and / or fragments thereof. Antibody fragments include Fab, F(ab')2, scFv, disulfide-linked Fv, Fc, or variants and / or mixtures. Antibodies can be chimeric, humanized, single-chain, or bispecific. All antibody isotypes are encompassed by the present disclosure, including IgA, IgD, IgE, IgG, and IgM. Suitable IgG subtypes include IgG1, IgG2, IgG3, and IgG4. An antibody light or heavy chain variable region consists of a framework interrupted by three hypervariable regions called complementarity-determining regions (CDRs). The CDRs of the antibody or antigen-binding portion may be of non-human or human origin. The framework of the antibody or antigen-binding portion of the invention may be human, humanized, non-human (e.g., a mouse framework modified to reduce antigenicity in humans), or synthetic (e.g., a consensus sequence).

[0035] The antibodies or antigen-binding portions of the invention are -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M or about 10 -12 A dissociation constant (K D The affinity of antibodies according to the present disclosure can be readily determined using conventional techniques (Scatchard et al., Ann. NY Acad. Sci. (1949) 51:660, and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0036] The terms "chimeric receptor," "chimeric antigen receptor," or "CAR" are used interchangeably throughout and refer to a recombinant polypeptide construct comprising at least an extracellular antigen-binding region, a transmembrane region, and a cytoplasmic signaling region (also referred to herein as an "intracellular signaling region") that includes a functional signaling region derived from a stimulatory molecule as defined below. Lee et al., Clin. Cancer Res. (2012) 18(10):2780; Jensen et al., Immunol Rev. (2014) 257(1):127; www.cancer.gov / about-cancer / treatment / research / car-t-cells. In one embodiment, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling region further comprises one or more functional signaling regions derived from at least one costimulatory molecule as defined below. The costimulatory molecule may also be 4-1BB (e.g., CD137), CD27, and / or CD28, or fragments of these molecules. In another embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition region, a transmembrane region, and an intracellular signaling region comprising a functional signaling region derived from a stimulatory molecule. The CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition region, a transmembrane region, and an intracellular signaling region comprising a functional signaling region derived from a costimulatory molecule and a functional signaling region derived from a stimulatory molecule. Alternatively, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition region, a transmembrane region, and an intracellular signaling region comprising two functional signaling regions derived from one or more costimulatory molecules and a functional signaling region derived from a stimulatory molecule. The CAR also comprises a chimeric fusion protein comprising an extracellular antigen recognition region, a transmembrane region, and at least two functional signaling regions derived from one or more costimulatory molecules and a functional signaling region derived from a stimulatory molecule. The antigen recognition portion of the CAR encoded by the nucleic acid sequence can comprise any lineage-specific antigen-binding antibody fragment. The antibody fragment can comprise one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or any combination of the above.

[0037] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through a defined signaling pathway, by generating second messengers, or by responding to such messengers and thereby functioning as an effector.

[0038] The terms "zeta" or "zeta chain," "CD3-zeta," or "TCR-zeta" are defined as the protein designated by GenBank Accession No. NP_932170, NP_000725, or XP_011508447, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "zeta stimulatory region," or "CD3-zeta stimulatory region," or "TCR-zeta stimulatory region" are defined as amino acid residues from the cytoplasmic region of the zeta chain that are suitable for functionally transmitting the initial signal required for T cell activation.

[0039] The term "genetically engineered" or "genetically modified" refers to a cell that has been manipulated by genetic engineering techniques, for example, by genome editing. That is, the cell contains a heterologous sequence that does not naturally occur in the cell. Generally, the heterologous sequence is introduced via a vector system or other means for introducing a nucleic acid molecule into a cell that contains a ribosome. The heterologous nucleic acid molecule can be integrated into the genome of the cell or can exist extrachromosomally, such as in the form of a plasmid. The term also includes embodiments in which an engineered and isolated CAR polypeptide is introduced into the cell.

[0040] The term "autologous" refers to any material from the same individual that is later reintroduced into the same individual.

[0041] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.

[0042] The term "cell lineage" refers to cells that share a common ancestor and develop into a specific identifiable / functional cell from identifiable cells of the same type. As used herein, cell lineage includes, but is not limited to, respiratory, prostate, breast, renal, intestinal, nervous, skeletal, vascular, hepatic, hematopoietic, muscle, or cardiac cell lineages.

[0043] The term "inhibition," when used in reference to gene expression or function of a lineage-specific antigen, refers to a reduction in the amount of gene expression or function of the lineage-specific antigen, where inhibition is the result of interference with gene expression or function. Inhibition can be complete, where there is no detectable expression or function, or inhibition can be partial. Partial inhibition can range from almost complete inhibition to almost no inhibition. By eliminating specific target cells, CAR T cells can effectively inhibit all expression of a particular cell lineage.

[0044] Cells, such as hematopoietic cells, that "lack a lineage-specific antigen" refer to cells that have a significantly reduced expression level of a lineage-specific antigen compared to their native counterparts, e.g., endogenous hematopoietic cells of the same type, or cells that do not express the lineage-specific antigen, i.e., are undetectable by routine measurements such as FACS. In some examples, the expression level of a lineage-specific antigen in "antigen-lacking" cells can be less than about 40% (e.g., 30%, 20%, 15%, 10%, 5%, or lower) of the expression level of the same lineage-specific antigen in its native counterpart. As used herein, the term "about" refers to a particular value ±5%. For example, an expression level of about 40% can include any amount between 35% and 45%.

[0045] Agents that target lineage-specific cell surface antigens Embodiments of the present disclosure provide agents that target lineage-specific cell surface antigens, for example, on target cancer cells (e.g., agents that target CD33, e.g., the agent comprises an antigen-binding fragment that binds CD33). Such agents can comprise an antigen-binding fragment that binds and targets the lineage-specific cell surface antigen. In some cases, the antigen-binding fragment can be a single-chain antibody (scFv) that specifically binds to the lineage-specific antigen. A. Lineage-specific cell surface antigens As used herein, the terms "lineage-specific cell surface antigen" and "cell surface lineage-specific antigen" are used interchangeably and refer to any antigen that is sufficiently present on the surface of a cell and associated with one or more populations of cell lineages. For example, an antigen may be present on one or more populations of cell lineages and absent (or present in reduced amounts) on the cell surface of other cell populations.

[0046] Generally, lineage-specific cell surface antigens can be classified based on a number of factors, including whether the antigen and / or the cell population presenting the antigen is required for the survival and / or development of the host organism. A summary of exemplary types of lineage-specific antigens is provided in Table 1 below. See also Figure 1. [Table 1]

[0047] As shown in Table 1 and Figure 1, type 0 lineage-specific cell surface antigens are required for tissue homeostasis and survival, and cell types bearing type 0 lineage-specific cell surface antigens may also be required for the survival of a subject. Thus, given the importance of type 0 lineage-specific cell surface antigens, or cells bearing type 0 lineage-specific cell surface antigens, in homeostasis and survival, targeting this category of antigens using conventional CAR T cell immunotherapy may be difficult, as inhibition or elimination of such antigens or cells bearing such antigens may be detrimental to the survival of the subject. As a result, lineage-specific cell surface antigens (such as type 0 lineage-specific antigens) and / or cells bearing such antigens may be required for survival, for example, because they perform non-redundant vital functions in a subject, and therefore this type of lineage-specific antigen may be a poor target for CAR T cell-based immunotherapy.

[0048] In contrast to type 0 antigens, type 1 cell surface lineage-specific antigens and cells bearing type 1 cell surface lineage-specific antigens are not required for tissue homeostasis or survival in a subject. Targeting type 1 cell surface lineage-specific antigens will not result in adverse consequences to the subject. For example, CAR T cells engineered to target CD307, a type 1 antigen specifically expressed on both normal plasma cells and multiple myeloma (MM) cells, result in the elimination of both cell types (Figure 2) (Elkins et al., Mol Cancer Ther. 10:2222 (2012)). However, because the plasma cell lineage may be sacrificed for the survival of the organism, CD307 and other type 1 lineage-specific antigens are suitable antigens for CAR T cell-based immunotherapy. Type 1 class lineage-specific antigens can be expressed in a wide variety of different tissues, including the ovary, testis, prostate, breast, endometrium, and pancreas. In some embodiments, a substance targets a cell surface lineage-specific antigen that is a type 1 antigen.

[0049] Targeting type 2 antigens presents significant challenges compared to type 1 antigens. Type 2 antigens are characterized by the following: (1) the antigen is not important for the survival of the organism (i.e., not required for survival), and (2) the cell lineage bearing this antigen is essential for the survival of the organism (i.e., a specific cell lineage is required for survival). For example, CD33 is a type 2 antigen expressed on both normal bone marrow cells and acute myeloid leukemia (AML) cells (Dohner et al., NEJM 373:1136 (2015)). As a result, CAR T cells engineered to target the CD33 antigen can result in the death of both normal cells and AML cells, which is incompatible with the survival of the subject (Figure 3). In some embodiments, the substance targets a cell surface lineage-specific antigen that is a type 2 antigen.

[0050] A wide variety of antigens can be targeted by the methods and compositions of the present disclosure.Monoclonal antibodies against these antigens can be commercially available, or can be produced using standard methods, including immunizing animals with the antigen of interest followed by standard monoclonal antibody methodology, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature (1975) 256:495, as described above.Antibodies or nucleic acids encoding antibodies can be sequenced using any standard DNA or protein sequencing technology.

[0051] In some embodiments, the cell surface lineage-specific antigen targeted using the methods and cells described herein is a cell surface lineage-specific antigen of a leukocyte or a subpopulation of leukocytes. In some embodiments, the cell surface lineage-specific antigen is an antigen associated with myeloid cells. In some embodiments, the cell surface lineage-specific antigen is a cluster of differentiation (CD). Examples of CD antigens include, but are not limited to, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, C D27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD4 2a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49 b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD61, CD6 2E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD7 3, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110,CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、C D120b、CD121a、CD121b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126 、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、C D138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、CD146、C D147、CD148、CD150、CD152、CD152、CD153、CD154、CD155、CD156a、CD156b、C D156c、CD157、CD158b1、CD158b2、CD158d、CD158e1 / e2、CD158f、CD158g、CD1 58h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD163、CD164 、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172 g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD1 80、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、 CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD202b、CD203c、CD20 4、CD205、CD206、CD207、CD208、CD209、CD210a、CDw210b、CD212、CD213a1、CD 213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD2 35a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271 , CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283 , CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD2 98, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, 306, CD307a, C D307b, CD307c, D307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362, and CD363. See www.bdbiosciences.com / documents / BD_Reagents_CDMarkerHuman_Poster.pdf.

[0052] In some embodiments, the cell surface lineage-specific antigen is CD19, CD20, CD11, CD123, CD56, CD34, CD14, CD33, CD66b, CD41, CD61, CD62, CD235a, CD146, CD326, LMP2, CD22, CD52, CD10, CD3 / TCR, CD79 / BCR, and CD26. In some embodiments, the cell surface lineage-specific antigen is CD33.

[0053] Alternatively, or in addition, the cell surface lineage-specific antigen can be a cancer antigen, e.g., a cell surface lineage-specific antigen that is differentially present on cancer cells. In some embodiments, the cancer antigen is a tissue- or cell lineage-specific antigen. Examples of cell surface lineage-specific antigens associated with particular types of cancer include, but are not limited to, CD20, CD22 (non-Hodgkin's lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (acute myeloid leukemia (AML)), CD10 (gp100) (common (precursor B-cell) acute lymphocytic leukemia and malignant myeloma), CD3 / T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79 / B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (lymphoid malignancies), RCAS1 (gynecological cancers, biliary adenocarcinoma, and ductal adenocarcinoma of the pancreas), and prostate-specific membrane antigen. In some embodiments, the cell surface antigen CD33 is associated with AML cells.

[0054] B. Antigen-binding fragments Any antibody or antigen-binding fragment thereof (e.g., one that binds to CD33) can be used to construct agents that target lineage-specific cell surface antigens as described herein. Such antibodies or antigen-binding fragments can be prepared by conventional methods, e.g., hybridoma technology or recombinant technology.

[0055] For example, antibodies specific to a lineage-specific antigen of interest can be produced using conventional hybridoma technology. A lineage-specific antigen, which can be conjugated to a carrier protein such as KLH, can be used to immunize a host animal to generate antibodies that bind to this conjugate. The route or schedule for immunization of the host animal typically follows established conventional techniques for antibody stimulation and production, as further described herein. General techniques for producing murine, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells derived therefrom can be engineered to serve as a basis for producing mammalian, including human, hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of immunogen, including those described herein.

[0056] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the standard somatic cell hybridoma technique described by Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or modified by Buck, D.W. et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those obtained from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used for hybridization. Generally, this technique involves fusing myeloma cells with lymphocytes using a fusing agent such as polyethylene glycol or by electrical methods well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parent cells. Any of the media disclosed herein, supplemented or unsupplemented with serum, can be used to culture hybridomas secreting monoclonal antibodies. As an alternative to cell fusion techniques, EBV-immortalized B cells can be used to produce the TCR-like monoclonal antibodies described herein. Hybridomas are expanded and subcloned as necessary, and supernatants are assayed for anti-immunogen activity by conventional immunoassay methods (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0057] Hybridomas that can be used as antibody sources include all derivatives that are descendants of parent hybridomas that produce monoclonal antibodies capable of binding to lineage-specific antigens. Hybridomas producing such antibodies can be grown in vitro or in vivo using known methods. If necessary, monoclonal antibodies can be separated from culture media or body fluids by conventional immunoglobulin purification methods such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by passing the preparation over an adsorbent consisting of the immunogen bound to a solid phase, thereby eluting or releasing the desired antibody from the immunogen. Immunization of host cells with a target antigen or fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or RN=C=NR (where R and R are different alkyl groups), can generate a population of antibodies (e.g., monoclonal antibodies).

[0058] If necessary, the antibody of interest (e.g., produced by a hybridoma) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in host cells, which can then be propagated and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic engineering to "humanize" the antibody or to improve the affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is to be used in clinical trials or human therapy, the constant region can be genetically engineered to more resemble a human constant region to avoid an immune response. It may be desirable to genetically engineer an antibody sequence to obtain high affinity for a lineage-specific antigen. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to an antibody while still maintaining its binding specificity for the target antigen.

[0059] In other embodiments, fully human antibodies can be obtained using commercially available mice that have been genetically engineered to express specific human immunoglobulin proteins. Transgenic animals designed to generate more desirable (e.g., fully human) or stronger immune responses can also be used to generate humanized or human antibodies. Examples of such technology are the Xenomouse™ from Amgen, Inc. (Fremont, Calif.) and the HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). Alternatively, antibodies can be produced recombinantly using phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winter et al. (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) region gene repertoires from unimmunized donors.

[0060] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by routine methods, for example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule and Fab can be generated by reducing the disulfide bridges of the F(ab')2 fragment.

[0061] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, by conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into one or more expression vectors, which are then introduced into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to synthesize the monoclonal antibody in the recombinant host cells. See, for example, International Publication No. WO 87 / 04462. The DNA can then be modified, for example, by substituting sequences encoding human heavy and light chain constant regions in place of the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies, can be produced that have the binding specificity of a target antigen.

[0062] Techniques developed for the production of "chimeric antibodies" are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

[0063] Methods for constructing humanized antibodies are well known in the art.See, for example, Queen et al., Proc.Natl.Acad.Sci.USA,86:10029-10033(1989).In one example, the variable regions of VH and VL of parent non-human antibody are subjected to three-dimensional molecular model analysis by methods known in the art.Then, the framework amino acid residues predicted to be important for forming accurate CDR structure are identified using the same molecular model analysis.In parallel, human VH and VL chains with amino acid sequences that are homologous to those of parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries.Human VH and VL acceptor genes are then selected.

[0064] The CDR regions in the selected human acceptor gene can be replaced with CDR regions from the parent non-human antibody or functional variants thereof. If necessary, residues in the framework regions of the parent chain that are predicted to be important in interactions with the CDR regions (see above) can be used to replace the corresponding residues in the human acceptor gene.

[0065] Single-chain antibodies can be produced recombinantly by linking a nucleotide sequence encoding a heavy-chain variable region with a nucleotide sequence encoding a light-chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques reported for producing single-chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to generate phage or yeast scFv libraries, and scFv clones specific for lineage-specific antigens can be identified from the libraries using routine procedures. Positive clones can be subjected to further screening to identify those that bind the lineage-specific antigen.

[0066] In some examples, the lineage-specific antigen of interest is CD33, and the antigen-binding fragment specifically binds CD33, for example, human CD33. The amino acid and nucleic acid sequences of exemplary heavy and light chain variable regions of anti-human CD33 antibodies are shown below. The CDR sequences are shown in bold and underlined in the amino acid sequences. Amino acid sequence of anti-CD33 heavy chain variable region (SEQ ID NO: 12) [ka] Nucleic acid sequence of anti-CD33 heavy chain variable region (SEQ ID NO: 2) CAGGTGCAGCTGCAGCAGCCCGGCGCCGAGGTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCCGGCCAGGGCCTGGAGTGGGTGGGCGTGATCTACCCCGGCAACGACGACATCAGC TACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCACCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGAGGTGAGGCTGAGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Amino acid sequence of anti-CD33 light chain variable region (SEQ ID NO: 13) [ka] Nucleic acid sequence of anti-CD33 heavy chain variable region (SEQ ID NO: 1) GAGATCGTGCTGACCCAGAGCCCCGGCAGCCTGGCCGTGAGCCCCGGCGAGAGGGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCAGCCAGAAGAACTACCTGGCCTGGTACCAGCAGATCCCCGGCCAGAGCCCCAGGCTGCTGATCTACTGGG CCAGCACCAGGGAGAGCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCAGGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGG

[0067] Anti-CD33 antibody binding fragments for use in constructing CD33-targeting agents described herein may contain the same heavy and / or light chain CDRs as SEQ ID NO: 12 and SEQ ID NO: 13. Such antibodies may contain amino acid residue mutations in one or more framework regions. In some examples, an anti-CD33 antibody fragment may contain a heavy chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or more) with SEQ ID NO: 12 and / or a light chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or more) with SEQ ID NO: 13.

[0068] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. When gaps exist between the two sequences, Gapped BLAST can be used as reported in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0069] C. Immune cells expressing chimeric receptors In some embodiments, the agents that target lineage-specific cell surface antigens described herein are immune cells that express a chimeric receptor comprising an antigen-binding fragment (e.g., a single-chain antibody) that can bind to the lineage-specific antigen (e.g., CD33). Recognition of a target cell (e.g., a cancer cell) bearing the lineage-specific antigen on its cell surface by the antigen-binding fragment of the chimeric receptor transmits an activation signal to the signaling domain (e.g., a costimulatory signaling domain and / or a cytoplasmic signaling domain) of the chimeric receptor, which can activate effector functions in the immune cell expressing the chimeric receptor.

[0070] As used herein, a chimeric receptor refers to a non-naturally occurring molecule that is expressed on the surface of a host cell and contains an antigen-binding fragment that binds to a cell surface lineage-specific antigen. Generally, a chimeric receptor contains at least two regions derived from different molecules. In addition to the antigen-binding fragment described herein, a chimeric receptor may further contain one or more of a hinge region, a transmembrane region, at least one costimulatory region, and a cytoplasmic signaling region. In some embodiments, a chimeric receptor contains, from the N-terminus to the C-terminus, an antigen-binding fragment that binds to a cell surface lineage-specific antigen, a hinge region, a transmembrane region, and a cytoplasmic signaling region. In some embodiments, a chimeric receptor further contains at least one costimulatory region.

[0071] In some embodiments, the chimeric receptors described herein comprise a hinge region, which may be located between the antigen-binding fragment and the transmembrane region. A hinge region is a segment of amino acids typically found between two regions of a protein, which may allow flexibility of the protein and movement of one or both regions relative to one another. Any amino acid sequence that provides such flexibility and movement of the antigen-binding fragment relative to another region of the chimeric receptor may be used.

[0072] The hinge region can comprise about 10 to 200 amino acids, e.g., 15 to 150 amino acids, 20 to 100 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge region can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length.

[0073] In some embodiments, the hinge region is that of a naturally occurring protein. Hinge regions of any protein known in the art to contain a hinge region are suitable for use in the chimeric receptors described herein. In some embodiments, the hinge region is at least a portion of the hinge region of a naturally occurring protein, conferring flexibility to the chimeric receptor. In some embodiments, the hinge region is that of CD8α or CD28α. In some embodiments, the hinge region is a portion of the hinge region of CD8α, e.g., a fragment comprising at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge region of CD8α or CD28α.

[0074] Hinge regions of antibodies, such as IgG, IgA, IgM, IgE, or IgD antibodies, are also suitable for use in the chimeric receptors described herein. In some embodiments, the hinge region is the hinge region connecting the CH1 and CH2 constant regions of the antibody. In some embodiments, the hinge region is of an antibody and comprises the hinge region of the antibody and the hinge region of one or more constant regions of the antibody. In some embodiments, the hinge region comprises the hinge region of the antibody and the hinge region of the CH3 constant region of the antibody. In some embodiments, the hinge region comprises the hinge region of the antibody and the hinge region of the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0075] Chimeric receptors comprising hinge regions that are non-naturally occurring peptides are also within the scope of this disclosure. In some embodiments, the hinge region between the C-terminus of the extracellular ligand binding region and the N-terminus of the transmembrane region of an Fc receptor is (Gly xSer) n linker (SEQ ID NO: 74), where x and n can independently be integers from 3 to 12 or more, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0076] Additional peptide linkers that can be used in the hinge region of the chimeric receptors described herein are known in the art. See, e.g., Wriggers et al., Current Trends in Peptide Science (2005) 80(6):736-746, and WO 2012 / 088461.

[0077] In some embodiments, the chimeric receptors described herein may include a transmembrane region. The transmembrane region for use in the chimeric receptor may be in any form known in the art. As used herein, "transmembrane region" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. A transmembrane region suitable for use in the chimeric receptors used herein is obtained from a naturally occurring protein. Alternatively, the transmembrane region may be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0078] Transmembrane regions are classified based on the topology of the transmembrane region, including the number of passes the transmembrane region makes across the membrane and the orientation of the protein. For example, a single-pass membrane protein crosses the cell membrane once, while a multi-pass membrane protein crosses the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). In some embodiments, the transmembrane region is a single-pass transmembrane region. In some embodiments, the transmembrane region is a single-pass transmembrane region in which the N-terminus of the chimeric receptor faces the extracellular side of the cell and the C-terminus of the chimeric receptor faces the intracellular side of the cell. In some embodiments, the transmembrane region is obtained from a single-pass transmembrane protein. In some embodiments, the transmembrane region is that of CD8α. In some embodiments, the transmembrane region is that of CD28. In some embodiments, the transmembrane region is that of ICOS.

[0079] In some embodiments, the chimeric receptors described herein comprise one or more costimulatory signaling regions. The term "costimulatory signaling region," as used herein, refers to at least a portion of a protein that mediates intracellular signaling to elicit an immune response, such as an effector function. The costimulatory signaling region of the chimeric receptors described herein can be a cytoplasmic signaling region from a costimulatory protein that transmits a signal to regulate responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

[0080] In some embodiments, the chimeric receptor comprises two or more (at least two, three, four, or more) costimulatory signaling regions. In some embodiments, the chimeric receptor comprises two or more costimulatory signaling regions obtained from different costimulatory proteins. In some embodiments, the chimeric receptor does not comprise a costimulatory signaling region.

[0081] Typically, many immune cells require costimulation in addition to antigen-specific signal stimulation to promote cell proliferation, differentiation, and survival, as well as to activate cell effector functions. Activation of costimulatory signaling regions in host cells (e.g., immune cells) can induce the cells to increase or decrease cytokine production and secretion, macrophage characteristics, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling region of any costimulatory protein can be adapted for use in the chimeric receptors described herein. The type of costimulatory signaling region is selected based on factors such as the type of immune cell in which the chimeric receptor is expressed (e.g., primary T cells, T cell lines, NK cell lines) and the desired immune effector function (e.g., cytotoxicity). Examples of costimulatory signaling regions for use in chimeric receptors can be the cytoplasmic signaling region of a costimulatory protein, including, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, Cd40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In some embodiments, the costimulatory region is derived from 4-1BB, CD28, or ICOS. In some embodiments, the costimulatory region is derived from CD28, and the chimeric receptor includes a second costimulatory region derived from 4-1BB or ICOS.

[0082] In some embodiments, the costimulatory region is a fusion region comprising two or more costimulatory regions or portions of two or more costimulatory regions, hi some embodiments, the costimulatory region is a fusion of costimulatory regions from CD28 and ICOS.

[0083] In some embodiments, the chimeric receptors described herein comprise a cytoplasmic signaling region. Any cytoplasmic signaling region can be used in the chimeric receptors described herein. Generally, the cytoplasmic signaling region transmits a signal, such as the interaction of an extracellular ligand-binding region with its ligand, to stimulate a cellular response, such as inducing a cellular effector function (e.g., cytotoxicity).

[0084] As will be apparent to those skilled in the art, a factor involved in T cell activation is phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic signaling domain. Any ITAM-containing domain known in the art can be used to construct the chimeric receptors described herein. Generally, an ITAM motif contains two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, where each x can independently be any amino acid, generating the conserved motif YxxL / Ix(6-8)YxxL / I. In some embodiments, the cytoplasmic signaling domain is derived from CD3ζ.

[0085] Exemplary chimeric receptors are shown in Tables 2 and 3 below. [Table 2]

[0086] Nucleic acid sequences of exemplary building blocks for the construction of chimeric receptors are provided below. CD28 intracellular signaling region-DNA-human (SEQ ID NO: 3) ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC ICOS intracellular signaling region - DNA - human (SEQ ID NO: 4)

[0087] CTATCAATTTTTGATCCTCCTCCTTTTAAAGTAACTCTTACAGGAGGATATTGCATATTATGAATCACAACTTTGTTGCCAGCTGAAGTTCTGGTTACCCATAGGATTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTCGACCTAAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCG CGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGGACAAGAGACGTGGCCGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAACCCTCAGGAAGGCCTTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGGAGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACAAGGACCACTACGACGCCCTTCCACATGCAGGCCTGCCCCCTCGC CD28 / ICAS

[0088] ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGC TATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0089] In some embodiments, the nucleic acid encodes an antigen-binding fragment that binds to CD33 and comprises a heavy chain variable region having CDRs identical to those in SEQ ID NO: 12 and a light chain variable region having CDRs identical to those in SEQ ID NO: 13. In some embodiments, the antigen-binding fragment comprises a heavy chain variable region set forth in SEQ ID NO: 12 and a light chain variable region set forth in SEQ ID NO: 13. In some embodiments, the chimeric receptor further comprises at least a transmembrane region and a cytoplasmic signaling region. In some embodiments, the chimeric receptor further comprises a hinge region and / or a costimulatory signaling region.

[0090] Table 3 provides exemplary chimeric receptors described herein. Exemplary constructs have, from N-terminus to C-terminus, an antigen-binding fragment, a transmembrane region, and a cytoplasmic signaling region. In some examples, the chimeric receptor further comprises a hinge region located between the antigen-binding fragment and the transmembrane region. In some examples, the chimeric receptor further comprises one or more costimulatory regions, which may be located between the transmembrane region and the cytoplasmic signaling region. [Table 3]

[0091] The amino acid sequences of the exemplary chimeric receptors described in Table 3 above are provided below: CART1 amino acid sequence (SEQ ID NO: 20) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTK LEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLR YFDVWGQGTTVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART2 amino acid sequence (SEQ ID NO: 21) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLE IKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFD VWGQGTTVTVSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART3 amino acid sequence (SEQ ID NO: 22) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGK PGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFS HFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART8 amino acid sequence (SEQ ID NO: 23) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSG SGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSALSNS IMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLTKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART4dual amino acid sequence (SEQ ID NO: 24) MWLQSLLLLGTVACSISIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF GGGTKLEIGSTSGSGKPGSGEGSTKGLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI YYCAKHYYYGGSYAMDYWGQGTSVTVSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFII FWVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART5dual amino acid sequence (SEQ ID NO: 25) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTIS SVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVI YPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CART6 amino acid sequence (SEQ ID NO: 26) MWLQSLLLLGTVACSISIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIGST SGSGKPGSGEGSTKGLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART7 amino acid sequence (SEQ ID NO: 27) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFG QGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVY YCAREVRLRYFDVWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0092] The nucleic acid sequences of the exemplary chimeric receptors described in Table 3 above are provided below: CART1 nucleic acid sequence (SEQ ID NO: 38) CART2 nucleic acid sequence (SEQ ID NO: 39) CART3 nucleic acid sequence (SEQ ID NO: 40) CART4dual nucleic acid sequence (SEQ ID NO: 41) CART5dual nucleic acid sequence (SEQ ID NO: 42) CART6 nucleic acid sequence (SEQ ID NO: 43) CART7 nucleic acid sequence (SEQ ID NO: 44)

[0093] Any of the chimeric receptors described herein can be prepared by routine methods, such as recombinant techniques. Methods for preparing chimeric receptors herein include generating nucleic acids encoding polypeptides comprising each region of the chimeric receptor, including an antigen-binding fragment and, optionally, a hinge region, a transmembrane region, at least one costimulatory signaling region, and a cytoplasmic signaling region. In some embodiments, the nucleic acids encoding each of the components of the chimeric receptor are linked to each other using recombinant techniques.

[0094] The sequence of each of the components of the chimeric receptor can be obtained by routine techniques, such as PCR amplification, from any one of a variety of sources known in the art. In some embodiments, the sequence of one or more of the components of the chimeric receptor is obtained from a human cell. Alternatively, the sequence of one or more components of the chimeric receptor can be synthesized. The sequences (e.g., regions) of each component can be linked directly or indirectly (e.g., using a nucleic acid encoding a peptide linker) using methods such as PCR amplification or ligation to form a nucleic acid sequence encoding the chimeric receptor. Alternatively, the nucleic acid encoding the chimeric receptor can be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0095] Mutation of one or more residues in one or more components (e.g., antigen-binding fragments, etc.) of a chimeric receptor, before or after linking the sequences of each of the components. In some embodiments, one or more mutations in a component of a chimeric receptor can be made to modulate (increase or decrease) the affinity of the component for its target (e.g., antigen-binding fragment for a target antigen) and / or modulate the activity of the component.

[0096] Any of the chimeric receptors described herein can be introduced into appropriate immune cells for expression by conventional techniques. In some embodiments, the immune cells are T cells, such as primary T cells or T cell lines. Alternatively, the immune cells can be NK cells, such as established NK cell lines (e.g., NK-92 cells). In some embodiments, the immune cells are CD8 (CD8 + ) or CD8 and CD4 (CD8 + / CD4 + In some embodiments, the T cells are T cells of an established T cell line, e.g., 293T cells or Jurkat cells.

[0097] Primary T cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, tissues such as spleen, lymph nodes, thymus, or tumor tissue. Suitable sources for obtaining the desired immune cell type will be apparent to those skilled in the art. In some embodiments, the immune cell population is derived from a human patient with a hematopoietic malignancy, such as from bone marrow or PBMCs obtained from the patient. In some embodiments, the immune cell population is derived from a healthy donor. In some embodiments, the immune cells are obtained from a subject to whom immune cells expressing a chimeric receptor will later be administered. Immune cells administered to the same subject from whom the cells were obtained are called autologous cells, while immune cells obtained from a subject other than the subject to whom the cells will be administered are called allogeneic cells.

[0098] The desired host cell type can be expanded within the resulting cell population by co-culturing the cells with stimulatory molecules, for example, anti-CD3 and anti-CD28 antibodies can be used for the expansion of T cells.

[0099] To construct immune cells expressing any of the chimeric receptor constructs described herein, vectors for stable or transient expression of the chimeric receptor can be constructed by conventional methods described herein and introduced into immune host cells. For example, a nucleic acid encoding the chimeric receptor can be cloned into an appropriate expression vector, such as a viral vector, in operably linked to a suitable promoter. The nucleic acid and vector can be contacted with a restriction enzyme under appropriate conditions to form complementary ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, a synthetic nucleic acid linker can be ligated to the ends of the nucleic acid encoding the chimeric receptor. The synthetic linker can contain a nucleic acid sequence corresponding to a specific restriction site in the vector. The choice of expression vector / plasmid / viral vector depends on the type of host cell for chimeric receptor expression, but should be suitable for integration and replication in eukaryotic cells.

[0100] Various promoters can be used for expression of the chimeric receptors described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate-early promoter, viral LTRs such as Rous sarcoma LTR, HIV-LTR, HTLV-1 LTR, Moloney murine leukemia virus (MMLV) LTR, myeloproliferative sarcoma virus (MPSV) LTR, spleen-limited focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) intermediate-early promoter, the herpes simplex tk virus promoter, and the elongation factor 1-α (EF1-α) promoter with or without the EF1-α intron. Additional promoters for expression of the chimeric receptor include any promoter that is constitutively active in immune cells. Alternatively, any regulatable promoter can be used, such that its expression can be regulated within immune cells.

[0101] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene, for selection of stable or transient transfectants in host cells; an enhancer / promoter sequence from the human CMV intermediate-early gene for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5'- and 3'-untranslated regions from highly expressed genes such as α-globin or β-globin for mRNA stability and translation efficiency; the SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" that, when induced, causes death in vector-harboring cells (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and a reporter gene for assessing expression of the chimeric receptor. See Section VI, below. Suitable vectors and methods for generating vectors containing transgenes are well known and available in the art. Examples of preparing vectors for expression of chimeric receptors can be found, for example, in U.S. Application Publication No. 2014 / 0106449, which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the nucleic acid encoding the chimeric receptor construct or chimeric receptor is a DNA molecule. In some embodiments, the nucleic acid encoding the chimeric receptor construct or chimeric receptor is a DNA vector, which can be introduced into immune cells by electroporation (see, e.g., Till et al., Blood (2012) 119(17):3940-3950). In some embodiments, the nucleic acid encoding the chimeric receptor is an RNA molecule, which can be introduced into immune cells by electroporation.

[0103] Any vector containing a nucleic acid sequence encoding the chimeric receptor constructs described herein is within the scope of the present disclosure. Such vectors can be delivered into host cells, such as host immune cells, by appropriate methods. Methods for delivering vectors to immune cells are well known in the art and include DNA, RNA, or transposon electroporation; gene transfer reagents such as liposomes or nanoparticles that deliver DNA, RNA, or transposons; delivery of DNA, RNA, or transposons or proteins by mechanical deformation (see, e.g., Sharei et al., Proc. Natl. Acad. Sci. USA (2013) 110(6):2082-2087); or viral transduction. In some embodiments, vectors for expression of chimeric receptors are delivered to host cells by viral transduction. Exemplary viral methods for delivery include, but are not limited to, recombinant retroviruses (see, e.g., WO 90 / 07936, WO 94 / 03622, WO 93 / 25698, WO 93 / 25234, WO 93 / 11230, WO 93 / 10218, WO 91 / 02805, U.S. Pat. Nos. 5,219,740 and 4,777,127, GB Patent No. 2,200,651, and EP Patent No. 0345242), Arifa virus-based vectors, and adeno-associated virus (AAV) vectors (see, e.g., WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655). In some embodiments, the vector for expression of the chimeric receptor is a retrovirus. In some embodiments, the vector for expression of the chimeric receptor is a lentivirus. In some embodiments, the vector for expression of the chimeric receptor is an adeno-associated virus.

[0104] In instances where the vector encoding the chimeric receptor is introduced into host cells using a viral vector, viral particles carrying the vector capable of infecting immune cells can be produced by any method known in the art and found, for example, in WO 1991 / 002805 A2, WO 1998 / 009271 A1, and U.S. Patent No. 6,194,191. Viral particles can be collected from cell culture supernatants and separated and / or purified before contacting the viral particles with immune cells.

[0105] Methods for preparing host cells expressing any of the chimeric receptors described herein can include activating and / or expanding immune cells in vitro. Activating host cells refers to stimulating the host cells to an activated state in which the cells can perform effector functions (e.g., cytotoxicity). The method for activating host cells depends on the type of host cells used for expression of the chimeric receptor. Expanding host cells can include any method that results in an increase in the number of cells expressing the chimeric receptor, such as growing the host cells or stimulating the host cells to proliferate. Methods for stimulating host cell expansion depend on the type of host cells used for expression of the chimeric receptor and will be apparent to one of skill in the art. In some embodiments, host cells expressing any of the chimeric receptors described herein are activated and / or expanded in vitro prior to administration to a subject.

[0106] In some embodiments, the agent targeting a cell surface lineage-specific antigen is an antibody-drug conjugate (ADC). As will be appreciated by those skilled in the art, the term "antibody-drug conjugate" is used interchangeably with "immunotoxin" to refer to a fusion molecule comprising an antibody (or an antigen-binding fragment thereof) conjugated to a toxin or drug molecule. Binding of the antibody to the corresponding antigen enables delivery of the toxin or drug molecule to cells that display the antigen on their cell surface (e.g., target cells), thereby resulting in the death of the target cell.

[0107] In some embodiments, the substance is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises an antigen-binding fragment and a toxin or drug that induces cytotoxicity in target cells. In some embodiments, the antibody-drug conjugate targets a type 2 antigen. In some embodiments, the antibody-drug conjugate targets CD33 or CD19.

[0108] In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain CDRs as the heavy chain variable region set forth in SEQ ID NO: 12 and the same light chain CDRs as the light chain variable region set forth in SEQ ID NO: 13. In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain variable region set forth in SEQ ID NO: 12 and the same light chain variable region set forth in SEQ ID NO: 13.

[0109] Toxins or drugs suitable for use in antibody-drug conjugates are well known in the art and will be apparent to those skilled in the art. See, e.g., Peters et al. Biosci. Rep. (2015) 35(4):e00225; Beck et al. Nature Reviews Drug Discovery (2017) 16:315-337; Marin-Acevedo et al. J. Hematol. Oncol. (2018) 11:8; Elgundi et al. Advanced Drug Delivery Reviews (2017) 122:2-19.

[0110] In some embodiments, the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker such as a cleavable linker) connecting the antibody and the drug molecule. Examples of antibody-drug conjugates include, but are not limited to, brentuximab vedotin, glembatumumab vedotin / CDX-011, depatuxizumab mafodotin / ABT-414, and PSMA. ADC, polatuzumab vedotin / RG7596 / DCDS4501A, denintuzumab mafodotin / SGN-CD19A, AGS-16C3F, CDX-014, RG7841 / DLYE5953A, RG7882 / DMUC406A, RG7986 / DCDS0780A, SGN-LIV1A, enfortumab vedotin / ASG-22ME, AG-15ME, AGS67E, telisotuzumab vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, tisotuzumab vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, polatuzumab vedotin / RG7593 / DCDT2980S, rifastuzumab vedotin / RG7599 / DNIB0600A, indusatumab vedotin / MLN-0264 / TAK-264, bundletuzumab vedotin / RG7450 / DSTP3086S, sofituzumab vedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC 5T4, trastuzumab emtansine / T-DM1, mirvetuximab soravtansine / IMGN853, coltuximab emtansine / SAR3419, naratuximab emtansine / IMGN529, indatuximab emtansine / BT-062, anetumab emtansine / BAY 94-9343, SAR408701, SAR428926, AMG 224, PCA062, HKT288, LY3076226, SAR566658, lorvotuzumab mertansine / IMGN901, cantuzumab mertansine / SB-408075, cantuzumab mertansine / IMGN242, laprituximab emtansine / IMGN289, IMGN388, bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP628, vadastuximab butarilin / SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, rovalpituzumab tesirin / SC16LD6.5, SC-002, SC-003, ADCT-301 / HuMax-TAC-PBD, ADCT-402, MEDI3726 / ADC-401, IMGN779, IMGN632, gemtuzumab ozogamicin, inotuzumab ozogamicin / CMC-544, PF-06647263, CMD-193, C MB-401, trastuzumab duocarmazine / SYD985, BMS-936561 / MDX-1203, sacituzumab govitecan / IMMU-132, labetuzumab govitecan / IMMU-130, DS-8201a, U3-1402, milatuzumab doxorubicin / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / pertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, lupartumab amadotin / BAY1129980, aprutumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, DSTA4637S / RG7861. In one example, the antibody-drug conjugate is gemtuzumab ozogamicin.

[0111] In some embodiments, binding of an antibody-drug conjugate to a cell surface lineage-specific protein induces internalization of the antibody-drug conjugate and the drug (or toxin) can be released. In some embodiments, binding of an antibody-drug conjugate to a cell surface lineage-specific protein induces internalization of a toxin or drug, which can kill cells expressing the lineage-specific protein (target cells). In some embodiments, binding of an antibody-drug conjugate to a cell surface lineage-specific protein induces internalization of a toxin or drug, which can modulate the activity of cells expressing the lineage-specific protein (target cells). The type of toxin or drug used in the antibody-drug conjugates described herein is not limited to any particular type.

[0112] The ADCs described herein may be used as a follow-on treatment to subjects who have received the combination therapy described herein.

[0113] Hematopoietic cells lacking lineage-specific cell surface antigens The present disclosure also provides hematopoietic cells, such as hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), that have been genetically modified to lack a lineage-specific cell surface antigen (e.g., CD33, e.g., using a gRNA described herein, e.g., wherein the gRNA comprises the nucleotide sequence AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67) or CCUCACUAGACUUGACCCAC (SEQ ID NO: 70)). In some embodiments, the cells comprise a genetic mutation at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50) or CCTCACTAGACTTGACCCAC (SEQ ID NO: 58). In some embodiments, the hematopoietic cells are HSCs, HPCs, or a combination thereof, referred to herein as "HSPCs" ("hematopoietic stem and / or progenitor cells"). In some embodiments, the cell populations described herein comprise a plurality of hematopoietic stem cells; in some embodiments, the cell populations described herein comprise a plurality of hematopoietic progenitor cells; and in some embodiments, the cell populations described herein comprise a plurality of hematopoietic stem cells and a plurality of hematopoietic progenitor cells. Hematopoietic stem cells (HSCs) can give rise to both myeloid and lymphoid progenitor cells, which further generate myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. HSCs express the cell surface marker CD34 (e.g., CD34) which can be used to identify and / or isolate HSCs. + ) and the absence of cell surface markers associated with lineage commitment. Thus, in some embodiments, HSCs are characterized by the expression of CD34 + is.

[0114] In some embodiments, the HSCs are obtained from a subject, such as a mammalian subject. In some embodiments, the mammalian subject is a non-human primate, a rodent (e.g., a mouse or rat), a cow, a pig, a horse, or a domestic animal. In some embodiments, the HSCs are obtained from a human patient, such as a human patient with a hematopoietic malignancy. In some embodiments, the HSCs are obtained from a healthy donor. In some embodiments, the HSCs are obtained from a subject to whom immune cells expressing a chimeric receptor will later be administered. HSCs administered to the same subject from which the cells were obtained are referred to as autologous cells, while HSCs obtained from a subject other than the subject to whom the cells will be administered are referred to as allogeneic cells.

[0115] HSCs can be obtained from any suitable source using conventional means known in the art. In some embodiments, HSCs are obtained from a subject-derived sample, such as a bone marrow sample or a blood sample. Alternatively, or in addition, HSCs can be obtained from the umbilical cord. In some embodiments, HSCs are obtained from bone marrow or peripheral blood mononuclear cells (PBMCs). Generally, bone marrow cells can be obtained from a subject's iliac crest, femur, tibia, vertebrae, ribs, or other bone marrow cavity. Bone marrow can be collected from a patient and isolated by various separation and washing procedures known in the art. An exemplary method for isolating bone marrow cells includes the following steps: a) extraction of a bone marrow sample; b) centrifugation of the bone marrow suspension into three fractions and collection of the intermediate fraction and buffy coat; c) centrifuging the buffy coat fraction from step (b) once more in a separation solution, usually Ficoll™, to collect the intermediate fraction containing bone marrow cells; and d) washing the fraction collected from step (c) to recover reinjectable bone marrow cells.

[0116] HSCs are normally present in the bone marrow, but can be mobilized into the circulating blood by administering a mobilization agent to harvest HSCs from peripheral blood. In some embodiments, the subject from whom HSCs are harvested is administered a mobilization agent such as granulocyte colony-stimulating factor (G-CSF). The number of HSCs collected by mobilization using a mobilization agent is usually greater than the number of cells obtained without the use of a mobilization agent. In some embodiments, the HSCs are peripheral blood HSCs.

[0117] In some embodiments, a sample is obtained from a subject and then analyzed for a desired cell type (e.g., CD34 + / CD33 - cells) are enriched, e.g., PBMCs and / or CD34 + Hematopoietic cells can be isolated from blood as described herein. Cells can also be isolated from other cells, for example, by isolation and / or activation using antibodies that bind to cell surface epitopes of the desired cell type. Another method that can be used involves negative selection using antibodies against cell surface markers to selectively enrich for specific cell types without receptor-mediated cell activation.

[0118] The HSC population may be expanded before or after genetically engineering the HSCs to lack lineage-specific cell surface antigens. The cells may be cultured under conditions including a growth medium containing one or more cytokines, such as stem cell factor (SCF), Flt-3 ligand (Flt3L), thrombopoietin (TPO), interleukin 3 (IL-3), or interleukin 6 (IL-6). The cells may be expanded for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days, or any desired range of time. In some embodiments, the HSCs are isolated from a desired cell population (e.g., CD34) from a sample obtained from a subject. + / CD33 -) are expanded after isolation and before genetic manipulation. In some embodiments, HSCs are expanded after genetic manipulation, thereby selectively expanding cells that have been genetically modified and lack lineage-specific cell surface antigens. In some embodiments, after genetic modification, one cell (a "clone") or several cells with a desired characteristic (e.g., phenotype or genotype) can be selected and independently expanded.

[0119] In some embodiments, hematopoietic cells are genetically engineered to lack (e.g., do not express) a cell surface lineage-specific antigen (e.g., CD33). In some embodiments, hematopoietic cells are genetically engineered to lack the same cell surface lineage-specific antigen targeted by the agent. As used herein, hematopoietic cells are considered to lack a cell surface lineage-specific antigen if they have significantly reduced expression of the cell surface lineage-specific antigen compared to naturally occurring hematopoietic cells of the same type as the genetically engineered hematopoietic cells (e.g., characterized by the presence of the same cell surface marker, such as CD34). In some embodiments, hematopoietic cells do not have detectable expression of the cell surface lineage-specific antigen (e.g., do not express the cell surface lineage-specific antigen). The expression level of a cell surface lineage-specific antigen can be assessed by any means known in the art. For example, the expression level of a cell surface lineage-specific antigen can be assessed by detecting the antigen using an antigen-specific antibody (e.g., flow cytometry, Western blotting).

[0120] In some embodiments, expression of cell surface lineage-specific antigens on the genetically engineered hematopoietic cells is compared to expression of cell surface lineage-specific antigens on native origin hematopoietic cells (e.g., wild-type counterparts). In some embodiments, the genetic engineering results in a reduction in expression levels of cell surface lineage-specific antigens of at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to expression of cell surface lineage-specific antigens on native origin hematopoietic cells. In some embodiments, the genetically engineered hematopoietic cells express less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a cell surface lineage-specific antigen (e.g., CD33) compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).

[0121] In some embodiments, the genetic engineering results in a reduction in the expression level of a wild-type cell surface lineage-specific antigen (e.g., CD33) by at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the expression level of the cell surface lineage-specific antigen on a native origin hematopoietic cell. That is, in some embodiments, the genetically engineered hematopoietic cell expresses less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the wild-type cell surface lineage-specific antigen (e.g., CD33) compared to a native origin hematopoietic cell (e.g., its wild-type counterpart).

[0122] In some embodiments, the hematopoietic cells lack the entire endogenous gene encoding the cell surface lineage-specific antigen. In some embodiments, the entire endogenous gene encoding the cell surface lineage-specific antigen has been deleted. In some embodiments, the hematopoietic cells comprise a portion of the endogenous gene encoding the cell surface lineage-specific antigen. In some embodiments, the hematopoietic cells express a portion (e.g., a truncated protein) of the cell surface lineage-specific antigen. In other embodiments, a portion of the endogenous gene encoding the cell surface lineage-specific antigen has been deleted. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the gene encoding the cell surface lineage-specific antigen has been deleted.

[0123] As will be appreciated by those skilled in the art, a portion of the nucleotide sequence encoding the cell surface lineage-specific antigen may be removed or may be one or more non-coding sequences such that the hematopoietic cell lacks the antigen (e.g., has significantly reduced expression of the antigen).

[0124] In some embodiments, the cell surface lineage-specific antigen is CD33. The predicted structure of CD33 includes two immunoglobulin domains, an IgV domain and an IgC2 domain. In some embodiments, a portion of the immunoglobulin C domain of CD33 is deleted.

[0125] Genetically engineered hematopoietic cells, such as HSCs, lacking cell surface lineage-specific antigens can be prepared by conventional methods or by the methods described herein. In some embodiments, genetic engineering is performed using genome editing. As used herein, "genome editing" refers to a method of modifying the genome, including any protein-coding or non-coding nucleotide sequence, of an organism to knock out the expression of a target gene. Generally, genome editing methods involve the use of endonucleases that can cleave nucleic acids in the genome, for example, at targeted nucleotide sequences. Double-strand breaks in the genome can be repaired by introducing mutations and / or foreign nucleic acids can be inserted into the targeted site.

[0126] Genome editing methods are generally classified based on the type of endonuclease involved in creating a double-strand break in the target nucleic acid. These methods include the use of zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), meganucleases, and CRISPR / Cas systems.

[0127] In one embodiment of the present disclosure, the replacement of tumor cells with a modified normal cell population is carried out using normal cells in which lineage-specific antigens are modified. Such modifications can include depletion or inhibition of any lineage-specific antigens using the CRISPR-Cas9 system, which is a genetically engineered non-naturally occurring CRISPR-Cas9 system (Figure 4).

[0128] The CRISPR-Cas system has been successfully used to edit the genomes of a variety of organisms, including but not limited to bacteria, humans, Drosophila, zebrafish, and plants. For example, Jiang et al.,Nature Biotechnology(2013)31(3):233;Qi et al,Cell(2013)5:1173;DiCarlo et al.,Nucleic Acids Res.(2013)7:4336;Hwang et al.,Nat.Biotechnol(2013),3:227);Gratz et al. al.,Genetics(2013)194:1029;Cong et al.,Science(2013)6121:819;Mali et al.,Science(2013)6121:823;Cho et al.Nat.Biotechnol(2013)3:230;and Jiang et al.,Nucleic Acids Please refer to Research(2013)41(20):el88.

[0129] The present disclosure utilizes a CRISPR / Cas9 system that hybridizes to a target sequence in a lineage-specific antigen polynucleotide, the CRISPR / Cas9 system comprising a Cas9 nuclease and an engineered crRNA / tracrRNA (or single-stranded guide RNA). The CRISPR / Cas9 complex can bind to the lineage-specific antigen polynucleotide, allowing cleavage of the antigen polynucleotide, thereby modifying the polynucleotide.

[0130] The CRISPR / Cas system of the present disclosure can bind to and / or cleave a region of interest within a cell surface lineage-specific antigen in a coding or non-coding region, within or near a gene, such as a leader sequence, trailer sequence, or intron, or within a non-transcribed region, either upstream or downstream of a coding region. The guide RNA (gRNA) used in the present disclosure can be designed so that the gRNA directs binding of the Cas9-gRNA complex to a predetermined cleavage site (target site) in the genome. The cleavage site can be selected to release a fragment containing an unknown sequence region or a region containing a SNP, nucleotide insertion, nucleotide deletion, rearrangement, etc.

[0131] The cleavage of the gene region can include single-strand or double-strand cleavage at the position of the target sequence by Cas enzyme. In one embodiment, such cleavage can result in reduced transcription of the target gene. In another embodiment, the cleavage further includes repairing the cleaved target polynucleotide by homologous recombination using an exogenous template polynucleotide, and the repair results in the insertion, deletion, or substitution of one or more nucleotides of the target polynucleotide.

[0132] The terms "gRNA," "guide RNA," and "CRISPR guide sequence" are used interchangeably throughout and refer to a nucleic acid containing a sequence that determines the specificity of the Cas DNA-binding protein of a CRISPR / Cas system. The gRNA hybridizes (complementary, partial, or complete) to a target nucleic acid sequence in the genome of a host cell. The gRNA, or the portion thereof, that hybridizes to the target nucleic acid can be 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid sequence is 10-30, or 15-25 nucleotides in length.

[0133] In addition to the sequence that binds to target nucleic acid, in some embodiments, gRNA also comprises scaffold sequence.Expression of gRNA that encodes both the sequence that is complementary to target nucleic acid and scaffold sequence has the dual function of binding (hybridizing) to target nucleic acid and recruiting endonuclease to target nucleic acid, which can produce site-specific CRISPR activity.In some embodiments, this chimeric gRNA is called single-stranded guide RNA (sgRNA).

[0134] In some embodiments, the gRNA is modified, e.g., chemically modified. The modified gRNA comprises at least one nucleotide having a modification in the chemical structure of at least one of the nucleobase, sugar, and phosphodiester bond or backbone moiety (e.g., nucleotide phosphate). Exemplary gRNA modifications will be apparent to those skilled in the art and can be found, for example, in Lee et al., Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife. 2017 May 2;6.pii:e25312.doi:10.7554 / eLife.25312 and US Publication 2016 / 0289675. Additional suitable modifications include phosphorothioate backbone modifications, 2'-O-Me-modified sugars, 2'F-modified sugars, substitution of the ribose sugar with the bicyclic nucleotide-cEt, 3'thioPACE (MSP), or combinations thereof. Suitable gRNA modifications are described, for example, in Rahdar et al. PNAS December 22, 2015 112(51)E7110-E7117 and Hendel et al., Nat Biotechnol. 2015 Sep;33(9):985-989, which are incorporated herein by reference in their entirety.

[0135] In some embodiments, the gRNA described herein is chemically modified. For example, the gRNA comprises one or more 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides. In some embodiments, the gRNA comprises 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides, at the 5' end of the gRNA. In some embodiments, the gRNA comprises 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides at the 3' end of the gRNA. In some embodiments, the gRNA comprises 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides at both the 5' and 3' ends of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., at the 5'-end nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., at the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-terminal end of the gRNA, and the third nucleotide from the 3'-terminal end of the gRNA, 2'-O-methyl modified. In some embodiments, the gRNA is 2'-O-modified, e.g., at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal end of the gRNA, the third nucleotide from the 5'-terminal end of the gRNA, the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-terminal end of the gRNA, and the third nucleotide from the 3'-terminal end of the gRNA, 2'-O-methyl modified. In some embodiments, the gRNA is 2'-O-modified, e.g., at the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-terminal end of the gRNA, the third nucleotide from the 3'-terminal end of the gRNA, and the fourth nucleotide from the 3'-terminal end of the gRNA, 2'-O-methyl modified. In some embodiments, the 3'-terminal nucleotide of the gRNA is not chemically modified. In some embodiments, the 3'-terminal nucleotide of the gRNA does not have a chemically modified sugar.In some embodiments, the gRNA is 2'-O-methyl modified, for example, at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal end of the gRNA, the third nucleotide from the 5'-terminal end of the gRNA, the second nucleotide from the 3'-terminal end of the gRNA, the third nucleotide from the 3'-terminal end of the gRNA, and the fourth nucleotide from the 3'-terminal end of the gRNA. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphate linkage to the adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphorothioate linkage to the adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a thioPACE linkage to the adjacent nucleotide.

[0136] In some embodiments, the gRNA comprises one or more 2'-O-modified nucleotides and 3' phosphorus-modified nucleotides, for example, 2'-O-methyl 3' phosphorothioate nucleotides. In some embodiments, the gRNA comprises a 2'-O-modified and a 3' phosphorus-modified nucleotide, for example, a 2'-O-methyl 3' phosphorothioate nucleotide at the 5'-end of the gRNA. In some embodiments, the gRNA comprises a 2'-O-modified and a 3' phosphorus-modified nucleotide, for example, a 2'-O-methyl 3' phosphorothioate nucleotide at the 3'-end of the gRNA. In some embodiments, the gRNA comprises a 2'-O-modified and a 3' phosphorus-modified nucleotide, for example, a 2'-O-methyl 3' phosphorothioate nucleotide at the 5'-end and 3'-end of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3' phosphorothioate-modified at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal end of the gRNA, and the third nucleotide from the 5'-terminal end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3' phosphorothioate-modified at the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-terminal end of the gRNA, and the third nucleotide from the 3'-terminal end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3' phosphorothioate-modified at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal end of the gRNA, the third nucleotide from the 5'-terminal end of the gRNA, the third nucleotide from the 3'-terminal end of the gRNA, and the second nucleotide from the 3'-terminal end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3' phosphorothioate modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA.In some embodiments, the 3'-terminal nucleotide of the gRNA is not chemically modified. In some embodiments, the 3'-terminal nucleotide of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA are 2'-O-methyl 3' phosphorothioate-modified.

[0137] In some embodiments, the gRNA may comprise one or more 2'-O-modifications and 3' phosphorus modifications, such as 2'-O-methyl 3'thioPACE nucleotides. In some embodiments, the gRNA comprises a 2'-O-modification and a 3' phosphorus modification, such as 2'-O-methyl 3'thioPACE nucleotides at the 5' end of the gRNA. In some embodiments, the gRNA comprises a 2'-O-modification and a 3' phosphorus modification, such as 2'-O-methyl 3'thioPACE nucleotides at the 3' end of the gRNA. In some embodiments, the gRNA comprises a 2'-O-modification and a 3' phosphorus modification, such as 2'-O-methyl 3'thioPACE nucleotides at the 5' and 3' ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom and one or more non-bridging oxygen atoms are replaced with an acetate group. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, for example, 2'-O-methyl 3'thioPACE modified at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal end of the gRNA, and the third nucleotide from the 5'-terminal end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, for example, 2'-O-methyl 3'thioPACE modified at the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-terminal end of the gRNA, and the third nucleotide from the 3'-terminal end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus modified, for example, 2'-O-methyl 3' phosphorothioate modified at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal end of the gRNA, the third nucleotide from the 5'-terminal end of the gRNA, the third nucleotide from the 3'-terminal end of the gRNA, and the second nucleotide from the 3'-terminal end of the gRNA. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3'thioPACE-modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA.In some embodiments, the 3'-terminal nucleotide of the gRNA is not chemically modified. In some embodiments, the 3'-terminal nucleotide of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3'thioPACE-modified at the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-terminal of the gRNA, the third nucleotide from the 5'-terminal of the gRNA, the second nucleotide from the 3'-terminal of the gRNA, the third nucleotide from the 3'-terminal of the gRNA, and the fourth nucleotide from the 3'-terminal of the gRNA.

[0138] In some embodiments, the gRNA comprises a chemically modified backbone. In some embodiments, the gRNA comprises phosphorothioate linkages. In some embodiments, one or more non-bridging oxygen atoms are replaced with sulfur atoms. In some embodiments, the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-end of the gRNA, and the third nucleotide from the 5'-end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-end of the gRNA, and the third nucleotide from the 3'-end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5'-end of the gRNA, the third nucleotide from the 5'-end of the gRNA, the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3'-end of the gRNA, and the third nucleotide from the 3'-end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the 5' end nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.

[0139] In some embodiments, the gRNA comprises a thioPACE linkage. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom and one or more non-bridging oxygen atoms are replaced with an acetate group. In some embodiments, the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a thioPACE linkage. In some embodiments, the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage. In some embodiments, the 5'-terminal nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the 3'-terminal nucleotide of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE linkage. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each contain a thioPACE bond. In some embodiments, the 5' end nucleotide of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each contain a thioPACE bond.

[0140] Chemical modifications of gRNAs are described, for example, in Hendel, A. et al., Nature Biotech., 2015, Vol. 33, No. 9, the entire contents of which are incorporated herein by reference.

[0141] As used herein, "scaffold sequence," also known as tracrRNA, refers to a nucleic acid sequence that recruits a Cas endonuclease to a target nucleic acid that is bound (hybridized) to a complementary gRNA sequence. Any scaffold sequence that includes at least one stem-loop structure and recruits an endonuclease can be used in the genetic elements and vectors described herein. Exemplary scaffold sequences are clear to those skilled in the art and can be found, for example, in Jinek et al., Science (2012) 337 (6096): 816-821; Ran et al., Nature Protocols (2013) 8: 2281-2308; WO 2014 / 093694; and WO 2013 / 176772.

[0142] In some embodiments, the gRNA sequence does not include a scaffold sequence, which is expressed as a separate transcript. In such embodiments, the gRNA sequence further includes an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence and recruit an endonuclease to the target nucleic acid.

[0143] In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the target nucleic acid (see also U.S. Patent No. 8,697,359, incorporated by reference, for teachings on complementarity of gRNA sequences with target polynucleotide sequences). It has been demonstrated that mismatches between CRISPR guide sequences and target nucleic acids near the 3' end of the target nucleic acid can abolish nuclease cleavage activity (Upadhyay, et al., Genes Genome Genetics (2013) 3(12):2233-2238). In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3' end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3' end of the target nucleic acid).

[0144] The target nucleic acid is flanked on the 3' side by a protospacer adjacent motif (PAM), which may interact with the endonuclease and further participate in targeting the endonuclease activity to the target nucleic acid. Generally, the PAM sequence flanking the target nucleic acid sequence will depend on the endonuclease and the source from which the endonuclease is derived. For example, in the Cas9 endonuclease derived from Streptococcus pyogenes, the PAM sequence is NGG. In the Cas9 endonuclease derived from Staphylococcus aureus, the PAM sequence is NNGRRT. In the Cas9 endonuclease derived from Neisseria meningitidis, the PAM sequence is NNNNGATT. In the Cas9 endonuclease derived from Staphylococcus thermophilus, the PAM sequence is NNAGAA. In the Cas9 endonuclease derived from Treponema denticola, the PAM sequence is NAAAAC. In the Cpf1 nuclease, the PAM sequence is TTN.

[0145] In some embodiments, genetically engineering the cell also includes introducing one or more (e.g., 1, 2, 3, or more) Cas endonucleases into the cell. In some embodiments, nucleic acids encoding the Cas endonuclease and gRNA are provided on the same nucleic acid (e.g., vector). In some embodiments, nucleic acids encoding the Cas endonuclease and gRNA are provided on different nucleic acids (e.g., different vectors). Alternatively, or additionally, the Cas endonuclease may be provided or introduced into the cell in protein form.

[0146] In some embodiments, the Cas endonuclease is a Cas9 enzyme or a variant thereof. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9), Neisseria meningitidis (NmCas9), Streptococcus thermophilus, Campylobacter jejuni (CjCas9), or Treponema denticola. In some embodiments, the nucleotide sequence encoding the Cas endonuclease can be codon-optimized for expression in a host cell. In some embodiments, the endonuclease is a Cas9 homolog or ortholog. In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease is further modified to alter the activity of the protein. In some embodiments, the Cas9 endonuclease is modified to inactivate one of the catalytic residues of the endonuclease, referred to as a "nickase" or "Cas9n." The Cas9 nickase endonuclease cleaves one DNA strand of a target nucleic acid. See, e.g., Dabrowska et al. Frontiers in Neuroscience (2018) 12(75). It has been shown that one or more mutations in the RuvC and HNH catalytic domains of the enzyme can improve Cas9 efficiency. See, e.g., Sarai et al. Currently Pharma. Biotechnol. (2017) 18(13). In some embodiments, the Cas9 endonuclease is a catalytically inactive Cas9. For example, dCas9 contains mutations in catalytically active residues (D10 and H840) and has no nuclease activity. Alternatively, or in addition, the Cas9 endonuclease can be fused to another protein or portion thereof. In some embodiments, dCas9 is fused to a repressor domain, such as a KRAB domain. In some embodiments, such dCas9 fusion proteins are used in the constructs described herein for multiplex gene suppression (e.g., CRISPR interference (CRISPRi)). In some embodiments, dCas9 is fused to an activator domain, such as VP64 or VPR.In some embodiments, such dCas9 fusion proteins are used in constructs described herein for gene activation (e.g., CRISPR activation (CRISPRa)). In some embodiments, dCas9 is fused to an epigenetic regulatory region, such as a histone demethylase region or a histone acetyltransferase region. In some embodiments, dCas9 is fused to LSD1 or p300, or portions thereof. In some embodiments, dCas9 fusions are used for CRISPR-based epigenetic regulation. In some embodiments, dCas9 or Cas9 is fused to a Fok1 nuclease region. In some embodiments, Cas9 or dCas9 fused to a Fok1 nuclease region is used for genome editing. In some embodiments, Cas9 or dCas9 is fused to a fluorescent protein (e.g., GFP, RFP, mCherry, etc.). In some embodiments, Cas9 / dCas9 proteins fused to fluorescent proteins are used to label and / or visualize genomic loci or to identify cells expressing the Cas endonuclease.

[0147] In some embodiments, the Cas endonuclease is engineered to increase the specificity of the enzyme (e.g., to maintain robust on-target cleavage while reducing off-target effects). In some embodiments, the Cas endonuclease is an enhanced-specificity Cas9 variant (e.g., eSPCas9). See, e.g., Slaymaker et al. Science (2016) 351(6268):84-88. In some embodiments, the Cas endonuclease is a high-fidelity Cas9 variant (e.g., SpCas9-HF1). See, e.g., Kleinstiver et al. Nature (2016) 529:490-495.

[0148] Cas enzymes, such as Cas endonucleases, are known in the art and can be obtained from various sources and / or engineered / modified to modulate one or more activities or specificities of the enzyme. In some embodiments, the Cas enzyme is engineered / modified to recognize one or more PAM sequences. In some embodiments, the Cas enzyme is engineered / modified to recognize one or more PAM sequences that are different from the PAM sequences that the Cas enzyme recognizes without the engineered / modified sequence. In some embodiments, the Cas enzyme is engineered / modified to reduce the off-target activity of the enzyme.

[0149] In some embodiments, the nucleotide sequence encoding the Cas endonuclease is further modified to alter the specificity of the endonuclease (e.g., to reduce off-target cleavage, reduce Cas endonuclease activity or life span in cells, enhance homologous recombination, and reduce non-homologous end joining). See, e.g., Komor et al. Cell (2017) 168:20-36. In some embodiments, the nucleotide sequence encoding the Cas endonuclease is modified to alter the PAM recognition of the endonuclease. For example, the Cas endonuclease SpCas9 recognizes the PAM sequence NGG, while relaxed variants of SpCas9 containing one or more endonuclease modifications (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) can recognize the PAM sequences NGA, NGAG, NGCG. The PAM recognition of a modified Cas endonuclease is considered "relaxed" if the Cas endonuclease recognizes a more cryptic PAM sequence than the unmodified Cas endonuclease. For example, the Cas endonuclease SaCas9 recognizes the PAM sequence NNGRRT, while a relaxed variant of SaCas9 containing one or more endonuclease modifications (e.g., KKH SaCas9) can recognize the PAM sequence NNNRRT. In one example, the Cas endonuclease FnCas9 recognizes the PAM sequence NNG, while a relaxed variant of FnCas9 containing one or more endonuclease modifications (e.g., RHA FnCas9) can recognize the PAM sequence YG. In one example, the Cas endonuclease is a Cpf1 endonuclease containing substitution mutations S542R and K607R and recognizes the PAM sequence TYCV. In one example, the Cas endonuclease is a Cpf1 endonuclease containing the substitution mutations S542R, K607R, and N552R, which recognizes the PAM sequence TATV. See, e.g., Gao et al. Nat. Biotechnol. (2017) 35(8):789-792.

[0150] In some embodiments, two or more (e.g., two, three, or more) Cas endonucleases are used. In some embodiments, at least one Cas endonuclease is a Cas9 enzyme. In some embodiments, at least one Cas endonuclease is a Cpfl enzyme. In some embodiments, at least one Cas9 endonuclease is from Streptococcus pyogenes. In some embodiments, at least one Cas9 endonuclease is from Streptococcus pyogenes and at least one Cas9 endonuclease is from an organism that is not Streptococcus pyogenes. In some embodiments, the endonuclease is a base editor. Base editor endonucleases typically comprise a catalytically inactive Cas endonuclease fused to a functional domain. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964; Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas endonuclease is dCas9. In some embodiments, the endonuclease comprises dCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises dCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)). In some embodiments, the catalytically inactive Cas endonuclease has reduced activity and is nCas9. In some embodiments, the endonuclease comprises nCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises nCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA.In some embodiments, the endonuclease comprises nCas9 fused to a cytodine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)).

[0151] Examples of base editors include, but are not limited to, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, and ABE7.10. * , xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE and CRISPR-SKIP.Additional examples of base editors can be found in, for example, US Patent Publication No. 2018 / 0312825A1, US Patent Publication No. 2018 / 0312828A1 and PCT International Publication No. 2018 / 165629A1.These patents are incorporated herein by reference in their entirety.

[0152] In some embodiments, the base editor is further modified to inhibit base excision repair and induce cellular mismatch repair at the target site. Any of the Cas endonucleases described herein are fused to a Gam domain (bacteriophage Mu protein) to protect the Cas endonuclease from degradation and exonuclease activity. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964.

[0153] In some embodiments, the Cas endonuclease belongs to Class 2 Type V of Cas endonucleases. Class 2 Type V Cas endonucleases are further classified into Type VA, Type VB, Type VC, and Type VU. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017). In some embodiments, the Cas endonuclease is a Type VA Cas endonuclease, e.g., Cpf1 nuclease. In some embodiments, the Cas endonuclease is a Type VB Cas endonuclease, e.g., C2c1 endonuclease. See, e.g., Shmakov et al. Mol Cell (2015) 60:385-397. In some embodiments, the Cas endonuclease is Mad7.

[0154] Alternatively or additionally, the Cas endonuclease is a Cpf1 nuclease or a variant thereof. As will be understood by those skilled in the art, the Cas endonuclease Cpf1 nuclease is also referred to as Cas12a. See, for example, Strohkendl et al. Mol. Cell (2018) 71:1-9. In some embodiments, the host cell expresses a Cpf1 nuclease from Prevotella or Francisella species, Acidaminococcus (AsCpf1), Lachnospira (LpCpf1), or Eubacterium lectare. In some embodiments, the nucleotide sequence encoding the Cpf1 nuclease can be codon-optimized for expression in the host cell. In some embodiments, the nucleotide sequence encoding the Cpf1 endonuclease is further modified to alter the activity of the protein.

[0155] A catalytically inactive variant of Cpf1 (Cas12a) may be referred to as dCas12a. As described herein, a catalytically inactive variant of Cpf1 may be fused to a functional domain to form a base editor. See, e.g., Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas endonuclease is dCas9. In some embodiments, the endonuclease comprises dCas12a fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises dCas12a fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas12a fused to a cytodine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)).

[0156] Alternatively or additionally, the Cas endonuclease can be a Cas14 endonuclease or a variant thereof. In contrast to the Cas9 endonuclease, the Cas14 endonuclease is derived from archaea and tends to be small in size (e.g., 400-700 amino acids). Furthermore, the Cas14 endonuclease does not require a PAM sequence. See, e.g., Harrington et al., Science (2018).

[0157] Any of the Cas endonucleases described herein can be adjusted to control the expression level and / or activity of the Cas endonuclease at a desired time. For example, it may be advantageous to increase the expression and / or activity level of the Cas endonuclease during a particular phase of the cell cycle. It has been shown that the level of homology-directed repair is reduced during the G1 phase of the cell cycle, and therefore increasing the expression and / or activity level of the Cas endonuclease during the S, G2, and / or M phases can enhance homology-directed repair after Cas endonuclease editing. In some embodiments, the expression and / or activity level of the Cas endonuclease is increased during the S, G2, and / or M phases of the cell cycle. In one example, the Cas endonuclease is fused to the N-terminal region of human geminin. See, e.g., Gutschner et al. Cell Rep. (2016) 14(6):1555-1566. In some embodiments, the expression and / or activity level of the Cas endonuclease is reduced during the G1 phase. In one example, the Cas endonuclease is regulated such that its activity is reduced during the G1 phase. See, e.g., Lomova et al. Stem Cells (2018).

[0158] Alternatively, or in addition, any of the Cas endonucleases described herein can be fused to an epigenetic modifier (e.g., a chromatin-modifying enzyme, e.g., a DNA methylase, a histone deacetylase). See, e.g., Kungulovski et al. Trends Genet. (2016) 32(2):101-113. A Cas endonuclease fused to an epigenetic modifier is referred to as an "epigenetic effector" and allows for temporary and / or transient endonuclease activity. In some embodiments, the Cas endonuclease is dCas9 fused to a chromatin-modifying enzyme.

[0159] In some embodiments, the present disclosure provides compositions and methods for inhibiting a cell surface lineage-specific antigen in hematopoietic cells using the CRISPR / Cas9 system, wherein the guide RNA sequence hybridizes to a nucleotide sequence encoding the cell surface lineage-specific antigen. In some embodiments, the cell surface lineage-specific antigen is CD33, and the gRNA hybridizes to a portion of the nucleotide sequence encoding CD33 (Figure 5). Examples of gRNAs that target CD33 are shown in Table 4, although additional gRNAs that hybridize to CD33 and can be used in the methods described herein can be developed. In some embodiments, the gRNA comprises SEQ ID NO:50 or SEQ ID NO:51.

[0160] Table 4 shows exemplary guide RNA sequences that hybridize, or are predicted to hybridize, to portions of CD33. Both RNA and DNA sequences of exemplary guide RNA sequences are provided, and one of skill in the art will understand that unless otherwise specified, the polynucleotide sequences described in this application use "T" in representative DNA sequences, but when sequences represent RNA, "T" is replaced with "U." [Table 4]

[0161] In some cases, a gRNA for use in the present disclosure may include a spacer sequence that is at least 90% (at least 93%, 95%, 96%, 97%, 98%, or 99%) identical to any of the exemplary guide RNA sequences in Table 4 above, e.g., SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:70.

[0162] The "percent identity" of two nucleic acids is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm has been incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length -12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. If gaps exist between the two sequences, gapped BLAST can be used as reported in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0163] In some embodiments, it may be desirable to further genetically engineer HSCs, particularly allogeneic HSCs, to reduce the graft-versus-host effect. For example, the standard treatment for relapsed AML is hematopoietic stem cell transplantation (HSCT). However, at least one of the factors limiting the success of HSCT is graft-versus-host disease (GVHD), which is related to the expression of the cell surface molecule CD45. See, for example, Van Besie, Hematology Am. Soc. Hematol Educ Program (2013) 56; Mawad Curr. Hematol. Malig. Rep. (2013) 8(2):132. CD45RA and CD45RO are isoforms of CD45 (found on all hematopoietic cells except erythrocytes). In T lymphocytes, CD45RA is expressed on naive cells, while CD45RO is expressed on memory cells. CD45RA T cells are more likely to be reactive to recipient-specific antigens after HSCT, resulting in GVHD. Therefore, there remains a need for efficient and safe AML treatments that also reduce the likelihood of transplant rejection or GVHD.CD45 is a type 1 lineage antigen because CD45-bearing cells are required for survival, but the antigen can be removed from stem cells using CRISPR.

[0164] In view of the complications arising from the progression of GVHD after HSCT, the present disclosure also provides compositions and methods that target CD45RA, which are intended to prevent and / or reduce the occurrence or severity of GVHD.

[0165] Thus, in the case of GVHD, treatment of a patient may include the following steps: (1) administering to the patient a therapeutically effective amount of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that targets the CD45RA lineage-specific antigen, and (2) infusing the patient with hematopoietic stem cells, wherein the hematopoietic cells have reduced expression of the CD45RA lineage-specific antigen.

[0166] Additionally, the present disclosure provides compositions and methods for the combined inhibition of both CD33 and CD45RA lineage-specific antigens. Such therapeutic regimens can include the steps of: (1) administering to a patient a therapeutically effective amount of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that targets the CD33 and CD45RA lineage-specific antigens, and (2) infusing or reinfusing the patient with hematopoietic stem cells, either autologous or allogeneic, wherein the hematopoietic cells have reduced expression of the CD33 and CD45RA lineage-specific antigens.

[0167] In some embodiments, the cell surface lineage-specific antigen CD45RA is also removed or inhibited in hematopoietic cells using the CRISPR / Cas9 system. In some embodiments, the gRNA sequence hybridizes to a portion of the nucleotide sequence encoding CD45RA (Figure 6). Examples of gRNAs that target CD45RA are shown in Table 5, although additional gRNAs can be developed that hybridize to CD45RA and can be used in the methods described herein.

[0168] Table 5 shows exemplary guide RNA sequences that hybridize or are predicted to hybridize to exon 4 or exon 5 of human CD45. [Table 5]

[0169] Also provided herein are methods for producing cells lacking a cell surface lineage-specific antigen, comprising providing a cell and introducing components of a CRISPR / Cas system into the cell for genome editing. In some embodiments, a nucleic acid comprising a CRISPR / Cas guide RNA (gRNA) that hybridizes, or is predicted to hybridize, to a portion of a nucleotide sequence encoding the lineage-specific cell surface antigen is introduced into the cell. In some embodiments, the gRNA is introduced into the cell in a vector. In some embodiments, a Cas endonuclease is introduced into the cell. In some embodiments, the Cas endonuclease is introduced into the cell as a nucleic acid encoding the Cas endonuclease. In some embodiments, the gRNA and the nucleotide sequence encoding the Cas endonuclease are introduced into the cell in the same nucleic acid (e.g., the same vector). In some embodiments, the Cas endonuclease is introduced into the cell in the form of a protein. In some embodiments, the Cas endonuclease and gRNA are preformed in vitro and introduced into the cell as a complex.

[0170] The present disclosure further provides engineered non-naturally occurring vectors and vector systems capable of encoding one or more components of a CRISPR / Cas9 complex, the vector comprising: (i) a (CRISPR)-Cas system guide RNA that hybridizes to a lineage-specific antigen sequence; and (ii) a polynucleotide encoding a Cas9 endonuclease.

[0171] The vectors of the present disclosure can be used to direct the expression of one or more sequences in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840) and pMT2PC (Kaufman et al., EMBO J. (1987) 6:187). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0172] The vectors of the present disclosure can direct the expression of a nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters, which can be tissue-specific or cell-specific. The term "tissue-specific," when applied to a promoter, refers to a promoter that can direct the selective expression of a nucleotide sequence of interest to a particular type of tissue (e.g., a seed) and relatively lack expression of the same nucleotide sequence of interest in a different type of tissue. The term "cell-type specific," when applied to a promoter, refers to a promoter that can direct the selective expression of a nucleotide sequence of interest to a particular cell type and relatively lack expression of the same nucleotide sequence of interest in different types of cells within the same tissue. The term "cell-type specific," when applied to a promoter, also refers to a promoter that can promote the selective expression of a nucleotide sequence of interest in a region within a single tissue. The cell-type specificity of a promoter can be assessed using methods well known in the art, such as immunohistochemical staining.

[0173] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding CRISPR / Cas9 into mammalian cells or target tissues, and such methods can be used to administer nucleic acids encoding components of the CRISPR-Cas system to cells in culture or to a host organism.

[0174] Non-viral delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with a delivery vehicle. In one embodiment, the non-viral vector delivery system is a preformed ribonucleoprotein complex (e.g., a complex comprising a Cas9 protein complexed with a targeting gRNA). The preformed ribonucleoprotein complex can then be introduced into cells by electroporation, biolistic bombardment, or other physical delivery methods. In one embodiment, electroporation is used to introduce the preformed ribonucleoprotein complex into cells.

[0175] Viral vector delivery systems include DNA and RNA viruses, which have episomal or integrated genomes after delivery to cells. Viral vectors can be administered directly to patients (in vivo), or they can be used to engineer cells in vitro or ex vivo, and the engineered cells can be administered to patients. In one embodiment, the present disclosure utilizes virus-based systems, including but not limited to retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses, for gene transfer. Additionally, the present disclosure provides vectors capable of integration into the host genome, such as retroviruses or lentiviruses. Preferably, the vector used for expression of the CRISPR-Cas system of the present disclosure is a lentiviral vector.

[0176] In one embodiment, the present disclosure provides for the introduction of one or more vectors encoding CRISPR-Cas into eukaryotic cells. The cells may be cancer cells. Alternatively, the cells are hematopoietic cells, such as hematopoietic stem cells. Examples of stem cells include pluripotent, multipotent, and unipotent stem cells. Examples of pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, and induced pluripotent stem cells (iPSCs). In a preferred embodiment, the present disclosure provides for the introduction of CRISPR-Cas9 into hematopoietic stem cells.

[0177] The vectors of the present disclosure are delivered to eukaryotic cells in a subject. The modification of the eukaryotic cells via the CRISPR / Cas9 system can be performed in cell culture, and the method includes isolating the eukaryotic cells from the subject before modification. In some embodiments, the method further includes returning the eukaryotic cells and / or cells derived therefrom to the subject. Combination therapy

[0178] As described herein, an agent comprising an antigen-binding fragment that binds to a cell surface lineage-specific antigen (e.g., CD33) can be administered to a patient in combination with hematopoietic cells lacking the cell surface lineage-specific antigen, e.g., hematopoietic stem or progenitor cells produced using a gRNA described herein (e.g., a gRNA comprising the nucleotide sequence AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67) or CCUCACUAGACUUGACCCAC (SEQ ID NO: 70)). In some embodiments, the cells comprise a genetic mutation at a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50) or CCTCACTAGACTTGACCCAC (SEQ ID NO: 58). As used herein, "subject," "individual," and "patient" are used interchangeably and refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, human primates, non-human primates, or murine, bovine, equine, canine, or feline species. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0179] In some embodiments, the substances and / or hematopoietic cells may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0180] To practice the methods described herein, an effective amount of a substance comprising an antigen-binding fragment that binds to a cell surface lineage-specific antigen and an effective amount of hematopoietic cells can be co-administered to a subject in need of treatment. As used herein, the term "effective amount" is used interchangeably with the term "therapeutically effective amount" and refers to the amount of a substance, cell population, or pharmaceutical composition (e.g., a composition comprising a substance and / or hematopoietic cells) sufficient to produce a desired activity upon administration to a subject in need thereof. In the context of the present disclosure, the term "effective amount" refers to the amount of a compound, cell population, or pharmaceutical composition sufficient to delay the onset, halt the progression, or alleviate or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is an effective amount when administered individually.

[0181] As will be appreciated by those skilled in the art, the effective amount will vary depending on the particular condition being treated, the severity of the condition, parameters including the age, physical condition, height, sex, and weight of the individual patient, the duration of treatment, the nature of concomitant therapy (if any), the particular route of administration, and factors in the knowledge and opinion of the medical professional. In some embodiments, the effective amount alleviates, relieves, cures, improves, reduces, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0182] As described herein, hematopoietic cells and / or immune cells expressing chimeric receptors can be autologous to a subject, i.e., cells are obtained from a subject in need of treatment, genetically engineered to lack the expression of cell surface lineage-specific antigens or chimeric receptor constructs, and then administered to the same subject.Administering autologous cells to a subject can result in reduced rejection of host cells compared to administering non-autologous cells.Alternatively, host cells can be allogeneic cells, i.e., cells are obtained from a first subject, genetically engineered to lack the expression of cell surface lineage-specific antigens or chimeric receptor constructs, and administered to a second subject that is different from the first subject but is of the same species.For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient that is different from the donor.

[0183] In some embodiments, the immune cells and / or hematopoietic cells are allogeneic and further genetically engineered to reduce graft-versus-host disease. For example, as described herein, hematopoietic stem cells can be genetically engineered (e.g., using genome editing) to have reduced expression of CD45RA.

[0184] In some embodiments, immune cells expressing any of the chimeric receptors described herein are administered to a subject in an amount effective to reduce the number of target cells (e.g., cancer cells) by at least 20%, e.g., 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more.

[0185] A typical amount of cells, i.e., immune or hematopoietic cells, administered to a mammal (e.g., a human) can range, for example, from 1 million to 100 billion cells, although amounts below or above this exemplary range are also within the scope of the present disclosure. For example, a daily dose of cells can be about 1 million to about 50 billion cells (e.g., about 5 million, about 25 million, about 500 million, about 1 billion, about 5 billion, about 20 billion, about 30 billion, about 40 billion, or a range defined by any two of the above values), preferably about 10 million to about 100 billion cells (e.g., about 20 million, about 30 million, about 40 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, or a range defined by any two of the above values). The number of amino acids present in the molecule may be about 0 billion, about 25 billion, about 50 billion, about 75 billion, about 90 billion, or a range defined by any two of the above values), more preferably about 100 million to about 50 billion (for example, about 120 million, about 250 million, about 350 million, about 450 million, about 650 million, about 800 million, about 900 million, about 3 billion, about 30 billion, about 45 billion, or a range defined by any two of the above values).

[0186] In one embodiment, a chimeric receptor (e.g., a nucleic acid encoding the chimeric receptor) is introduced into immune cells, and a subject (e.g., a human patient) receives an initial administration or dose of immune cells expressing the chimeric receptor. One or more subsequent administrations of a substance (e.g., immune cells expressing the chimeric receptor) can be administered to the patient at intervals of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. More than one dose of a substance may be administered to a subject per week, for example, two, three, four, or more administrations of the substance. A subject may receive more than one administration of a substance (e.g., immune cells expressing a chimeric receptor) per week, followed by a week without administration of the substance, and finally more than one booster administration of the substance (e.g., more than one administration of immune cells expressing the chimeric receptor per week). Immune cells expressing the chimeric receptor can be administered every other day for three doses per week for 2, 3, 4, 5, 6, 7, 8, or more weeks.

[0187] For the purposes of this disclosure, the terms "treat," "treatment," and similar terms, insofar as they relate to any of the disease conditions described herein, mean alleviating or alleviating at least one symptom associated with such condition, or slowing or reversing the progression of such condition. In the sense of this disclosure, the term "treat" also means halting or delaying the onset (i.e., the period before clinical symptoms of disease) and / or reducing the risk of developing or worsening the disease. For example, in the context of cancer, the term "treat" can mean eliminating or reducing a patient's tumor burden, or preventing, delaying, or inhibiting metastasis, etc.

[0188] In some embodiments, a substance comprising an antigen-binding fragment that binds a cell-surface lineage-specific antigen and a hematopoietic cell population lacking the cell-surface lineage-specific antigen is used. Thus, in such treatment methods, the substance recognizes (binds to) target cells that express the cell-surface lineage-specific antigen in order to target them for death. The hematopoietic cells lacking the antigen allow for repopulation of the cell type targeted by the substance. In some embodiments, treating a patient can include the following steps: (1) administering to the patient a therapeutically effective amount of a substance that targets the cell-surface lineage-specific antigen, and (2) infusing or reinfusing the patient with either autologous or allogeneic hematopoietic stem cells, wherein the hematopoietic cells have reduced expression of the lineage-specific disease-associated antigen. In some embodiments, treating a patient may include the following steps: (1) administering to the patient a therapeutically effective amount of immune cells expressing a chimeric receptor, wherein the immune cells comprise a nucleic acid sequence encoding a chimeric receptor that binds a cell surface lineage-specific disease-associated antigen, and (2) infusing or reinfusing the patient with either autologous or allogeneic hematopoietic cells (e.g., hematopoietic stem cells), wherein the hematopoietic cells have reduced expression of the lineage-specific disease-associated antigen.

[0189] The efficacy of a treatment method using a substance comprising an antigen-binding fragment that binds a cell surface lineage-specific antigen and a hematopoietic cell population lacking the cell surface lineage-specific antigen can be assessed by any method known in the art, which will be apparent to a skilled medical professional. For example, the efficacy of a treatment can be assessed by the survival of the subject or by the cancer burden in the subject or its tissues or samples. In some embodiments, the efficacy of a treatment is assessed by quantifying the number of cells belonging to a particular population or lineage of cells. In some embodiments, the efficacy of a treatment is assessed by quantifying the number of cells presenting the cell surface lineage-specific antigen.

[0190] In some embodiments, the substance comprising the antigen-binding fragment that binds to the cell surface lineage-specific antigen and the hematopoietic cell population are administered simultaneously.

[0191] In some embodiments, a substance comprising an antigen-binding fragment that binds a cell surface lineage-specific antigen (e.g., immune cells expressing a chimeric receptor described herein) is administered prior to administration of the hematopoietic cells. In some embodiments, a substance comprising an antigen-binding fragment that binds a cell surface lineage-specific antigen (e.g., immune cells expressing a chimeric receptor described herein) is administered at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, or more prior to administration of the hematopoietic cells. In some embodiments, a substance comprising an antigen-binding fragment that binds a cell surface lineage-specific antigen (e.g., immune cells expressing a chimeric receptor described herein) is administered to a subject multiple times prior to administration of the hematopoietic cells.

[0192] In some embodiments, the hematopoietic cells are administered prior to the administration of the substance comprising an antigen-binding fragment that binds a cell surface lineage-specific antigen (e.g., immune cells expressing a chimeric receptor described herein). In some embodiments, the hematopoietic cell population is administered at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months or more prior to the administration of the substance comprising an antigen-binding fragment that binds a cell surface lineage-specific antigen.

[0193] In some embodiments, the substance targeting a cell surface lineage-specific antigen and the hematopoietic cell population are administered substantially simultaneously. In some embodiments, the substance targeting a cell surface lineage-specific antigen is administered, the patient is evaluated for a period of time, and then the hematopoietic cell population is administered. In some embodiments, the hematopoietic cell population is administered, the patient is evaluated for a period of time, and then the substance targeting a cell surface lineage-specific antigen is administered.

[0194] Multiple administrations (e.g., dosages) of the substance and / or hematopoietic cell population are also within the scope of the present disclosure. In some embodiments, the substance and / or hematopoietic cell population is administered to the subject once. In some embodiments, the substance and / or hematopoietic cell population is administered to the subject more than once (e.g., at least 2, 3, 4, 5, or more times). In some embodiments, the substance and / or hematopoietic cell population is administered to the subject at regular intervals, e.g., every 6 months.

[0195] In some embodiments, the subject is a human subject with a hematopoietic malignancy. As used herein, a hematopoietic malignancy refers to a malignant disorder involving hematopoietic cells (e.g., blood cells, including progenitors and stem cells). Examples of hematopoietic malignancies include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. Leukemia includes acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia or chronic lymphocytic leukemia, and chronic lymphocytic leukemia.

[0196] In some embodiments, the leukemia is acute myeloid leukemia (AML). AML is characterized as a heterogeneous, clonal tumor disease originating from transformed cells with continuously acquired significant genetic alterations that disrupt key differentiation and proliferation control pathways (Dohner et al., NEJM, (2015) 373:1136). CD33 glycoprotein is expressed on most myeloid leukemia cells, as well as on normal bone marrow and monocyte precursors, and is considered an attractive target for AML therapy (Laszlo et al., Blood Rev. (2014) 28(4):143-53). Clinical trials using anti-CD33 monoclonal antibody-based therapy have shown improved survival in a subset of AML patients when combined with standard chemotherapy, but these effects have also been accompanied by safety and efficacy issues.

[0197] Other attempts to target AML cells have involved generating T cells expressing chimeric antigen receptors (CARs) that selectively target CD33 in AML. Buckley et al., Curr. Hematol. Malig. Rep. (2):65 (2015). However, data are limited, and it is uncertain how effective this approach is in treating patients (whether all targeted cells are eliminated). Furthermore, because myeloid lineage cells are essential for survival, depleting a subject of myeloid lineage cells may have a detrimental effect on patient survival. The present disclosure aims, at least in part, to solve these problems associated with AML treatment.

[0198] Alternatively, or in addition, the methods described herein may be used to treat non-hematopoietic cancers, including, but not limited to, lung cancer, ear, nose, and pharyngeal cancer, colon cancer, melanoma, pancreatic cancer, breast cancer, prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, pharyngeal cancer, liver cancer, fibroma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, and other carcinomas and sarcomas.

[0199] Carcinomas are cancers of epithelial origin. Carcinomas contemplated for treatment by the methods of the present disclosure include, but are not limited to, acinar carcinoma, lobular carcinoma, follicular adenocarcinoma (also called adenocystic carcinoma, adenomyoepithelioma, cribriform carcinoma, and cylindroma), adenomatous carcinoma, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma (also called bronchiolar carcinoma, alveolar cell tumor, and pulmonary adenomatosis), basal cell carcinoma, and pulmonary carcinoma. basocellulare) (also called basaloma, or basiloma, and pilomatrix carcinoma), basaloid carcinoma, basosquamous carcinoma, breast carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma (also called bile duct carcinoma and cholangiocellular carcinoma), choriocarcinoma, colloid carcinoma, comedo carcinoma, uterine carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, columnar cell carcinoma, and columnar Cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonal carcinoma, encephalomatous carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, fibrocarcinoma, glial carcinoma, glial carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, pilomatrix carcinoma, hematoid carcinoma, hepatocellular carcinoma (also called hepatocellular carcinoma, malignant hepatocellular carcinoma, and hepatocarcinoma), Hürthle cell carcinoma, hyaline carcinoma, adrenal carcinoma, infantile embryonal carcinoma, carcinoma in situ In situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Klutzycki cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipocarcinoma, lymphoepithelial carcinoma, breast carcinoma, medullary carcinoma, medullary carcinoma, carcinoma melanodes, melanotic carcinoma, mucinous carcinoma, mucinous secretory carcinoma, mucous cell carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, melanotic carcinomanigrum, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of the kidney (also called renal adenocarcinoma and adrenoid carcinoma), reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, cord carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, nodular carcinoma These include tumors of the breast, cervix, ovary, prostate, lung, colon or rectum, pancreas, stomach, or kidney.

[0200] Sarcomas are mesenchymal neoplasms that arise in bone and soft tissue. Various types of sarcomas are recognized, including liposarcoma (such as myxoid liposarcoma and pleomorphic liposarcoma), leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor (also called malignant schwannoma, neurofibrosarcoma, or neurogenic sarcoma), Ewing's tumor (including Ewing's sarcoma of bone, extraosseous (i.e., non-bone) Ewing's sarcoma, and primitive neuroectodermal tumor [PNET]), synovial sarcoma, angiosarcomas, and hemangiosarcomas. arcoma), lymphangiosarcoma, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, desmoid tumor (also called invasive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolar soft part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small round cell tumor, gastrointestinal stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma) - skeletal and extraskeletal, and chondrosarcoma.

[0201] In some embodiments, the cancer being treated may be a refractory cancer. As used herein, a "refractory cancer" refers to a cancer that is resistant to prescribed standard treatment. These cancers may initially appear responsive to treatment (then recur), or may be completely unresponsive to treatment. The usual standard treatment varies depending on the cancer type and how advanced the cancer is in the subject. It may be chemotherapy, surgery, radiation, or a combination of these. Those skilled in the art are aware of such standard treatments. Subjects whose refractory cancers are treated according to the present disclosure may therefore have already received another treatment for their cancer. Alternatively, if the cancer is likely to be refractory (e.g., based on analysis of the cancer cells or the subject's medical history), the subject may not have previously received another treatment. Examples of refractory cancers include, but are not limited to, leukemia, melanoma, renal cell carcinoma, colon cancer, liver cancer (hepatoma), pancreatic cancer, non-Hodgkin's lymphoma, and lung cancer.

[0202] Any of the immune cells expressing the chimeric receptors described herein can be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier or excipient.

[0203] The phrase "pharmaceutically acceptable" as used in connection with the compositions and / or cells of the present disclosure refers to molecular entities or other ingredients of a composition that are physiologically tolerated and generally do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in mammals, more preferably humans. "Acceptable" means that the carrier is compatible with the active ingredients of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to whom the composition is administered. Any of the pharmaceutical compositions and / or cells used in the present methods may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.

[0204] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; other carbohydrates; metal complexes; and / or non-ionic surfactants. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0205] Kits for therapeutic use Also within the scope of the present disclosure are kits for use of substances that target cell-surface lineage-specific antigens in combination with hematopoietic cell populations lacking the cell-surface lineage-specific antigen. Such kits may include one or more containers containing a first pharmaceutical composition comprising any substance (e.g., immune cells expressing a chimeric receptor described herein) containing an antigen-binding fragment that binds the cell-surface lineage-specific antigen and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising a hematopoietic cell (e.g., hematopoietic stem cell) population lacking the cell-surface lineage-specific antigen and a pharmaceutically acceptable carrier.

[0206] In some embodiments, the kits described herein include a gRNA that binds to a site having the sequence ATCCCTGGCACTCTAGAACC (SEQ ID NO: 50) or CCTCACTAGACTTGACCCAC (SEQ ID NO: 58). In some embodiments, the gRNA includes CCUCACUAGACUUGACCCAC (SEQ ID NO: 70) or AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67).

[0207] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering the first and second pharmaceutical compositions to a subject to achieve a desired activity in the subject. The kit further includes instructions for selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. In some embodiments, the instructions include instructions for administering the first and second pharmaceutical compositions to a subject in need of the treatment.

[0208] The instructions for use of the substance targeting a cell surface lineage-specific antigen and the first and second pharmaceutical compositions described herein typically include information about the dosage, administration schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. The instructions for use provided with the kit of the present disclosure are generally the instructions written on the label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0209] The kits provided herein are suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Packaging for use with specific devices, such as inhalers, nasal administration devices, or infusion containers, is also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile access port. At least one active ingredient in the pharmaceutical composition is a chimeric receptor variant described herein.

[0210] The kit may optionally provide additional components such as buffers and determining information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article of manufacture containing the contents of the kit.

[0211] General methods The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are well explained in the literature and can be found in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R.I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds. 1993-98) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984>>; Animal Cell Culture (R.I. Freshney, ed. (1986); Immobilized Cells and Enzymes (lRL Press, (1986); and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.) etc. are described.

[0212] Without further elaboration, it is believed that one skilled in the art can, based on the foregoing, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0213] Example 1: In vitro depletion of CD33 in human leukemia cell lines To test the ability of the CRSPR-Cas9 system to target CD33 in vitro, human leukemia cells K-562 were co-transfected with Cas9-GFP (PX458, Streptococcus pyogenes) and a guide RNA containing an NGG PAM sequence using Neon™ (Termo Fisher Scientific) (Figure 4). The guide RNA was designed to target the hCD33 genomic sequence. Forty-eight hours after transfection, cells expressing Cas9 were identified and isolated using FACS sorting for GFP. The cells were then incubated for 96 hours and tested for CD33 expression by flow cytometry (Figure 5). Flow cytometry plots using an anti-CD33 antibody show CD33 expression by K-562 cells before (top graph) and after (bottom graph) delivery of the Cas9 vector and guide RNA. As shown in Figure 5, 98% of the cells lacked CD33 expression after transfection. This example demonstrates the efficient depletion of CD33 using the CRISPR-Cas9 system in human leukemia cells.

[0214] Example 2: In vitro depletion of CD45 in human leukemia cell lines We used the CRISPR-Cas9 system to target CD45RA in vitro. Briefly, TIB-67 reticulum cell sarcoma mouse macrophage-like cells were co-transfected with Cas9-GFP (PX458, Streptococcus pyogenes) and a CRISPR gRNA (containing the "NGG" PAM sequence) targeting the hCD45RA genomic sequence using Neon™ Reagent (Termo Fisher Scientific). Forty-eight hours after transfection, cells expressing the CRISPR-Cas9 system were identified and isolated using FACS sorting for GFP. The cells were then incubated for 96 hours and tested for CD45RA expression (Figure 6). Flow cytometry plots using a CD45RA antibody show CD45RA expression before (top graph) and after (bottom graph) delivery of the Cas9 vector and guide RNA. Similar to Example 1, in which CD33 expression was successfully reduced in leukemia cells, the results of this example demonstrate efficient targeting of CD45RA using the CRISPR-Cas9 system.

[0215] Example 3: Targeting cell surface lineage-specific CD33 in acute myeloid leukemia (AML) This example involves targeting the CD33 antigen in AML. The specific steps of this example are outlined in Table 6. [Table 6]

[0216] I. CD33-targeted chimeric antigen receptor (CAR) T cell therapy A. Generation of anti-CD33 CAR constructs The CD33-targeting chimeric antigen receptor described herein can consist of, in 5' to 3' order, the pHIV-Zsgreen lentiviral backbone (www.addgene.org / 18121 / ), a peptide signal, a CD33 scFv, a hinge, the transmembrane domain of the CD28 molecule, the intracellular domain of CD28, and the signaling domain of the TCR-ζ molecule. First, the peptide signal, anti-CD33 light chain (SEQ ID NO: 1), flexible linker and anti-CD33 heavy chain (SEQ ID NO: 2) are cloned into the EcoRI site of pHIV-Zsgreen along with an optional Kozak sequence. The nucleic acid structure of an exemplary chimeric receptor that binds CD33, having the basic structure light chain-linker-heavy chain-hinge-CD28 / ICOS-CD3ζ, is shown below.

[0217] Part 1: Light chain-linker-heavy chain (SEQ ID NO: 16): The Kozak initiation site is shown in bold. The peptide signal L1 is shown in italics. The anti-CD33 light and heavy chains are shown in bold italics and are separated by a linker. [ka]

[0218] Part 2: Hinge-CD28 / ICOS-CD3ζ The NotI restriction enzyme recognition site is shown in uppercase. The translation stop site is shown in bold. The BamHI restriction cleavage site is underlined. CD28 costimulatory region (SEQ ID NO: 17) [ka] ICOS costimulatory region (SEQ ID NO: 18) [ka] Fusion (hybrid) CD28 and ICOS costimulatory domain (SEQ ID NO: 19) [ka]

[0219] In the next step, the hinge region, CD28 region (SEQ ID NO: 3), and cytoplasmic components of TCR-ζ are cloned into the NotI and BamHI sites of pHIV-Zsgreen (which already contains the peptide signal and CD33 scFv). Alternatively, the CD28 region may be replaced with the ICOS region (SEQ ID NO: 4).

[0220] In addition to the CD28 and ICOS regions, a fusion region containing fragments of the CD28 and ICOS intracellular signaling regions is engineered (SEQ ID NO: 5) and used to generate additional chimeric receptors. In this configuration, the chimeric receptor contains an antigen-binding fragment, an anti-CD33 light chain variable region, a linker, an anti-CD33 heavy chain variable region, a CD28 / ICOS hybrid region (including the TM region of CD28), and the signaling region of the TCR-ζ molecule.

[0221] Exemplary amino acid sequences of components that can be used to create chimeric receptors are provided herein, such as the CD28 region (SEQ ID NO: 6), the ICOS region (SEQ ID NO: 7), the CD28 / ICOS hybrid region (SEQ ID NO: 8), and TCR-ζ. Alternatively, chimeric receptors can also be created (Section B).

[0222] B. Alternative Methods for Generation of Anti-CD33 CAR Constructs A schematic of an exemplary chimeric receptor is shown in Figure 7, panels A-D. The chimeric receptor is created using an extracellular humanized scFv that recognizes the CD33 antigen linked to the extracellular CD8 hinge region, transmembrane and cytoplasmic signaling regions, and the CD3ζ signaling chain (Figure 7, panel B). DNA encoding the anti-CD33 chimeric receptor is generated by using a humanized scFv (Essand et al., J Intern Med. (2013) 273(2):166). Alternatives include CAR T cells containing OX-1 or 41-BB instead of CD28 or CD28 / OX1 or CD28 / 4-1-BB hybrids (Figure 7, panels C and D).

[0223] To generate the anti-CD33 scFv sequence, the coding regions of the heavy and light chains of the variable region of the aforementioned anti-CD33 antibody (SEQ ID NOs: 1 and 2) are amplified with specific primers and cloned into the pHIV-Zsgreen vector for cellular expression. To evaluate the binding strength of the scFv (single-chain variable fragment) to the target antigen, the scFv is expressed in Hek293T cells. For this purpose, the vector (pHIV-Zsgreen containing the coding region) is transformed into E. coli Top10F to prepare a plasmid. The resulting expression vector encoding the scFv antibody is transfected into Hek293T cells. After culturing the transfected cells for 5 days, the supernatant is removed and the antibody is purified.

[0224] The resulting antibodies can be humanized using framework replacements by protocols known in the art, see, for example, the protocol provided by BioAtla (San Diego), in which a library of synthetic CDR-encoding fragments derived from a template antibody is ligated to fragments encoding human framework regions from a pool of human frameworks restricted to germline sequences from functionally expressed antibodies (bioatla.com / applications / express-humanization / ).

[0225] To improve antigen binding affinity, affinity maturation can be performed. This can be performed using common techniques known in the art, such as phage display (Schier R., J. Mol. Biol (1996), 263:551). Variants can be screened for their biological activity (e.g., binding affinity) using, for example, Biacore analysis. To identify hypervariable region residues that may be good candidates for modification, alanine systematic mutagenesis can be performed to identify hypervariable region residues that are significantly involved in antigen binding. Furthermore, the above-mentioned combinatorial library can be used to improve antibody affinity (Rajpal et al., PNAS (2005) 102(24):8466). Alternatively, BioAtla has developed a platform for rapid and efficient affinity maturation of antibodies, which can also be used for antibody optimization (bioatla.com / applications / functional-maturation / ).

[0226] (C) Assembly of the CAR construct Next, the anti-CD33 scFv is linked to the extracellular CD8 hinge region, transmembrane and cytoplasmic CD28 signaling regions, and the CD3ζ signaling chain. Briefly, primers specific to the anti-CD33 scFv sequence are used to amplify the scFv as previously described. The CD8 hinge and transmembrane regions (amino acids 135–205) are amplified using a plasmid (pUN1-CD8) (www.invivogen.com / puno-cd8a) containing the complete human CD8 coding sequence. The CD3ζ fragment is amplified from the Invivogen plasmid pORF9-hCD247a (http: / / www.invivogen.com / PDF / pORF9-hCD247a_10E26v06.pdf), which contains the complete human CD3ζ coding sequence. Finally, CD28 (amino acids 153–220, corresponding to the TM and signaling domains of CD28) was amplified from cDNA generated using RNA collected by Trizol from activated T cells. A fragment containing anti-CD33-scFv-CD8-hinge+TM-CD28-CD3ζ was assembled using splice overlap extension (SOE) PCR. The resulting PCR fragment was then cloned into the pELPS lentiviral vector. pELPS is a derivative of the third-generation lentiviral vector pRRL-SIN-CMV-eGFP-WPRE, in which the CMV promoter was replaced with the EF-1α promoter and the HIV central polypurine tract was inserted 5′ of the promoter (Milone et al., Mol Ther. (2009)(8):1453, Porter et al., NEJM (2011)(8):725). All constructs were verified by sequencing.

[0227] Alternatively, CARs containing ICOS, CD27, 41BB, or OX-40 signaling regions in place of the CD28 region are generated, introduced into T cells, and tested for their ability to eliminate CD33-positive cells (Figure 7, Panel C). The generation of "third-generation" chimeric receptors is also being considered (Figure 7, Panel D), which combine multiple signaling regions, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance potency (Sadelain et al., Cancer Discov. (2013) 4:388).

[0228] (D) Anti-CD33 CAR T cell preparation Primary human CD8 + T cells were isolated from patients' peripheral blood by immunomagnetic separation (Miltenyi Biotec). T cells were cultured in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 U / mL penicillin, 100 μg / mL streptomycin sulfate, and 10 mM HEPES) and stimulated with anti-CD3 and anti-CD28 mAb-coated beads (Invitrogen) as previously described (Levine et al., J. Immunol. (1997) 159(12):5921).

[0229] A packaging cell line is used to generate viral vectors that can transduce target cells and contain an anti-CD33 chimeric receptor. To generate lentiviral particles, the CAR generated in section (1) of this example is co-transfected into immortalized normal fetal kidney 293T packaging cells. The cells are cultured in high-glucose DMEM containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. 48-72 hours after transfection, the supernatant is collected, and the recombinant lentivirus is concentrated in DMEM without FBS. Next, primary CD8 +T cells are transduced at a multiplicity of infection (MOI) of approximately 5-10 in the presence of polybrene. Human recombinant IL-2 (R&D Systems) is added (50 IU / mL) every other day. T cells are cultured for approximately 14 days after stimulation. Transduction efficiency of human primary T cells is assessed by expression of the ZsGreen reporter gene (Clontech, Mountain View, CA).

[0230] E. Infusion of CAR T cells into patients Prior to intravenous infusion of anti-CD33 CAR T cells into patients, the cells are washed and concentrated in phosphate-buffered saline. A cell processor such as the Haemonetics CellSaver (Haemonetics Corporation, Braintree, MA), which provides a closed, sterile system, is used for the washing and concentration steps before compounding. The final T cells expressing the anti-CD33 chimeric receptor are compounded in 100 mL of sterile saline supplemented with human serum albumin. Finally, patients receive 1-10x10 T cells over 1-3 days. 7 The number of T cells expressing anti-CD33 chimeric receptors injected depends on many factors, including the patient's condition, age, and previous treatments.

[0231] Furthermore, immune cells expressing chimeric receptors that target CD45RA in addition to chimeric receptors that target CD33 in AML patients are also contemplated herein. This can be achieved in two different ways: 1) separately generating immune cells that express anti-CD33 chimeric receptors and immune cells that express anti-CD47RA chimeric receptors, and injecting both types of immune cells into the patient separately, or 2) generating immune cells that simultaneously target both CD33 and CD45RA (Kakarla et al., Cancer (2): 151 (2014)).

[0232] II.CD34 + CD33 - Autologous hematopoietic stem cell transplantation (HSCT) using cells It is understood that protocols for isolating stem cells from a patient, conditioning treatment, and injecting the stem cells into the patient vary greatly depending on the patient's age, condition, and medical history, and the facility where the treatment is performed. Therefore, the protocols described below are merely examples and are routinely optimized by those skilled in the art.

[0233] A. Isolation of hematopoietic stem cells using peripheral blood stem cell (PBSC) mobilization followed by adoptive transfer of anti-CD33 CAR T cells AML patients are stimulated with granulocyte colony-stimulating factor (G-CSF) at 10 mg / kg per day intravenously. + Cell positive selection is performed using immunomagnetic beads and an immunomagnetic enrichment device. + 2x10 of cells 6 A minimum of 100 cells / kg body weight is expected to be collected using a Fenwall CS 3000+ cell separator (Park et al., Bone Marrow Transplantation (2003) 32:889).

[0234] B. Patient Conditioning Conditioning for autologous peripheral blood stem cell transplantation (PBSCT) is performed using etoposide (VP-16) + cyclophosphamide (CY) + total body irradiation (TBI). Briefly, the dosing regimen is 1.8 g / m over 26 hours as a single dose. 2 It consisted of etoposide (VP-16) by continuous intravenous infusion (civ), followed by cyclophosphamide (CY) intravenously at 60 mg / kg body weight per day over 2 hours for 3 days, followed by total body irradiation (TBI) at 300 cGy per day for the next 3 days.

[0235] To calculate the dosage, use the lesser of ideal body weight or actual body weight. As previously mentioned, factors such as the patient's condition, age, and prior treatment, as well as the type of facility where the treatment will be performed, are all considered when determining the correct conditioning regimen.

[0236] C. Plasmid construction of the CD33-targeting CRISPR-Cas9 system LentiCRISPRv2 containing insert Cas9 and puromycin resistance was obtained from Addgene (plasmid #52961) (Sanjana et al., Nat Methods (2014) (8):783). To clone the single guide RNA (sgRNA) CD33 guide sequence, lentiCRISPRv2 was cleaved and dephosphorylated with FastDigest BsmBI and FastAP (Fermentas) at 37°C for 2 hours. A gRNA targeting CD33 was designed using the online optimized design tool at crispr.mit.edu. Alternatively, the gRNA had the sequence shown in Figure 12 (SEQ ID NO: 11). CD33 gRNA oligonucleotides were obtained from Integrated DNA Technologies (IDT) and phosphorylated with polynucleotide kinase (Fermentas) at 37°C for 30 minutes, followed by annealing at 95°C for 5 minutes and cooling to 25°C at 1.5°C / min. The oligonucleotides are annealed using T7 ligase, and then the annealed oligonucleotides are ligated into a gel-purified vector (Qiagen) for 5 minutes at 25° C. The resulting plasmid can then be amplified using an endotoxin-free midi-prep kit (Qiagen) (Sanjana et al., Nat Methods (2014) (8): 783).

[0237] Alternatively, a two-vector system can be used (where the gRNA and Cas are expressed from separate vectors) according to a previously reported protocol (Mandal et al., Cell Stem Cell (2014) 15(5):643), where Mandal et al. achieved efficient gene ablation in human hematopoietic stem cells using a CRISPR-Cas system expressed from a non-viral vector.

[0238] Briefly, a human codon-optimized Cas9 gene containing a C-terminal SV40 nuclear localization signal is cloned into a CAG expression plasmid containing 2A-GFP. To direct Cas9 to cleave the CD33 sequence of interest, a guide RNA (gRNA (SEQ ID NO: 11)) is separately expressed from a plasmid containing a human U6 polymerase III promoter. gRNA sequence oligonucleotides are obtained from Integrated DNA Technologies (IDT), annealed, and introduced into the plasmid using the BbsI restriction site. Due to the requirement for a "G" base for transcription initiation in the human U6 promoter, as well as the requirement for a PAM (protospacer adjacent motif), the genomic target is GN. 20 It contains the GG nucleotide sequence.

[0239] In addition to infusing patients with CD33-depleted hematopoietic stem cells (HSCs), a protocol will be developed in which patients are subsequently infused with CD45RA-depleted HSCs. Alternatively, the inventors will use the CRISPR-Cas9 system to inject CD34 + CD33 - CD45RA - Cells are generated to simultaneously reduce both the CD33 and CD45RA genes. Example guide RNA sequences for CD45RA and CD33 are shown in Tables 4 and 5.

[0240] D.CD34 + CD33 - CD34 for generating cells + Gene transduction of HSC cells Freshly isolated peripheral blood-derived CD34 + Cells (from step 4) were cultured at 1x10 cells per well in serum-free CellGro SCGM medium in the presence of cell culture grade stem cell factor (SCF) 300ng / ml, FLT3-L 300ng / ml, thrombopoietin (TPO) 100ng / ml, and IL-360ng / ml. 6 24 hours after pre-stimulation, CD34 +HSCs were transduced with lenti-CRISPRv2 containing Cas9 and CD33 gRNA using the Amaxa Human CD34 cell Nucleofector kit (U-008) (#VPA-1003) (Mandal et al., Cell Stem Cell (2014) 15(5):643). 24-48 hours after transfection, CD34 + CD33 - After puromycin selection, CD34 + CD33 - Cells are maintained in puromycin-free medium for 2-3 days.

[0241] E. CD34 in patients + CD33 - Re-injection of cells CD34 transduced ex vivo by CRISPR-Cas9-CD33 + cells (CD34 + CD33 - The cells are immediately reinfused through the Hickman catheter using a standard blood administration set without a filter (Hacein-Bey Abina et al., JAMA (2015) 313(15):1550).

[0242] Typically, patients undergoing the treatment protocols outlined above are monitored for the reappearance of circulating blasts and cytopenias. Furthermore, depending on the underlying mechanism of AML in a particular patient, success of treatment is monitored by examining the reappearance of informative molecular or cytogenetic markers or informative flow cytometry patterns. For example, the reappearance of BCR-ABL signals in Philadelphia chromosome-positive AML is detected using fluorescence in situ hybridization (FISH) with probes for BCR (on chromosome 22) and ABL (on chromosome 9).

[0243] To evaluate the success of CD33 depletion via the CRISPR-Cas9 system, peripheral blood CD34 +Cells are isolated from the patient (post-transplant) and assessed for CD33 expression using, for example, flow cytometry, Western blotting, or immunohistochemistry.

[0244] As described herein, the HSCT reported in this example can be either autologous or allogeneic; both approaches are suitable and may be incorporated into the methods described in this disclosure.

[0245] III. Optional Step: Continued Treatment of the Patient with a CD33 Antibody Conjugated to a Toxin A. Treatment of patients with the CD33 immunotoxin gemtuzumab ozogamicin (GO) Patients were treated with 9 mg / m as intravenous infusion in two doses, 2 weeks apart. 2 The patients were treated with the anti-CD33 antibody gemtuzumab ozogamicin (GO) (Larson et al., Cancer (2005), 104(7):1442-52). GO consists of a humanized monoclonal antibody against CD33 conjugated to the cytostatic agent calicheamicin (Figure 8).

[0246] Alternatively, anti-CD33 antibodies can be conjugated to various toxins, such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A (PE), or ricin toxin A chain (RTA) (Wayne et al., Blood (2014) 123(16):2470). Similarly, anti-CD45RA antibodies can be conjugated to toxins and included in therapeutic regimens.

[0247] Example 4: T cell and NK cell lines expressing anti-CD33 chimeric receptors induce cell death of CD33-expressing target cells Binding of chimeric receptors to CD33 Chimeric receptors that bind CD33 (e.g., CART1, CART2, CART3) were generated using conventional recombinant DNA technology and inserted into the pHIV-Zsgreen vector (Addgene; Cambridge, MA). The vectors containing the chimeric receptors were used to generate lentiviral particles, which were used to transduce different cell types, such as T cell lines (e.g., 293T cells) and NK cell lines (e.g., NK92 cells). Expression of the chimeric receptors was detected by Western blotting (Figure 9, panel A) and flow cytometry (Figure 9, panel B).

[0248] Cells expressing the chimeric receptors were selected by fluorescence-activated cell sorting (FACS) and assessed for their ability to bind CD33. Briefly, lysates of 293T cells expressing the chimeric receptors were co-incubated with CD33 or CD33-allophycocyanin (APC) complexes. Samples were subjected to protein electrophoresis and stained with Ponceau protein stain (Figure 10, panel A) or transferred to membranes and probed with anti-CD3ζ primary antibodies (Figure 10, panel B). In both cases, binding occurs between the chimeric receptors and their target, CD33.

[0249] K562 cells expressing the chimeric receptors were also assessed for binding to CD33 by flow cytometry using CD33 as a probe (Figure 10, panel C). The number of cells positive for expression of the chimeric receptor (CART1, CART2, or CART3) and CD33 binding was increased compared to cells containing the empty vector control, indicating that the chimeric receptors bind to CD33.

[0250] Cytotoxicity induced by cells expressing chimeric receptors NK-92 cells expressing the chimeric receptor were functionally characterized for their ability to induce cytotoxicity of target cells displaying CD33 on their cell surface (e.g., K562 cells, a human chronic myeloid leukemia line that is CD33+). To perform the cytotoxicity assay, effector cells (immune cells, such as NK-92 cells) were infected with lentiviral particles encoding the chimeric receptor and expanded. Several days after infection, cells expressing the chimeric receptor were selected by FACS analysis for a fluorescent marker (e.g., GFP+ or Red+) also encoded by the chimeric receptor-encoding vector. The selected cells expressing the chimeric receptor were expanded for one week. 14 days after infection, a cytotoxicity assay was performed, which involved staining target cells (cells expressing the target cell surface lineage-specific antigen, CD33) with carboxyfluorescein succinimidyl ester (CFSE) and counting both the target cells and the cells expressing the chimeric receptor. Various ratios of target cells and cells expressing the chimeric receptor were co-incubated in round-bottom 96-well plates for 4.5 hours, after which 7-aminoactinomycin D (7-AAD) was added to stain nonviable cells. Flow cytometry was performed to enumerate viable and nonviable target cell populations. As shown in Figure 11, panels A and B, NK92 cells expressing the chimeric receptors CART1, CART2, or CART3 induced significant cell death in target K562 cells at each of the cell ratios evaluated.

[0251] To determine whether K562 cell death depended on specific targeting of the chimeric receptor to CD33, K562 cells were genetically engineered to lack CD33 using the CRISPR / Cas system. Briefly, human codon-optimized Cas9 endonuclease and gRNA targeting a portion of the IgC region of CD33 were expressed in K562 cells to generate a CD33-deficient K562 cell population. The cells were expanded and co-incubated with NK92 cells expressing the chimeric receptor, and cytotoxicity assays were performed as described above. As shown in Figure 12, panel A, pooled CD33-deficient K562 cells showed a slight reduction in cell death upon co-incubation with NK92 cells expressing the chimeric receptor. However, when a single clone of CD33-deficient K562 cells was isolated, expanded, and used to perform cytotoxicity assays, a more significant reduction in cytotoxicity was observed (Figure 12, panel B).

[0252] Expression of chimeric receptors on primary T cells Primary T cell populations were isolated from PMBCs obtained from donors by FACS by positively selecting for CD4+, CD8+, or CD4+ / CD8+ cells, resulting in highly pure populations (Figure 13, panels A and B). + Each of the CD4+ T cell populations (CD8+ or CD4+ / CD8+ cells) was transduced with a lentiviral vector containing a chimeric receptor (e.g., CART1 or CART8), and primary T cells expressing the resulting chimeric receptor were used to perform a cytotoxicity assay as described above. Co-incubation of the chimeric receptor-expressing CD4+ T cell population with K562 cells (1000 target K562 cells) did not result in cytotoxicity of the K562 cells (Figure 14, panel A). In contrast, in a cytotoxicity assay using either CD8+ or CD4+ / CD8+ cells expressing the chimeric receptor and 1000 target K562 cells, the CD8+ or CD4+ / CD8+ cells were able to induce cell death of K562 cells at a low percentage (Figure 14, panel B).

[0253] Genetic manipulation of human hematopoietic stem cells Several gRNAs were designed to hybridize to the IgC region of CD33 (see, e.g., Table 4, SEQ ID NOs: 11 or 28-31). Each of the gRNAs was expressed in K562 cells along with Cas9 endonuclease. CD33 expression was assessed by flow cytometry (Figure 15). As shown for Crispr3 (SEQ ID NO: 28) and Crispr5 (SEQ ID NO: 29), a significant reduction in CD33 was observed in cells expressing the CD33-targeting CRISPR / Cas system compared to control cells expressing CD33.

[0254] We also evaluated various characteristics of CD33-deficient hematopoietic stem cells, including proliferation, erythropoietic differentiation, and colony formation. Briefly, CD33-deficient hematopoietic stem cells and control cells were induced to differentiate by exposing them to hemin, and expression of CD71, a marker for erythroid progenitors, was assessed by flow cytometry at various time points (Figure 16, panels A and B). CD33-deficient hematopoietic stem cells underwent erythropoietic differentiation, and their flow cytometry profile appeared similar to that of control cells (CD33+). Cells were also subjected to an MTT assay to measure the metabolic activity of CD33-deficient hematopoietic stem cells. As shown in panel C of Figure 16, CD33-deficient hematopoietic stem cells performed comparably to control cells. Finally, the ability of the cells to proliferate and form cell colonies was observed by microscopic colony formation assay. Again, CD33-deficient hematopoietic stem cells were able to form colonies to a similar extent as control cells (Figure 18). These results indicate that partial CRISPR / Cas ablation of CD33 does not significantly affect the cells' ability to proliferate, differentiate, or form colonies.

[0255] Example 5: Gene-edited stem cells enable CD33-directed immunotherapy for myeloid malignancies. Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-T) or antibody-drug conjugates (ADC), are associated with severe toxicity due to the lack of unique targetable antigens that can distinguish leukemia cells from normal myeloid cells or myeloid progenitor cells. Here, a novel method for treating AML by targeting the lineage-specific myeloid antigen CD33 is provided. This method combines CD33-targeted CAR-T cells and / or the ADC, gemtuzumab ozogamicin, with transplantation of hematopoietic stem cells (HSCs) genetically engineered to eliminate CD33 expression using genome engineering techniques. Highly effective genetic ablation of the CD33 antigen using CRISPR / Cas9 technology has been demonstrated in human stem / progenitor cells (HSPCs), providing evidence that deletion of CD33 in HSPCs does not impair their engraftment capacity or their ability to repopulate the multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA-seq analysis revealed no detectable off-target mutagenesis or functional p53 pathway degradation. Using a human AML cell line (HL-60), we modeled post-remission bone marrow with minimal residual disease and demonstrated that transplantation of CD33-depleted HSPCs with CD33-targeted immunotherapy led to leukemia elimination without myelosuppression, as demonstrated by the engraftment and recovery of multilineage progeny of CD33-depleted HSPCs. Therefore, this study contributes to the advancement of targeted immunotherapy and may be easily replicated in other malignancies.

[0256] Acute myeloid leukemia (AML) represents an unmet need for effective treatments, particularly for post-remission patients. Immunotherapies directed against lineage-specific antigens (LASs), such as CD33, have demonstrated on-target efficacy but are limited by toxicity because normal bone marrow cells and hematopoietic progenitor cells also express CD33. Here, we demonstrate that genetic ablation of CD33 in HSPCs using CRISPR technology enables immunotherapy of leukemia using anti-CD33 CAR-T or antibody therapy. Post-remission human bone marrow with minimal leukemia was modeled in mice, demonstrating effective ablation of AML and reconstitution of CD33-deficient human grafts. This study provides a novel approach to treat myeloid leukemia and may be extendable to other cancers and antigens.

[0257] Materials and Methods Cell lines and primary human cells The immortalized human acute myeloid cell line, HL-60, was obtained from ATCC and cultured in IMDMEM containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin. To track leukemia engraftment and progression over time, HL-60 cells were transduced with lentiviral particles expressing dTomato fluorescent protein under the EF1α promoter. Lentiviral vectors and particles were produced by Vectalys (Toulouse, France). Human bone marrow or umbilical cord blood CD34 + Stem cells were purchased from StemExpress (Folsom, CA, USA) and maintained in StemSpan SFEM II (STEMCELL Technologies Inc.) containing 1% penicillin-streptomycin, 100 ng / mL TPO, 100 ng / mL SCF, 100 ng / mL IL6, and 100 ng / mL FLT3L, and 0.35 nM UM171 (Xcessbio, San Diego, CA, USA). All human cytokines were purchased from BioLegend (San Diego, CA, USA). Human T cells were purified from fresh peripheral blood normal donor leukopaks purchased from the New York Blood Center. Briefly, leukopaks were diluted with 2-4 volumes of phosphate-buffered saline (1X) supplemented with 2 mM EDTA and stored at 4°C. 35 mL of diluted leukopaks were carefully layered onto 15 mL of Ficoll-Paque™ Premium (GE) and centrifuged at 400 g for 30 minutes at 25°C in a swinging bucket rotor. The mononuclear cell layer was then transferred to a new tube, diluted 1:1 with 2 mM EDTA in PBS (1X), and centrifuged at 400 g for 15 minutes at 25°C. The red blood cell pellet was then removed with 1X ACK lysis buffer (Gibco), incubated at room temperature for 5-8 minutes, washed with 2 mM EDTA in PBS (1X), and centrifuged again at 400 g for 10 minutes at 25°C. CD4 + and CD8 +T cells were then transfected into Miltenyi Biotec CD4 + and CD8 + Positive selection from mononuclear cell pellets with microbeads. CD4 + and CD8 + T cells were then activated on the same day using CD3 / CD28 Dynabeads (1:1 bead:cell ratio) (Gibco) and expanded separately in Gibco OpTmizer™ CTS™ T-Cell Expansion SFM medium containing 10 ng / mL IL7 and 5 ng / mL IL15.

[0258] CAR constructs, lentiviral production, and transduction An anti-CD3 chimeric antigen receptor was generated by cloning the light and heavy chains of a human anti-human CD33 scFv (clone My96) fused in-frame to the CD8 alpha hinge region, CD8 transmembrane domain, 4-1BB signaling domain, and CD3 zeta intracellular domain into the lentiviral plasmid pHIV-Zsgreen (a gift from Bryan Welm & Zena Werb (Addgene plasmid # 18121) (Welm et al. Cell Stem Cell. 2008;2(1):90-102)). All cDNA fragments were codon-optimized and synthesized by GeneArt (Regensburg, Germany). Lentiviral particles were produced by Vectalys (Toulouse, France). 24 hours after activation, CD4 + T cells were transduced with lentiviral particles at an MOI of 30 and in parallel with CD8 + T cells were transduced with lentiviral particles at an MOI of 40.

[0259] CRISPR / Cas9-mediated CD33 genomic targeting Human bone marrow or umbilical cord blood CD34 +Stem cells were maintained in StemSpan SFEM II (STEMCELL Technologies Inc.) containing 1% penicillin-streptomycin and the following human cytokines: 100 ng / mL TPO, 100 ng / mL SCF, 100 ng / mL IL6, and 100 ng / mL FLT3L, and 0.35 nM UM171 (Xcessbio, San Diego, CA, USA). All human cytokines were purchased from BioLegend (San Diego, CA, USA).

[0260] TrueCut Cas9 Protein V2 was purchased from Invitrogen. Chemically modified CD33-targeting sgRNA was designed using the Synthego CRISPR Gene KO design tool and purchased from Synthego. 3 μg of TrueCut Cas9 Protein A and 200,000 CD34 + 1.5 μg of sgRNA for cells was mixed in P3 buffer (Lonza, Amaxa P3 Primary Cell 4D-Nucleofector Kit) and incubated at 37°C for 10 minutes. Cells were then washed with PBS, resuspended in P3 buffer, mixed with the Cas9 / sgRNA RNP complex, and electroporated with the 4D-Nucleofector. After electroporation, cells were cultured at 37°C until analysis. Deletion efficiency was assessed 7 days after electroporation using the following antibodies from BioLegend: hCD34-PerCp / Cy5.5 and hCD33-FITC.

[0261] Whole genome and RNA sequencing After electroporation with Cas9 alone or the RNP complex Cas9 / sgRNA, CD34 +Cells were maintained in vitro for 10 days, and their DNA or RNA was isolated as follows: DNA was purified with the QIAAmp DNA Mini Kit according to the manufacturer's protocol, then eluted in 30 μl, and DNA concentration was measured using a NanoDrop and QuBit dsDNA BR assay. RNA was purified with the miRNeasy Micro Kit according to the manufacturer's protocol, then eluted in 18 μl. NanoDrop and Bioanalyzer Pico chip assays were performed to measure concentration and quality.

[0262] For whole-genome sequencing, the NEBNext® Ultra™ II DNA Library Prep Kit was used with Illumina, clustering, and sequencing reagents. Briefly, genomic DNA was fragmented by acoustic shearing, purification, and end repair. Adapters were ligated, and DNA libraries were generated. The DNA libraries were also quantified by real-time PCR (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions, clustered on two lanes of a flow cell, and loaded onto an Illumina Hiseq instrument. Samples were sequenced using a 2x150 paired-end (PE) configuration. Image analysis and base calling were performed using the HiSeq Control software (HCS) on the HiSeq instrument. DNA sequences were processed with Illumina HiSeq Analysis Software v2.1 (HAS 2.1) using default parameters.

[0263] For RNA sequencing, cDNA synthesis and amplification were performed using the SMART-Seq v4 Ultra Low Input Kit for Sequencing (Clontech, Mountain View, CA). Sequencing libraries were generated using a Nextera XT (Illumina). Samples were sequenced using a 2x150 paired-end (PE) configuration. After examining the quality of the raw data, trimmed reads were mapped to the Homo sapiens reference genome available on ENSEMBL using STAR Aligner v.2.5.2b. A BAM file was generated as a result of this step. Unique gene hit counts were calculated using feature counts from the Subread package v.1.5.2. Only unique reads falling within exon regions were counted. After extraction of gene hit counts, the gene hit count table was used for downstream differential expression analysis using the edgeR package in the SARTool package (Varet et al. PLoS One. 2016;11(6):e0157022). Genes were considered significantly differentially expressed if the p-value was >0.05.

[0264] Flow cytometry and sorting In vitro After transduction, CAR-T cells were expanded for up to 15 days and then analyzed for GFP using a BioRad S3e sorter. + (Dead cells were excluded using propidium iodide) and mixed 1:1 for in vitro and in vivo experiments. CAR expression and their ability to recognize and bind CD33 were assessed by incubating CAR-T cells with biotinylated human CD33 protein (ACRO biosystems) for 20 min at 4°C, followed by staining with fluorescent dye-labeled streptavidin. CD34 + Stem cells were analyzed 5 to 7 days after electroporation using hCD34-PerCp / Cy5.5 and hCD33-FITC antibodies.

[0265] In vivo Engraftment and repopulation of the hematopoietic system over time was assessed by analysis of peripheral blood, bone marrow aspirates, and whole bone marrow (from anesthetized mice) using the following resulting antibodies from BioLegend (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA): Ter119-PeCy5, Ly5-BV711, H2kd-BV711, hCD45-BV510, hCD3-PacificBlue, hCD123-BV605, hCD33-APC, hCD14-APC / Cy7, hCD10-BUV395, hCD19-BV650, CD34-BV421, CD90-PeCy7, hCD38-BUV661, and hCD45RA-BUV737. CAR-T cells stably expressed the fluorescent protein zsGreen, leukemia cells stably expressed dTomato, and dead cells were removed using propidium iodide. - dtomato + Gating was performed using CD34. + Injection-derived human cells were cloned into Ter119 - dtomato - , Ly5 - / H2kd - Human CD45 + CART - All data were acquired using a BioRad ZE5 flow cytometry analyzer in high-throughput mode, and analysis was performed using FlowJo 10.4.2. Leukemia progression was also assessed by fluorescence imaging using a PerkinElmer IVIS Spectrum optical imaging system. Images were acquired and analyzed using Living Image 4.4 Optical Imaging Analysis Software.

[0266] In vitro cytotoxicity assay Effector-selected CAR-T cells stably expressing zsGreen were incubated with the following target cells: HL-60 cells stably expressing dTomato and / or CD34 cells stained with Celltrace blue. + CD33 WTCells and / or CD34 stained with Celltrace Violet (Invitrogen) + CD33 Del CART33 cells were mixed with CFSE-positive T cells at various ratios. After 16-24 hours of incubation, data were acquired using a BioRad ZE5 flow cytometry analyzer in high-throughput mode to assess cytotoxicity using 7-AAD or Sytox Red as a cell viability assay. After subtracting spontaneous lysis in the negative control, the specific cytotoxicity (%) of CART33 cells was calculated as cells positive for both CFSE and 7-AAD or Sytox Red using the following formula: ((% positive cells with CART33 T cells) - (% positive cells with control T cells)) / (100 - (% positive cells with control T cells) x 100).

[0267] In vivo experiments NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav / MloySzJ(NSG-SGM3) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were conditioned with a sublethal dose (1.2 Gy) of total body irradiation (TBI). + CD33 Del Bone marrow or umbilical cord blood stem cells (5 * 10 5 ~1 * 10 6 5 pieces) * 10 5 Mice were intravenously injected with dTomato-HL-60 cells within 8–24 hours of TBI. One to two weeks later, mice were treated with 2–3 i.v. * 10 6 They were treated with 100 μg of anti-CD3 or control CAR-T cells (premixed CD4:CD8 = 1:1) or 6 μg of GO (gemtuzumab ozogamicin) or PBS.

[0268] Hematopoietic engraftment and repopulation over time was assessed by analysis of peripheral blood, bone marrow aspirates, and whole bone marrow (from anesthetized mice) using the following resulting antibodies from BioLegend (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA): Ter119-PeCy5, Ly5-BV711, H2kd-BV711, hCD45-BV510, hCD3-PacificBlue, hCD123-BV605, hCD33-APC, hCD14-APC / Cy7, hCD10-BUV395, hCD19-BV650, CD34-BV421, CD90-PeCy7, hCD38-BUV661, and hCD45RA-BUV737. Dead cells were removed using propidium iodide. + Injection-derived human cells were cloned into Ter119 - , Ly5 - / H2kd - Human CD45 + Gated with.

[0269] All experiments were conducted under protocols approved by the Columbia University Animal Care and Use Committee. statistics All statistical analyses were performed using GraphPad Prism version 7. For continuous variables, unpaired two-tailed t-tests were performed. Differences between means were considered significant if p-value < 0.05, otherwise they were considered non-significant (not significant; p > 0.05).

[0270] result CRISPR / Cas9-mediated gene ablation of CD33 antigen Using CD33-expressing HL-60 cells as donor hematopoietic stem and progenitor cells (HPSCs), we isolated myeloid leukemia and primary CD34 cells either from umbilical cord blood (CB) or from adult bone marrow (BM). +Surface expression of CD33 was measured using flow cytometry on HL-60 cells and CD34 cells as previously described (Taussig et al. Blood. 2005;106(13):4086-4092; Haubner et al. Leukemia. 2018;33(1):64-74; Wisniewski et al. Blood Cancer J. 2011;1(9):e36; Krupka et al. Blood. 2014;123(3):356-365). + This was confirmed in both guinea pig and guinea pig cells (Figure 19B). Using CRISPR / Cas9 (Haubner et al. Leukemia. 2018;33(1):64-74), a recently developed general-purpose RNA-guided DNA editing technology, the CD33 genomic locus was gene-edited to eliminate its expression. Guides were designed to target exon 3 of the genomic locus (Figure 19C shows the sgRNA811 spacer sequence of SEQ ID NO:58, Figure 25A shows the spacer sequence of SEQ ID NO:29, and Figure 25B shows the sgRNA846 spacer sequence of SEQ ID NO:50), because exon 3 is common to all CD33 transcripts and bears little to no similarity to the Siglec family pseudogenes of which CD33 is a member. Plasmid, lentivirus, and ribonucleoprotein (RNP)-based delivery systems were tested to investigate the efficiency of several guides in cell lines and primary cells, and the RNP system combined with chemically modified guides was found to be the most efficient in primary cells (Figures 19B and 19D). Under optimal conditions, loss of CD33 expression was measured by flow cytometry using the anti-CD33 clone HIM34, which recognizes an epitope located in the C2 region common to all CD33 isoforms, and subsequently CD33 Del It is called CD34 +This was observed in more than 80% of HSPCs (Figures 19B and 19D). This reduction in CD33 expression was accompanied by the presence of insertions / deletions (indels) at the predicted cleavage site of Cas9 in the DNA, as determined by DNA Sanger sequencing (Figure 19E, bottom chromatogram). Two other sgRNAs, also targeting exon 3, were tested and showed similar efficiency (Figures 25A and 25B). As expected, electroporation with Cas9 alone, in the absence of sgRNA, did not induce any indels at the target site (Figure 19E, top chromatogram). CD33 Del The cells maintained high expression of CD34 and CD90 (Fig. 19D, lower panel). Consistently high deletion efficiency was observed in several independent experiments (Fig. 19F), with CD33 WT An average of 85% of cells expressed CD33, which declined to less than 10% 5–7 days after RNP electroporation. Additionally, B cells lacking CD33 expression were used as a negative control to confirm the reduction in expression after Cas9-mediated deletion. The remaining 10% of CD33 expression observed likely comes from unedited (both wild-type alleles) or partially edited (only one allele harboring an indel) cells.

[0271] CD34 + CD33 Del HSPCs exhibit engraftment and multilineage differentiation in vivo. This method utilizes CD33 gene-edited stem cells (CD33) as a platform for the delivery of CAR-T and ADC targeting the CD33 antigen (CART33). Del ) may be involved in the transplantation of CD33, which contributes to engraftment and myelopoiesis and lymphopoiesis. Del It is important to test the ability of bone marrow and cord blood-derived CD34 cells. + Both CD33 and CD43 cells were tested (Figure 20). Del HSPCs were injected via the tail vein into sublethally irradiated NSG-SGM3 mice, and bone marrow and blood were analyzed (Figure 20A). In mice injected with bone marrow-derived cells, peripheral blood analysis at 7 weeks post-transplantation revealed the presence of human CD45 + Cell and bone marrow (CD14 +cells) and lymphoid (CD19 + Analysis of 15-week bone marrow aspirates (FIG. 26A) and whole bone marrow from 21-week anesthetized mice (FIG. 20C), summarized in FIG. 26B, demonstrated the presence of human CD45 + The results demonstrated chimerism, including sustained cell contribution over time. In all tissues examined, there was multilineage engraftment, with the presence of progenitor and mature cells of both myeloid (monocyte) and lymphoid (B cell) origin. All cells remained CD33 negative (Figures 26A and 26C). Del No significant differences in multilineage engraftment of cells were observed when compared to wild-type cells.

[0272] At the same time, CD34 + Similar methods were performed using cord blood derived cells, with similar results. Multilineage engraftment was observed in peripheral blood 9 weeks after transplantation (Fig. 20D), in bone marrow aspirates 16 weeks after transplantation (Fig. 26C), and in whole bone marrow 21 weeks after transplantation (Fig. 20E). The above results suggest that CD33 is a marker of umbilical cord blood (CB) or bone marrow (BM) CD34. + This suggests that it is not essential for cell engraftment or for sustained repopulation of a fully human hematopoietic system in animal models.

[0273] CD33 Del The cells are competent for myeloid differentiation and function. Since the goal of this method is its transfer to the clinic, bone marrow CD33 Del The functional capacity of the cells was assessed in vitro and in vivo. First, myeloid lineage diversity was assessed by CD34+CD33 WT Compared to humanized mice, CD34+CD33 DelThe phagocytic capacity of monocytic CD34+CD33WT and CD34+CD33WT cells to phagocytose E. coli bioparticles was examined in vitro. No significant differences were observed. LPS-induced cytokine production by monocytes / macrophages in NSGS mice transplanted with CD34+CD33WT or CD34+CD33Del cells was also analyzed, and plasma concentrations of TNFα, IL6, and IL8 were comparable after induction. Finally, to assess the phagocytic function of CD33-deficient cells in vivo, the peritoneal cavities of humanized mice were analyzed 2 hours after intraperitoneal injection of E. coli bioparticles. Flow cytometry analysis demonstrated similar phagocytic uptake by the hCD45+hCD11b+hCD14-hCD16- subsets in both CD34+CD33WT and CD34+CD33Del humanized mice. All these findings indicate an intact function of CD33Del myeloid cells.

[0274] No detectable off-target mutagenesis or loss of functional p53 pathway was observed in the gene-edited cells. To assess whether the two guides used in this study could introduce indels at off-target sites in HSP cells, we performed indel analysis using the Cas9 protein alone (CD33 WT ) compared to cells electroporated with the Cas9 / sgRNA RNP complex (CD33 Del ) electroporated human umbilical cord blood CD34 + Whole-genome sequencing data from HSP cells was evaluated. Over 629 million pass-filter reads were obtained with over 93% of reads exceeding Q30 base quality (Figure 28). The average read depth was over 26X. Reads were aligned to the human hg38 reference genome to identify single-nucleotide variants (SNVs) and small indels.

[0275] A summary of the variants detected in both samples is shown in Figure 28. Strong on-target activity was observed in over 90% of reads aligned to the predicted cleavage sites chr19:51225811 and chr19:51225846 (Figure 21A). Importantly, all indels were located within the predicted cleavage sites of the two sgRNAs used. The few small indels observed outside the targeted region in the entire CD33 locus were consistent with the presence of cleavage sites in electroporated CD33. Del These indels were not unique to the sgRNAs used and were also present in cells electroporated with Cas9 alone. The data were then examined for indels in predicted off-target sites that showed a high degree of similarity with up to four mismatches to the sgRNA used (Figures 21B, 29, and 30). Again, no indels were found at any of the off-target loci examined (Figures 29 and 30). Indels were also examined at the TP53 locus, revealing a similarity to CD33 Del Nothing cell-specific was observed.

[0276] To assess whether the reduction in CD33 expression leads to changes in the expression of other genes, CD33-deficient (n=5) and control (n=5) CD34 clones were obtained from four different donors. + The gene expression profiles of the cells were compared. Gene expression profiles of each sample were obtained using RNA sequencing, and comparisons between groups were performed using edgeR. Equivalent gene expression profiles were observed between the two groups, with a Pearson correlation coefficient of 0.9948 (Figure 21C), and no significant differences were observed based on adjusted p-values ​​(Figure 31). Fourteen genes were found to be significantly different based on p-values, with the most significant difference being CD33 compared to the control. Del These results suggest that the expression of CD34 in vitro was significantly reduced in the IL-16 / ... +This confirms that the absence of CD33 in cells does not significantly affect downstream gene expression.Among the 13 genes that showed expression changes based on p-value, there was no enrichment in any one pathway or cellular process.Notably, gene expression profiles did not suggest that the TP53 pathway or other DNA damage pathways were activated, which may impair HSC function or gradually reduce long-term potential.Therefore, it is concluded that CD33 removal in umbilical cord blood and adult HSCs using gene editing technology described herein does not appear to impair their subsequent function.

[0277] The data were also manually inspected for indels in reads mapping to exon 3 of CD33 and all coding exons of the TP53 transcript in the RNA-sequencing data using the Integrated Genome Viewer (IGV). As expected, indels were present in over 95% of reads in CD33 exon 3 (Figure 27). A small number of reads with indels were found in TP53, but they were within repetitive sequences and were also present in the control samples, suggesting they were sequencing artifacts. Collectively, these data suggest that CRISPR / Cas9-mediated genome editing at the CD33 locus using the guides used in this study does not result in detectable off-target indels in stem cell systems.

[0278] Expression of CD33-specific CAR in T cells CARs are classified into different generations based on the number of costimulatory domains. A second-generation CAR was designed containing a single-chain variable domain of anti-CD33 (clone My96) paired with a CD28 transmembrane domain, a 4-1BB (CD137) costimulatory domain, and the CD3 zeta chain derived from the CD3 TCR as the intracellular domain (Figure 22A). The CAR cDNA was inserted into the pHIV-Zsgreen lentiviral vector under the control of the EF1-α promoter, enabling bicistronic expression with Zs-green. Peripheral blood obtained from a normal donor was fractionated to obtain PBMCs, which were transduced with vector alone or lentiviral particles carrying the CAR construct (Figure 22B). The transduction efficiency of CD4 and CD8 cells in PBMCs was not the same; higher transduction of CD4 cells compared to CD8 cells was observed; similar observations have been made in other studies (Blaeschke et al. Cancer Immunol Immunother. 2018;67(7):1053-1066). Considering the uneven transduction of CD4 and CD8 cells in PBMCs and to obtain a more clearly defined composition of CD4 and CD8 cells, purified CD4 and CD8 cells were transduced separately, sorted based on GFP expression from the downstream IRES element, and mixed in an equimolar ratio. CAR expression was confirmed by measuring the surface expression and binding of CAR to purified biotinylated CD33 protein bound to streptavidin fluorescent dye. Strong expression of CAR and its binding to CD33 molecules were observed (Figure 22B).

[0279] CAR-expressing T cells exhibit CD33-dependent cytotoxicity in vitro The cytotoxicity of CART33 cells was first evaluated against targets with varying CD33 expression. High killing of CD33 myeloid leukemia cells, HL-60, was confirmed, while CD33 cells expressing reduced levels of CD33 were also observed. WT Lower stem cell death was observed. In particular, the cytotoxicity of CART33 was observed in the CD33 Del CD34 + The absence of CD33 expression (due to Cas9 / sgRNA-mediated deletion) was not observed when cells were incubated with CD34 +This initial experiment (Figure 22C) also confirmed the correlation between the level of CART33 cytotoxicity and the level of CD33 expression on target cells, i.e., CART33 cytotoxicity is proportional to the expression level of CD33 on target cells.

[0280] A triple culture assay was then designed to assess CART33 cell killing activity when co-incubated with targets of varying CD33 expression. WT HL-60 cells and CD34 + CD33 WT When cells were co-incubated with CART33, a correlated cytotoxicity of CART33 cells was observed in both cell types (Fig. 22D), whereas CD33 WT HL-60 cells and CD34 + CD33 Del When cells were co-incubated with CART33 cells, only HL-60 cells were killed (Figure 22E). Similarly, all three cell types, CD34 + CD33 WT , CD34 + CD33 Del Co-incubation of IL-16 with IL-16 and CART33 cells (Figure 22F) resulted in the upregulation of CD33 at levels proportional to their CD33 expression. WT Furthermore, CART33 cytotoxicity was tested in another acute myeloid cell line, KG1, and similar CD33-dependent cytotoxicity of CART33 was observed in vitro.

[0281] Anti-CD33 immunotherapy demonstrates CD33-dependent leukemia elimination in a cell line-derived xenograft (CDX) mouse model. For in vivo experiments, we designed a method that represents the human therapeutic environment in the setting of minimal residual disease (Figure 23A). In this model, leukemia is first initiated by injecting 500,000 HL-60 cells into sublethally irradiated mice. Mice are simultaneously injected with 500,000 CD33 Del CD34 +Cells were injected to mimic an AML relapse model. Preliminary experiments suggested that a 1-week period was sufficient to allow homing and engraftment of AML cells, as 100% of mice became leukemic after an additional 2 weeks. This recapitulates the post-remission bone marrow, where AML cells still persist but are clinically undetectable. Leukemia and CD34 + One week after the stem cell co-injection, mice were divided into various groups and treated with the indicated substances (Fig. 23A). Leukemic burden and CD34 + Cell engraftment was monitored over time using imaging and flow cytometry (Figures 23B-23F). By week 3, high tumor burden was observed in bone marrow aspirates of PBS and control CAR-T cell-treated mice (i.e., vector-only transduced T cells lacking the CAR-T construct), and by weeks 3 and 4, all mice in these two groups had succumbed to their disease (Figure 23A). Two control T-treated mice had relatively low leukemia burden at week 3 but progressed to extremely high leukemia burden in the BM at the time of death. In contrast, over 12 weeks, CD33 WT Leukemic cells were not observed in bone marrow aspirates (Figure 23B) or on imaging at 3.5 or 8 weeks (Figures 23C-23E) from mice treated with CART33 cells, anti-CD33 ADC GO, or the combination of GO and CART33. These results suggest that CART33 and GO are potent agents against CD33-expressing leukemia in this model.

[0282] CD34 + CD33 Del HSPCs exhibit multilineage engraftment and differentiation in therapeutic models. At the same time, mice were treated with the above-mentioned treatment model using CD34 + CD33 Del Multilineage engraftment of the cells was monitored. Engraftment was confirmed by CAR-T and CD33-negative human CD45+ cells in bone marrow aspirates from all groups. + As indicated by the presence of CD34 cells (Fig. 23F), + CD33 DelThis suggests that CD33 cells can also be engrafted in this therapeutic model. Del Human CD45 + Although a decrease in the percentage of cells was observed at week 6 compared with the early time point of week 3 in the CART33-treated groups (CART33+PBS and CART33+GO), these levels recovered to their initial levels by week 9 and were maintained until the final time point of week 12. The relatively low engraftment and subsequent decline in the two mouse groups may reflect treatment-related stress (including antileukemic responses by CAR-T cells in the bone marrow), which was more pronounced in the CAR-T group and persisted longer than in the GO group. Alternatively, different CD34 + The use of a T cell donor may have induced an alloreaction, which may explain the delayed repopulation observed in mice injected with CART33 cells. Notably, this phenomenon was reversible, as engraftment levels were restored beyond 8 weeks.

[0283] The pluripotency of the transplanted cells was then investigated by analyzing myelopoiesis and lymphopoiesis (Figure 24A). CD33-negative myeloid and lymphoid progenitor cells, as well as mature myeloid and lymphoid cells, were observed at all time points analyzed (Figure 24A). Complete hematopoietic repopulation was observed over time in all treated mice, while delayed lymphoid repopulation was observed in CAR-T cell-injected mice. This may be the result of an alloresponse, as lymphoid progenitor cells are known to be more susceptible to allospecific effects.

[0284] At the same time, CD34 + CD33 WT To demonstrate the specificity of CART33 and GO for primary HSPCs, sublethally irradiated NSG-SGM3 mice were inoculated with 500,000 HL-60 cells and 500,000 CD34 cells. + CD33 WTOne week later, a group of mice was treated with CART33 cells, and BM aspirates were taken at week 3. On the same day, another group of mice was injected with GO, and their BM aspirates were analyzed four days later. As observed in the therapeutic model, complete leukemia elimination was confirmed after treatment (Fig. 24C). Furthermore, CD33 WT Complete elimination of cells was observed (Fig. 24D). WT The cells remain sensitive to GO and CART33 treatments, whereas CD33-depleted cells are insensitive.

[0285] Consideration The success of any antigen-dependent immunotherapy using agents such as CAR-T or mAbs depends on the presence of a unique antigen on the surface of cancer cells that is absent from normal or other cells in the body. Unfortunately, such antigens are rare in cancer. One possible outcome would be to generate stem / progenitor cells that are resistant to antigen-dependent immunotherapy by using genome engineering techniques to remove LSA in stem cells, thereby maximizing immunotherapy. After demonstrating that such antigen-depleted cells are functionally similar to wild-type cells, they can be used to replace diseased cells. Careful selection of lineage-specific antigens that are not required for the normal function of that lineage is crucial for this approach. Alternatively, if LSA is essential, instead of completely eliminating LSA expression, gene editing techniques can be used to modify the epitope recognized by the antigen-dependent immunotherapy agent on LSA while maintaining LSA function (termed "functionally redundant epitope switching," or FRES).

[0286] In this study, we demonstrate that combining stem cells lacking the lineage antigen, CD33, with allogeneic engineered T cells or ADCs can enable leukemia elimination and complete hematopoietic repopulation. This approach was demonstrated using acute myeloid leukemia, a disease with unmet need in the therapeutic area. Because CD33 is an LSA and targeting CD33 in AML with CAR-T or CD33 mAbs results in severe myelosuppression and lymphodepletion due to the removal of stem / progenitor cells and myeloid lineage cells, a proposed approach to treat AML is to reconstitute the hematopoietic system with cells lacking CD33. Using CRISPR-based methods, we were able to target umbilical cord blood or bone marrow CD34 + One of the cells disrupted CD33 expression in the donor stem cells, rendering them "resistant" to CAR-T cell attack.

[0287] Recently, two groups made similar observations and independently reported the method described in this study (Kim et al. Cell. 2018;173(6):1439-1453 e1419; Humbert et al. Leukemia. 2019;33(3):762-808). The present data strengthen the observations made in those studies and also provide novel insights using complementary methods. Mice were the first to develop CD33 gene-edited CD34 +Unlike the study by Kim et al., in which cells were infused to allow complete engraftment before leukemia induction and treatment, this method more closely mimics the situation of AML relapse with minimal residual disease, as leukemia cells and gene-edited stem cells are co-infused followed by CAR-T or ADC therapy. Furthermore, by rigorously selecting CD33 guide RNAs with high on-target and low off-target activity, highly efficient ablation of CD33 expression was observed in HSCs without off-target indels observed in other genes, thus enabling confidence in the safety of this method in human trials (Kim et al. Cell. 2018;173(6):1439-1453 e1419). Indeed, no indels were found within any of the genes tested, including the Siglec family of pseudogenes. Kim et al. observed off-target activity in Siglec-22P, including deletion of a 14 kb fragment, likely due to the 100% homology of the sgRNA designed for CD33 with Siglec-22P (Kim et al. Cell. 2018;173(6):1439-1453 e1419). The selection of a location within exon 3 and the lack of homology between the selected sgRNA and other genes allowed for specific ablation of CD33 alone. The absence of indels or other genomic rearrangements in the TP53 gene (as analyzed using whole-genome sequencing) and the presence of any deregulated genes related to the p53 pathway or p53 itself suggest that the method developed herein may provide efficient genome editing with high specificity without compromising HSC function. Finally, the use of Cas9 RNP (which is only transiently present in HSCs during their ex vivo manipulation), in contrast to viral-mediated expression of Cas9, which is constitutive and continuous in HSCs in vivo, avoids future problems with pre-existing immunity to Cas9, which is known to be present in over 50% of the population (Charlesworth et al. Nat Med. 2019;25(2):249-254; Crudele et al. Nat Commun 2018;9(1):3497).

[0288] Furthermore, in contrast to Kim et al., our approach involves alloBMT using CD33-edited HSCs (derived from umbilical cord blood or adult bone marrow) followed by ADC or CAR-T therapy with T cells from an allogeneic donor. This approach is more practical in clinical settings. Patients with hematological malignancies who are heavily pretreated with cytotoxic chemotherapy often have poor autologous T cell yields, limiting the efficiency and effectiveness of autologous CAR-T therapy. This issue is circumvented by using alloBMT and allogeneic T cells, in which yield and quality are not an issue. More importantly, by using ADCs (rather than CAR-Ts) to target the disease, humoral therapy can act in concert with or as a substitute for CAR-T cells, further expanding the scope of anti-leukemia therapy using humoral approaches.

[0289] It is also noteworthy that the GO drug (consisting of the anti-CD33 antibody clone P67.6) recognizes an epitope in exon 2. Two isoforms of CD33 are found in humans. The more common isoform is the full-length protein, including exon 2, which is sensitive to GO. The less common isoform lacks exon 2. Approximately 30% of the population harbors a homozygous single nucleotide polymorphism (SNP, T / T), resulting in exclusive expression of the less common CD33 variant lacking exon 2. This population could also be considered a potential pool of HSCT donors for combined targeted CD33 immunotherapy as described in this work, thereby obviating the need for Cas9-directed ablation. However, this could dramatically limit the donor pool, making this method practically impractical for human trials. In this regard, Humbert et al. (Humbert et al. Leukemia. 2019;33(3):762-808) used CRISPR / Cas9 technology to target adjacent introns with two different sgRNAs to delete exon 2. It is unclear whether selective removal of V regions offers any advantage over total CD33 disruption, as no engraftment or functional loss was observed in mice or rhesus monkeys with total CD33 ablation (Kim et al. Cell. 2018;173(6):1439-1453 e1419). Furthermore, the use of multiple guides increases potential off-targeting, and guide efficiency will likely be limited by the least efficient guide in the pool.

[0290] In this study, human CD34 + Deletion of CD33 in the BM and CB did not result in any significant side effects. More than 21 weeks after transplantation, NSG-SGM3 mice did not exhibit any abnormal phenotypes. The absence of observable CD33 deletion-associated phenotypes may be explained by functional redundancy or compensation among Siglec members.

[0291] Despite the increasing number of clinical trials involving engineered immune cells, few improved outcomes have been achieved for patients, primarily due to on-target and off-tissue toxicities in normal tissues. CAR-T is a recent approach whose long-term efficacy has not been fully established, while the use of mAbs and ADCs is routine and generally safe in cancer treatment. The combination of engineered stem cells with CAR-T cells and / or ADCs, such as GO, has been shown to protect normal tissues from on-target and off-tissue toxicities and can result in complete remission and complete hematopoietic reconstitution in animal models. Despite the demonstrated benefits, virtually all GO-treated patients experience a significant reduction in normal myeloid lineage cells, which can lead to fatal febrile neutropenia and abnormal bleeding disorders due to GO-induced bone marrow suppression (Amadori et al. J Clin Oncol. 2016;34(9):972-979). These severe adverse events limit the use of GO during chemotherapy induction to brief exposure and substantially preclude its long-term use. This study also suggests that lower GO doses, combined with or without CART33 infusion, could represent a fundamentally new approach to treating AML patients. Finally, the recent relicensing of GO and current clinical trials of novel CD33-directed reagents (including novel anti-CD33 CAR-Ts, anti-CD33 ADCs, and CD33 bispecific T cell engagers or BiTEs) may enable the translation of this approach into the clinic in the near future.

[0292] A readily available pipeline is also being designed to test novel potential targets (e.g., CD123, CLL-1, or CD244) that share the properties that make CD33 an attractive target—i.e., a functionally redundant lineage marker expressed strictly by hematopoietic cells and also by cancer cells. This antigen becomes "cancer-specific" through CRISPR-mediated ablation of HSC-derived antigens (Haubner et al. Leukemia. 2018;33(1):64-74). This strategy could also be replicated in solid tumors, where functional organoids could be generated from embryonic or induced pluripotent stem cells that have been edited to ablate expression, or where the primary organ has already been ablated (i.e., to target normal prostate lineage antigens in patients after radical prostatectomy). Finally, the possibility of "epitope modification" of specific antigens using DNA base editing methods is proposed. "Epitope modification" could allow proteins to maintain their function but alter small antigenic determinants. In this strategy, stem cells retain the functional protein but no longer have the binding site for immunotherapy, while cancer cells with the unmodified protein remain specifically sensitive to immunotherapy.

[0293] Example 6: Gemtuzumab ozogamicin (GO)-targeted immunotherapy eliminates primary acute myeloid leukemia (AML) and upregulates CD33 Del Rescued cells CD34 + CD33 Del The cells were generated by contacting a CD34+ cell population with gRNA sgRNA811 (guide region: 5' CCUCACUAGACUUGACCCAC 3'; having SEQ ID NO: 70) and sgRNA846 (guide region: 5' AUCCCUGGCACUCUAGAACC 3'; having SEQ ID NO: 67). CD34 upon GO treatment + CD33 Del To assess cell engraftment and hematopoietic repopulation, 0.5x10 6Primary human AML cells were intravenously injected into NSG-SGM3 (NSGS) mice via the tail vein on day 1. Two months after AML cell injection, AML burden (minimal residual disease) was assessed by flow cytometry of bone marrow (BM) aspirates. The cohort was then divided into two equal groups (graph in Figure 32A: pre-treatment): a control group (circles)—in which mice received no treatment—and a treatment group (triangles)—in which mice received chronic GO administration. Leukemia cells were isolated using the following antibodies: Ter119 - H2kd / Ly5 - hCD45 + hCD33 + Gating was performed on cKit: Ter119 Pecy5, Ly5 / H2kd BV711, hCD45 BV510, hCD33 APC, cKit BV650. Once minimal residual disease was assessed, mice from the treatment group received a first injection of 1 μg of GO. Ten days after the first GO injection, both groups of mice (untreated and treated) received 0.5x10 6 CD34 + CD33 Del The cells were transplanted. CD34 + CD33 Del Ten days after cell transplantation, the treatment group began receiving long-term GO administration (1 μg of GO injected every 10 days). + CD33 Del Three weeks after transplantation, AML burden and CD34 in BM aspirates from both mouse groups were assessed. + CD33 Del Cell engraftment was assessed by flow cytometry. In control mice, AML levels were greater than 70%, while in treated mice, AML levels were less than 5%. In control mice, hCD33 Del The percentage of hCD33 cells was less than 20% in treated mice, whereas in untreated mice, Del The percentage of cells was over 70%, indicating successful engraftment (Fig. 32B, top graph). + CD33 Del The hematopoietic repopulating capacity of the cells was assessed by analyzing myeloid / lymphoid progenitor and mature cells by flow cytometry as shown in Figure 32 (continued). +CD33 Del population (cells, Ter119 - , Ly5 - / H2kd - , hCD45 + , hCD33 - (gated by ) + cells, CD14+ cells, CD10 + cells, and CD19 + The levels of CD33 cells were not significantly different between the control and treatment cohorts. As shown in Figure 32C, upper left panel, none of the control mice survived for 150 days, while all treated mice survived for at least 150 days. AML burden was comparable between the BM, spleen, and peripheral blood of control mice at the time of death (lower left panel). Furthermore, CD33 expression was significantly higher in gated CD33 cells derived from each organ or tissue. + As indicated by the cells, it was most prominent in the BM and spleen (right panel).

[0294] Example 7: CD34 + CD33 Del CLL1 Del Cell generation CD34 + CD33 Del CLL Del The double-deleted cells were cloned into CD34 +Cells were generated by transfection with various combinations of gRNAs, including sgRNA 811 (with a spacer sequence of 5' CCUCACUAGACUUGACCCAC 3' (SEQ ID NO: 70) for CD33), sgRNA 846 (with a spacer sequence of 5' AUCCCUGGCACUCUAGAACC 3' (SEQ ID NO: 67) for CD33), CLL-1 gRNA (with a spacer sequence of 5' GUUGUAGAGAAAUAUUUCUC 3' (SEQ ID NO: 115)), and a second CLL-1 gRNA (with a spacer sequence of 5' GGAGAGGUUCCUGAUCUUGU 3' (SEQ ID NO: 116)). On day 1, cells were transfected with sgRNAs using nucleofection to obtain CRISPR / Cas9-mediated ablation of the target gene. Four days after transfection, CD33 and CLL1 expression was assessed by flow cytometry. On day 5, CD34 +WT , CD34 + CD33 Del , CD34 + CLL1 Del or CD34 + CD33 Del CLL1 Del The cells were intravenously injected into NSGS mice. As shown in Figure 33A, CD33 and / or CLL1 levels were successfully reduced using these gRNAs individually and in combination. Mutations at the des...

Claims

1. A genetically engineered hematopoietic stem or progenitor cell comprising a genetic mutation in exon 3 of the endogenous CD33 gene, said genetic mutation being at a site targeted by a gRNA comprising the nucleotide sequence CCUCACUAGACUUGACCCAC (SEQ ID NO: 70), and said genetic mutation resulting in a reduced expression level of CD33 compared to a wild-type counterpart cell.

2. The genetically engineered hematopoietic stem or progenitor cells of claim 1, which express less than 20% of the CD33 expressed by their wild-type counterparts.

3. The genetically engineered hematopoietic stem or progenitor cells of claim 2, which do not express CD33.

4. The genetically engineered hematopoietic stem or progenitor cells of any one of claims 1 to 3, which are CD34+.

5. The genetically engineered hematopoietic stem or progenitor cells of any one of claims 1 to 4, which are derived from bone marrow cells or peripheral blood mononuclear cells of a subject.

6. The genetically engineered hematopoietic stem or progenitor cells of claim 5 , wherein the subject is a human patient with a hematopoietic malignancy.

7. The genetically engineered hematopoietic stem or progenitor cells of claim 5 , wherein the subject is a healthy human donor.

8. The genetically engineered hematopoietic stem or progenitor cell of any of claims 1 to 7, which does not contain a mutation in any predicted off-target site, for example, in any site listed in Figure 30.

9. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells according to any one of claims 1 to 8.

10. 1. A method for producing genetically engineered hematopoietic stem or progenitor cells, comprising: (i) providing hematopoietic stem or progenitor cells; and (ii) introducing into the cell (a) a guide RNA (gRNA) comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 70; and (b) a Cas9 endonuclease, thereby producing a genetically engineered hematopoietic stem or progenitor cell; A method comprising:

11. The method of claim 10, wherein the genetically engineered hematopoietic stem or progenitor cells express less than 20% of the CD33 expressed by their wild-type counterparts.

12. The method of claim 11, wherein the genetically engineered hematopoietic stem or progenitor cells do not express CD33.

13. The method according to any one of claims 10 to 12, wherein (a) and (b) are encoded by a single vector that is introduced into the cell.

14. The method of claim 13 , wherein the vector is a viral vector.

15. The method of any of claims 10 to 12, wherein (a) and (b) are introduced into the cell as a preformed ribonucleoprotein complex.

16. 16. The method of claim 15, wherein the ribonucleoprotein complex is introduced into the cells by electroporation.

17. 13. The method of any of claims 10 to 12, wherein the Cas9 endonuclease is introduced into the cell by delivering an mRNA molecule encoding the Cas9 endonuclease into the cell.

18. 18. The method of any one of claims 10 to 17, wherein the gRNA is a single-molecule guide RNA (sgRNA).

19. 19. The method of Claim 18, wherein the gRNA is a chemically modified sgRNA.

20. The method of any one of claims 10 to 19, wherein the hematopoietic stem or progenitor cells are CD34+.

21. The method of any one of claims 10 to 20, wherein the hematopoietic stem or progenitor cells are derived from the subject's bone marrow cells or peripheral blood mononuclear cells (PBMCs).

22. 22. The method of claim 21, wherein the subject has a hematopoietic disorder.

23. A genetically engineered hematopoietic stem or progenitor cell produced by the method of any one of claims 12 to 22.

24. 26. A method for treating a hematopoietic disorder, comprising administering to a subject in need thereof an effective amount of the genetically engineered hematopoietic stem or progenitor cells of any one of claims 1 to 8 and 23, or the cell population of claim 9.

25. 25. The method of claim 24, wherein the hematopoietic disorder is a hematopoietic malignancy.

26. The method of claim 24 or claim 25, further comprising administering to the subject an effective amount of a substance that targets CD33, and wherein the substance comprises an antigen-binding fragment that binds CD33.

27. 27. The method of claim 26, wherein the CD33-targeting agent is an immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment that binds CD33.

28. 28. The method of claim 27, wherein the immune cell is a T cell.

29. 29. The method of any one of claims 24 to 28, wherein the immune cells, the genetically engineered hematopoietic stem or progenitor cells, or both, are allogeneic.

30. 30. The method of any one of claims 24 to 29, wherein the immune cells, the genetically engineered hematopoietic stem or progenitor cells, or both, are autologous.

31. The method of any one of claims 27 to 30, wherein the antigen-binding fragment in the chimeric receptor is a single-chain antibody fragment (scFv) that specifically binds human CD33.

32. 32. The method of any one of claims 24 to 31, wherein the subject is a human patient with Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma.

33. 33. The method of claim 32, wherein the subject is a human patient with leukemia that is acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia.

34. A guide ribonucleic acid (gRNA) comprising a spacer sequence that is at least about 90% identical to SEQ ID NO:

70.

35. 35. The gRNA of Claim 34, wherein the gRNA is a single-molecule gRNA (sgRNA).

36. 36. The gRNA of claim 34 or claim 35, wherein the gRNA is chemically modified.

37. The gRNA of any one of claims 34 to 36, wherein the spacer sequence is SEQ ID NO:

70.

38. 38. The gRNA of any one of claims 34-37, wherein the gRNA does not direct gene editing at any predicted off-target sites in the target cell, e.g., does not direct gene editing at any of the sites listed in Figure 30 in the target cell.

39. A genetically engineered hematopoietic stem or progenitor cell comprising a genetic mutation in exon 3 of the endogenous CD33 gene, wherein the genetic mutation is at a site targeted by a gRNA comprising the nucleotide sequence AUCCCUGGCACUCUAGAACC (SEQ ID NO: 67).

40. A guide ribonucleic acid (gRNA) comprising a spacer sequence that is at least about 90% identical to SEQ ID NO:67.

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