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US-12622929-B2 - Genetically engineered hematopoietic stem cells and uses thereof

US12622929B2US 12622929 B2US12622929 B2US 12622929B2US-12622929-B2

Abstract

Genetically engineered hematopoietic cells such as hematopoietic stem cells having one or more genetically edited genes of lineage-specific cell-surface proteins and therapeutic uses thereof, either alone or in combination with immune therapy that targets the lineage-specific cell-surface proteins.

Inventors

  • Joseph Bolen
  • Aleksandar Filip Radovic-Moreno
  • John Lydeard

Assignees

  • SYZYGYMED INC.

Dates

Publication Date
20260512
Application Date
20220531

Claims (19)

  1. 1 . A method of treating a hematopoietic malignancy, the method comprising administering to a subject in need thereof: a plurality of genetically engineered hematopoietic cells (HCs) comprising: (i) a gene encoding CD33 that is genetically engineered to have reduced or eliminated expression of CD33, and (ii) a gene encoding CLL-1 that is genetically engineered to have reduced or eliminated expression of CLL-1, wherein the hematopoietic malignancy is characterized by cells expressing CD33 and/or CLL-1, wherein the method comprises administering and/or the subject has been administered a therapeutically effective amount of at least one agent comprising an anti-CD33 binding domain, at least one agent comprising an anti-CLL-1 binding domain, or a combination thereof.
  2. 2 . The method of claim 1 , wherein the at least one agent comprising the anti-CD33 binding domain and/or the at least one agent comprising the anti-CLL-1 binding domain comprises an immune cell comprising a chimeric antigen receptor (CAR), wherein the immune cell is a cytotoxic T cell or a Natural Killer (NK) cell.
  3. 3 . The method of claim 1 , wherein the at least one agent comprising the anti-CD33 binding domain and/or the at least one agent comprising the anti-CLL-1 binding domain comprises an antibody-drug conjugate.
  4. 4 . The method of claim 1 , wherein the genetically engineered gene encoding CD33 or the genetically engineered gene encoding CLL-1 comprises a frameshift mutation.
  5. 5 . The method of claim 1 , wherein the genetically engineered gene encoding CD33 and/or the genetically engineered gene encoding CLL-1 is modified such that an exon is skipped.
  6. 6 . The method of claim 1 , wherein the genetically engineered gene encoding CD33 and/or the genetically engineered gene encoding CLL-1 comprises an indel mutation.
  7. 7 . The method of claim 1 , wherein the expression level of the genetically engineered gene encoding CD33 and/or the genetically engineered gene encoding CLL-1 is less than 50% of the expression level of the corresponding wild-type gene.
  8. 8 . The method of claim 1 , wherein the genetically engineered HCs are genetically engineered using a CRISPR/Cas system comprising a guide nucleic acid.
  9. 9 . The method of claim 1 , wherein: exon 3 of the gene encoding the CD33 antigen is genetically engineered; and/or exon 4 of the gene encoding the CLL-1 antigen is genetically engineered.
  10. 10 . The method of claim 1 , wherein the plurality of genetically engineered HCs retains the capacity to differentiate normally compared to a population of HCs that are not genetically engineered.
  11. 11 . The method of claim 1 , wherein each cell of the plurality of genetically engineered HCs comprises the genetically engineered gene encoding CD33, the genetically engineered gene encoding CLL-1, and has reduced or no binding to the at least one agent comprising the anti-CD33 binding domain and to the at least one agent comprising the anti-CLL-1 binding domain as compared to binding of the agent to HCs that are not genetically engineered.
  12. 12 . The method of claim 1 , wherein the plurality of genetically engineered HCs comprises HCs with reduced or no binding to the at least one agent comprising the anti-CD33 binding domain and/or to the at least one agent comprising the anti-CLL-1 binding domain as compared to binding of the agent to HCs that are not genetically engineered.
  13. 13 . The method of claim 1 , wherein the plurality of genetically engineered HCs is capable of expanding by at least 2-fold over 8 days in culture.
  14. 14 . The method of claim 1 , wherein the method further comprises providing HCs from a biological sample from the subject, and genetically engineering the HCs from the biological sample from the subject, thereby forming the plurality of genetically engineered HCs.
  15. 15 . The method of claim 1 , wherein the plurality of HCs comprises HCs obtained from a healthy donor, or descendants of HCs obtained from a healthy donor.
  16. 16 . The method of claim 1 , wherein (i) the genetically engineered gene encoding CD33 comprises a genetic modification resulting in reduced expression of CD33 as compared with expression of the corresponding wild-type gene encoding CD33 in a cell that is not genetically engineered; and (ii) the genetically engineered gene encoding CLL-1 comprises a genetic modification resulting in reduced expression of CLL-1 as compared with expression of the corresponding wild-type gene encoding CLL-1 in a cell that is not genetically engineered.
  17. 17 . The method of claim 16 , wherein (i) the expression level of the genetically engineered gene encoding CD33 is less than 50% of the expression level of the corresponding wild-type gene encoding CD33 in a cell that is not genetically engineered; and/or (ii) the expression level of the genetically engineered gene encoding CLL-1 is less than 50% of the expression level of the corresponding wild-type gene encoding CLL-1 in a cell that is not genetically engineered.
  18. 18 . The method of claim 16 , wherein (i) the genetic modification in the genetically engineered gene encoding CD33 results in elimination of expression of CD33; and/or (ii) the genetic modification in the genetically engineered gene encoding CLL-1 results in elimination of expression of CLL-1.
  19. 19 . The method of claim 1 , wherein (i) the genetically engineered gene encoding CD33 comprises a genetic modification resulting in disruption of the gene encoding CD33 at a region encoding an epitope of CD33; and/or (ii) the genetically engineered gene encoding CLL-1 comprises a genetic modification resulting in disruption of the gene encoding CLL-1 at a region encoding an epitope of CLL-1.

Description

RELATED APPLICATIONS This application is a continuation of U.S. Ser. No. 17/222,855, which is a continuation of U.S. Ser. No. 17/176,610, filed Feb. 16, 2021, which is a continuation of U.S. Ser. No. 16/701,818, now U.S. Pat. No. 10,925,902, filed Dec. 3, 2019, which is a continuation of U.S. Ser. No. 16/554,520, filed Aug. 28, 2019, which claims priority to U.S. Ser. No. 62/723,993 filed Aug. 28, 2018, U.S. Ser. No. 62/728,061 filed Sep. 6, 2018, U.S. Ser. No. 62/789,440 filed Jan. 7, 2019, and U.S. Ser. No. 62/809,202 filed Feb. 22, 2019, the entire contents of each of which is incorporated herein by reference. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 26, 2022, is named V029170002US12-SEQ-CEW and is 93,443 bytes in size. BACKGROUND OF THE INVENTION A major challenge in designing targeted therapies is the successful identification of proteins that are uniquely expressed on cells that would be therapeutically relevant to eliminate (e.g., abnormal, malignant, or other target cells) but not present on cells that one does not wish to eliminate (e.g., normal, healthy, or other non-target cells). For example, many cancer therapeutics struggle to effectively target cancer cells while leaving normal cells unharmed. An alternative strategy that has emerged involves targeting an entire cell lineage, which includes targeting normal cells, cancer cells, and pre-cancerous cells. For example, CD19-targeted chimeric antigen receptor T cells (CAR T cells) and anti-CD20 monoclonal antibodies (e.g. Rituximab) each target B cell lineage proteins (CD19 and CD20, respectively). While potentially effective in treating B cell malignancies, use of such therapies is limited as elimination of B cells is detrimental. Similarly, targeting lineage-specific proteins of other cell populations, for example, myeloid lineage cells (e.g., cancers arising from myeloid blasts, monocytes, megakaryocytes, etc) is not feasible, as these cell populations are necessary for survival. Thus, there remains an unmet need to effectively target cells of interest, e.g., cancer cells, without targeting or harming normal cell populations. SUMMARY OF THE INVENTION Provided herein are compositions, e.g., engineered cells, and methods that provide the ability to target one or more cells or cell populations of interest while allowing non-targeted cell populations to escape such targeting. For example, provided herein are genetically engineered hematopoietic cells such as hematopoietic stem cells (HSCs) having genetically modified or edited genes of one or more lineage-specific cell-surface antigens. In some embodiments, the modified, e.g., edited genes are able to produce the lineage-specific cell surface proteins in modified form, which retain, at least partially, the biological activity of the lineage-specific cell-surface antigens in the HSCs or in descendant cells expressing such, but can escape targeting by cytotoxic agents that are specific to the wild-type lineage-specific cell-surface antigens. In some embodiments, the modified, e.g., edited, genes do not produce the lineage-specific cell surface protein(s) or produce a truncated version of the lineage-specific cell surface protein(s) that, while able to escape targeting by cytotoxic agents that are specific to the wild-type lineage-specific cell-surface antigen(s), may not retain biological activity of the lineage-specific cell-surface antigen(s) in the HSCs or in descendant cells expressing such. Thus, provided herein are genetically engineered hematopoietic cells, such as hematopoietic stem cells (HSCs), having one or more modified lineage-specific cell-surface antigen. In some embodiments, the one or more modified lineage-specific cell surface proteins are modified such that one or more of the lineage-specific cell surface proteins retain at least partially its biological activity of the lineage-specific cell-surface antigens in the HSCs or in descendant cells expressing such, but can escape targeting by cytotoxic agents that are specific to the corresponding wild-type lineage-specific cell-surface antigen(s). In some embodiments, the one or more modified lineage-specific cell surface proteins are modified such that all of the modified lineage-specific cell surface proteins (e.g., one, two, three, four, etc.) retain at least partial biological activity. In some embodiments, the one or more modified lineage-specific cell surface proteins are modified such that at least one, but not all, of the modified lineage-specific cell surface protein(s) retain at least partial biological activity. In some embodiments, the one or more modified lineage-specific cell surface proteins are modified such that one or more of the lineage-specific cell surface proteins do not retain at least partial bi