Compositions and methods for inhibiting series-specific antigens using a CRISPR-based base editor system

Abstract

Disclosed herein are methods for administering an agent that targets a lineage-specific cell surface antigen, such as CD33 or EMR2, and a population of hematopoietic cells having altered expression of the lineage-specific cell surface antigen, such as CD33 or EMR2, for immunotherapy of hematologic malignancies. Also disclosed herein are methods for administering an agent that targets multiple lineage-specific cell surface antigens and a population of hematopoietic cells having altered expression of the multiple lineage-specific cell surface antigens for immunotherapy of hematologic malignancies. Cells containing mutations in CD33, or EMR2, or multiple lineage-specific cell surface antigens are also provided, as are methods for producing such cells using a CRISPR-based base editor system.

Description

[Technical Field]

[0001] This application claims priority to U.S. Patent No. 63 / 033,966, filed on 3 June 2020, U.S. Patent No. 63 / 033,970, filed on 3 June 2020, and U.S. Patent No. 63 / 183,791, filed on 4 May 2021, the entire contents of each of these patents incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. [Background technology]

[0003] The emergence of targeted immunotherapy has opened up new possibilities and shown great success in patients with lymphoblastic leukemia. However, clinical trials of chimeric antigen T-cell therapy (CAR-T) have shown that its success is limited compared to CD19-targeted immunotherapy due to the lack of unique, targetable cell surface antigens. In fact, the success of CD19 immunotherapy depends on B-cell dysplasia that can be managed with immunoglobulin supplementation.

[0004] However, all targeted immunotherapy approaches require antigens that are uniquely or preferentially expressed in cancer cells. In fact, for an antigen to be an ideal candidate for immunotherapy, it must be specific to cancer cells, essential for their survival, and not expressed in normal cells. To date, truly tumor-specific antigens against cancer cells have not been found. For cancers where such ideal antigens do not exist, a novel approach has been explored that combines targeting of lineage-specific antigens overexpressed by malignant cells with the transplantation of genetically engineered stem cells lacking lineage-specific antigens (LSAs).

[0005] Previously published research and patents (U.S. No. 10,137,155) have shown that an approach using CD33-targeting CAR-T cells and / or AD gemtuzumab ozogamicin (GO) can rescue CD33+ acute myeloid leukemia-carrying mice without affecting the engraftment and lipopulation of CD33 knockout (KO) hematopoietic stem cells engineered using CRISPR / Cas9 technology to delete lineage-specific antigens.

[0006] However, genome editing using CRISPR / WT Cas9 induces DNA double-strand breaks (DSBs), which are associated with p53-mediated DNA damage responses and chromosomal translocations.

[0007] Therefore, a different approach is needed to generate targeting-resistant hematopoietic stem cells that do not cause off-target effects and debilitating immunosuppressive side effects. [Overview of the project]

[0008] This disclosure is at least partially based on the finding that drugs containing antigen-binding fragments that bind to lineage-specific cell surface antigens (e.g., immune cells expressing chimeric receptors that target lineage-specific cell surface antigens) selectively induce cell death in cells expressing lineage-specific cell surface antigens, while cells lacking the antigen (e.g., genetically engineered hematopoietic cells) avoid the resulting cell death. Based on these findings, it was anticipated that immunotherapy using a combination of lineage-specific cell surface antigen-targeting drugs, such as CAR-T cells targeting CD33, and hematopoietic cells lacking or modified with the lineage-specific cell surface antigen (e.g., CD33) would provide an effective treatment method for hematopoietic malignancies.

[0009] This book describes the use of CRISPR-based base editors to generate targeting-resistant hematopoietic stem cells (HSCs) that lack or contain modified epitopes targeted by therapeutic agents including antibodies and chimeric antigen receptor T cells (CAR-T). This approach further enables the treatment of hematological malignancies by combining targeted immunotherapy with the transplantation of targeting-resistant hematopoietic stem cells generated using base editors, thereby avoiding the potential genotoxicity of CRISPR / WT Cas9 genome editing. This novel approach enables high editing efficiency of HSCs / HSPCs using CRISPR-based cytosine and adenine base editors (CBE and ABE). CBE and ABE are Cas9 nickase fused to cytidine deaminase or adenosine deaminase, respectively, enabling precise base substitution in the target region without generating double-spin roots (DSBs). Because base editors avoid DSBs, they are considered safe editing tools that eliminate unwanted indels, translocations, or rearrangements caused by DSBs.

[0010] In one embodiment, the disclosure provides genetically engineered hematopoietic cells (e.g., HSCs) that lack a lineage-specific cell surface antigen present on the hematopoietic cells prior to the genetic engineering. In some embodiments, all or part of an endogenous gene encoding a lineage-specific cell surface antigen is deleted by genome editing, for example, using a base editor (e.g., using a CRISPR-based base editor system). In some embodiments, the CRISPR-based base editor system deletes an exon from the endogenous gene encoding the lineage-specific cell surface antigen. In some embodiments, the CRISPR-based base editor system results in a nucleotide substitution in the endogenous gene encoding the lineage-specific cell surface antigen. In some embodiments, the nucleotide substitution is within a sequence encoding a splice element, and the nucleotide substitution results in alternative splicing of the transcript encoded by the gene. In some embodiments, the CRISPR-based base editor system targets a splice element in the endogenous gene, and the CRISPR-based base editor system results in alternative splicing of the transcript encoded by the gene. In some embodiments, the alternative splicing causes the exon encoding the epitope to be skipped. In some embodiments, alternative splicing causes the exon encoding the epitope to be extended. In some embodiments, alternative splicing induces early codon termination. In some embodiments, the splice element is a splice donor, splice acceptor, splice enhancer, or splice silencer. In some embodiments, the base editor is a cytosine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C".

[0011] In some embodiments, the lineage-specific cell surface antigen is CD33. In some embodiments, the splice acceptor or exon splicing enhancer site within exon 2 of CD33 is modified. In some embodiments, the nucleotide sequence of the intron 1 / exon 2 junction of CD33 is modified. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C". In some embodiments, the gRNA sequence hybridizes to a portion of the nucleotide sequence encoding CD33. In some embodiments, the gRNA sequence targets the splice acceptor or exon splicing enhancer site within exon 2 of the nucleotide sequence encoding CD33. In some embodiments, the gRNA sequence targets rs12459419, which is a SNP of CD33. In some embodiments, the gRNA sequence targets the intron 1 / exon 2 junction of CD33. In some embodiments, the gRNA sequence targets the nucleotide sequence containing SEQ ID NO: 37. In some embodiments, the gRNA has a sequence containing any one of SEQ ID NOs: 1-3.

[0012] In some embodiments, the lineage-specific cell surface antigen is EMR2. In some embodiments, the splice donor site within exon 13 of EMR2 is modified. In some embodiments, the nucleotide sequence of the intron 12 / exon 13 junction of EMR2 is modified. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C". In some embodiments, the gRNA sequence hybridizes to a portion of the nucleotide sequence encoding EMR2. In some embodiments, the gRNA sequence targets the splice donor site within exon 13 of the nucleotide sequence encoding EMR2. In some embodiments, the gRNA sequence targets the intron 12 / exon 13 junction of EMR2. In some embodiments, the gRNA sequence targets the nucleotide sequence containing SEQ ID NO: 40. In some embodiments, the gRNA has a sequence containing either sequence number 4 or one of sequences 46-47.

[0013] In some embodiments, hematopoietic cells are hematopoietic stem cells (e.g., CD34 + / CD33 Δ2 Cells or CD34 + / EMR Δ13 )

[0014] In some embodiments, hematopoietic stem cells can be obtained from bone marrow cells or peripheral blood mononuclear cells (PBMCs).

[0015] In some embodiments, the present disclosure provides compositions and methods for inhibiting a combination of a first lineage-specific cell surface antigen and at least one further lineage-specific cell surface antigen, i.e., a second lineage-specific cell surface antigen, a third lineage-specific cell surface antigen, a fourth lineage-specific cell surface antigen, and so on.

[0016] In some embodiments, additional lineage-specific cell surface antigens are also deleted or inhibited in hematopoietic cells using a CRISPR-based base editor system. In some embodiments, all or part of an endogenous gene encoding a lineage-specific cell surface antigen(s) is deleted by genome editing, for example, using a base editor (e.g., a CRISPR-based base editor system). In some embodiments, a CRISPR-based base editor system deletes an exon from an endogenous gene encoding a lineage-specific cell surface antigen(s). In some embodiments, a CRISPR-based base editor system results in a nucleotide substitution in an endogenous gene encoding a lineage-specific cell surface antigen(s). In some embodiments, the nucleotide substitution is within a sequence encoding a splice element, and the nucleotide substitution results in alternative splicing of the transcript encoded by the gene(s). In some embodiments, a CRISPR-based base editor system targets a splice element in an endogenous gene(s), and the CRISPR-based base editor system results in alternative splicing of the transcript encoded by the gene(s). In some embodiments, alternative splicing causes the exon encoding the epitope to be skipped. In some embodiments, alternative splicing causes the exon encoding the epitope to be extended. In some embodiments, alternative splicing induces early codon termination. In some embodiments, the splice element is a splice donor, splice acceptor, splice enhancer, or splice silencer. In some embodiments, the base editor is a cytosine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G".In some embodiments, the nucleotide substitution is from "T" to "C".

[0017] In some embodiments, the first lineage-specific cell surface antigen is CD33. In some embodiments, at least one further lineage-specific cell surface antigen or a second lineage-specific cell surface antigen is EMR2.

[0018] In some embodiments, multiple additional lineage-specific cell surface antigens are deleted or inhibited using a CRISPR-based base editor system. In some embodiments, these additional lineage-specific cell surface antigens are distinct from CD33 and EMR2.

[0019] In some embodiments, hematopoietic cells are hematopoietic stem cells (e.g., CD34 + / CD33 Δ2 / EMR2 Δ13 It is a cell.

[0020] In some embodiments, hematopoietic stem cells can be obtained from bone marrow cells or peripheral blood mononuclear cells (PBMCs).

[0021] In some embodiments, the Disclosure provides genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site within exon 2 of the endogenous CD33 gene. One embodiment of the Disclosure provides genetically engineered hematopoietic stem cells and / or progenitor cells in which the expression level of the epitope encoded by exon 2 of CD33 is reduced compared to a wild-type equivalent. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells express less than 10% of the CD33 epitope expressed by a wild-type equivalent. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells do not express the CD33 epitope. In some embodiments, the splice acceptor or exon splicing enhancer site within exon 2 of CD33 is modified. In some embodiments, the nucleotide sequence of the intron 1 / exon 2 junction of CD33 is modified. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C". In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells are CD34 + In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells are derived from bone marrow cells or peripheral blood mononuclear cells of the subject (e.g., a human patient with hematopoietic malignancy, or a healthy donor).

[0022] In some embodiments, the present disclosure provides genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site within exon 13 of the endogenous EMR2 gene. One embodiment of the present disclosure provides genetically engineered hematopoietic stem cells and / or progenitor cells, wherein the genetically engineered hematopoietic stem cells and / or progenitor cells have a reduced expression level of an epitope encoded by exon 13 of EMR2 when compared to the wild-type equivalent. One embodiment of the present disclosure provides genetically engineered hematopoietic stem cells and / or progenitor cells, wherein the modified splice donor site induces early codon termination and the production of a mutant or truncated EMR2 when compared to the wild-type equivalent. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells do not express an epitope of EMR2. In some embodiments, the splice donor site within exon 13 of EMR2 is modified. In some embodiments, the nucleotide sequence of the intron 12 / exon 13 junction of EMR2 is modified. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C". In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells are CD34 + positive. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells are derived from bone marrow cells or peripheral blood mononuclear cells of a subject (e.g., a human patient having a hematological malignancy or a healthy donor).

[0023] In some embodiments, the Disclosure provides genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, and a modified splice element in the exon of at least one further lineage-specific cell surface antigen. One embodiment of the Disclosure provides genetically engineered hematopoietic stem cells and / or progenitor cells which have reduced expression levels of the epitope encoded by the CD33 and / or at least one further lineage-specific cell surface antigen exon compared to their wild-type equivalent. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells express less than 10% of the epitope encoded by the CD33 and / or at least one further lineage-specific cell surface antigen exon expressed by their wild-type equivalent. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells express CD34 + In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells are derived from bone marrow cells or peripheral blood mononuclear cells of the subject (e.g., a human patient with hematopoietic malignancy, or a healthy donor).

[0024] In some embodiments, the Disclosure provides genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene and a modified splice donor site in exon 13 of the endogenous EMR2. One embodiment of the Disclosure provides genetically engineered hematopoietic stem cells and / or progenitor cells that have reduced expression levels of the epitope encoded by exon 2 of CD33 and / or the epitope encoded by exon 13 of EMR2 compared to their wild-type counterparts. In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells express less than 10% of the epitope encoded by exon 2 of CD33 and / or the epitope encoded by exon 13 of EMR2 expressed by their wild-type counterparts. In some embodiments, alternative splicing induces early codon termination of EMR2 and the production of mutant or truncated EMR2 compared to the wild-type equivalent. In some embodiments, genetically engineered hematopoietic stem cells and / or progenitor cells are CD34 + In some embodiments, the genetically engineered hematopoietic stem cells and / or progenitor cells are derived from bone marrow cells or peripheral blood mononuclear cells of the subject (e.g., a human patient with hematopoietic malignancy, or a healthy donor).

[0025] In some embodiments, this disclosure also provides cell populations comprising multiple genetically engineered hematopoietic stem cells and / or progenitor cells as described herein.

[0026] In another aspect, the present disclosure provides a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells, comprising (i) preparing hematopoietic stem cells and / or progenitor cells, and (ii) introducing into the cells (a) a guide RNA (gRNA) that targets a nucleotide sequence encoding a lineage-specific cell surface antigen, and (b) a catalytically impaired Cas protein fused to a DNA modifying enzyme, i.e., Cas9 nickase, fused to a cytosine deaminase or adenosine deaminase (base editor), thereby providing a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells.

[0027] In another aspect, the present disclosure provides a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells, comprising (i) preparing hematopoietic stem cells and / or progenitor cells, and (ii) introducing into the cells (a) a guide RNA (gRNA) containing a targeting domain that targets nucleotide sequences in the genome of hematopoietic stem cells or progenitor cells containing splice elements, and (b) a catalytically impaired Cas protein fused to a DNA modifying enzyme i.e., Cas9 nickase fused to a cytosine deaminase or adenosine deaminase (base editor), thereby providing a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells.

[0028] In some embodiments, this method deletes an exon from an endogenous gene encoding a lineage-specific cell surface antigen. In some embodiments, this method results in a nucleotide substitution in the endogenous gene encoding a lineage-specific cell surface antigen. In some embodiments, the nucleotide substitution is within a sequence encoding a splice element, and the nucleotide substitution results in alternative splicing of the transcript encoded by the gene. In some embodiments, this method targets a splice element in an endogenous gene, and this method results in alternative splicing of the transcript encoded by the gene. In some embodiments, alternative splicing causes the exon encoding the epitope to be skipped. In some embodiments, alternative splicing causes the exon encoding the epitope to be extended. In some embodiments, the splice element is a splice donor, splice acceptor, splice enhancer, or splice silencer. In some embodiments, the base editor is a cytosine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C".

[0029] In some embodiments, the lineage-specific cell surface antigen is CD33. In some embodiments, the gRNA targets a nucleotide sequence that flanks exon 2 of CD33. In some embodiments, the gRNA contains a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. In some embodiments, the gRNA sequence targets the intron 1 / exon 2 junction of CD33. In some embodiments, the gRNA sequence targets a nucleotide sequence including SEQ ID NO: 37. In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are encoded in a single vector introduced into the cell. In some embodiments, the vector is a viral vector. In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are introduced into the cell as a pre-formed ribonucleoprotein complex. In some embodiments, the ribonucleoprotein complex is introduced into the cell via electroporation.

[0030] In some embodiments, the lineage-specific cell surface antigen is EMR2. In some embodiments, the gRNA targets a nucleotide sequence that flanks exon 13 of EMR2. In some embodiments, the gRNA contains a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4, SEQ ID NO: 46, and / or SEQ ID NO: 47. In some embodiments, the gRNA sequence targets the intron 12 / exon 13 junction of EMR2. In some embodiments, the gRNA sequence targets a nucleotide sequence including SEQ ID NO: 40. In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are encoded in a single vector introduced into the cell. In some embodiments, the vector is a viral vector. In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are introduced into the cell as a pre-formed ribonucleoprotein complex. In some embodiments, the ribonucleoprotein complex is introduced into the cell via electroporation.

[0031] In another aspect, the present disclosure provides a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells, comprising (i) preparing hematopoietic stem cells and / or progenitor cells, and (ii) introducing into the cells a catalytically impaired Cas protein fused to (a) a guide RNA (gRNA) that targets a nucleotide sequence encoding a first lineage-specific cell surface antigen, and (b) a DNA modifying enzyme fused to cytosine deaminase or adenosine deaminase (base editor), i.e., Cas9 nickase, and further comprising introducing into the cells a second guide RNA (gRNA) that targets at least one further lineage-specific cell surface antigen, and (b) a catalytically impaired Cas protein fused to a DNA modifying enzyme fused to cytosine deaminase or adenosine deaminase (base editor), i.e., Cas9 nickase, thereby providing a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells.

[0032] In another aspect, the present disclosure is a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells, comprising: (i) preparing hematopoietic stem cells and / or progenitor cells; and (ii) fertilizing the cells with (a) a guide RNA (gRNA) comprising a targeting domain that targets a nucleotide sequence in the genome of a hematopoietic stem cell or progenitor cell containing a splice element of a first lineage-specific cell surface antigen; and (b) a catalytically impaired Ca fused to a DNA modifying enzyme i.e., Cas9 nickase fused to a cytosine deaminase or adenosine deaminase (base editor). The present invention provides a method for producing genetically engineered hematopoietic stem cells and / or progenitor cells, comprising (b) introducing an s protein, and further comprising introducing into cells a second guide RNA (gRNA) containing a targeting domain that targets a nucleotide sequence in the genome of at least one further lineage-specific cell surface antigen of hematopoietic stem cells or progenitor cells, and (b) introducing a catalytically impaired Cas protein fused to a DNA modifying enzyme i.e., Cas9 nickase fused to a cytosine deaminase or adenosine deaminase (base editor).

[0033] In some embodiments, the lineage-specific cell surface antigen is CD33. In some embodiments, the gRNA targets a nucleotide sequence that flanks exon 2 of CD33. In some embodiments, the gRNA contains a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. In some embodiments, the gRNA sequence targets the intron 1 / exon 2 junction of CD33. In some embodiments, the gRNA sequence targets a nucleotide sequence that includes SEQ ID NO: 37.

[0034] In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are encoded in a single vector introduced into the cell. In some embodiments, the vector is a viral vector. In some embodiments, the gRNA and the catalytically impaired Cas protein fused to a DNA-modifying enzyme are introduced into the cell as a pre-formed ribonucleoprotein complex. In some embodiments, the ribonucleoprotein complex is introduced into the cell via electroporation.

[0035] In some embodiments, at least one further lineage-specific cell surface antigen is EMR2. In some embodiments, the gRNA targets a nucleotide sequence that flanks exon 13 of EMR2. In some embodiments, the gRNA contains a nucleotide sequence that is at least 90% identical to SEQ ID NO: 4, SEQ ID NO: 46 and / or SEQ ID NO: 47. In some embodiments, the gRNA sequence targets the intron 12 / exon 13 junction of EMR2. In some embodiments, the gRNA sequence targets a nucleotide sequence including SEQ ID NO: 40. In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are encoded in a single vector introduced into the cell. In some embodiments, the vector is a viral vector. In some embodiments, the gRNA and a catalytically impaired Cas protein fused to a DNA-modifying enzyme are introduced into the cell as a pre-formed ribonucleoprotein complex. In some embodiments, the ribonucleoprotein complex is introduced into the cell via electroporation.

[0036] In some aspects, this disclosure also provides the use of gRNAs described herein to reduce the expression of lineage-specific cell surface antigen epitopes in a sample of hematopoietic stem cells or progenitor cells using a CRISPR-based base editor system.

[0037] The disclosure also provides, in some embodiments, the use of a CRISPR-based base editor system to reduce the expression of lineage-specific cell surface antigen epitopes in a sample of hematopoietic stem cells or progenitor cells.

[0038] This disclosure also, in some aspects, provides the use of the gRNAs described herein to reduce the expression of the CD33 epitope in a sample of hematopoietic stem cells or progenitor cells using a CRISPR-based base editor system.

[0039] This disclosure also provides, in some embodiments, the use of a CRISPR-based base editor system to reduce the expression of the CD33 epitope in a sample of hematopoietic stem cells or progenitor cells.

[0040] This disclosure also provides, in some aspects, the use of gRNAs described herein to reduce the expression of the EMR2 epitope in a sample of hematopoietic stem cells or progenitor cells using a CRISPR-based base editor system.

[0041] This disclosure also provides, in some embodiments, the use of a CRISPR-based base editor system to reduce the expression of the EMR2 epitope in a sample of hematopoietic stem cells or progenitor cells.

[0042] This disclosure also provides, in some embodiments, the use of gRNAs described herein to reduce the expression of CD33 and at least one further lineage-specific cell surface antigen in a sample of hematopoietic stem cells or progenitor cells using a CRISPR-based base editor system.

[0043] This disclosure also provides, in some embodiments, the use of a CRISPR-based base editor system to reduce the expression of CD33 and at least one further lineage-specific cell surface antigen in a sample of hematopoietic stem cells or progenitor cells.

[0044] In some embodiments, at least one further series-specific antigen is EMR2.

[0045] In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA).

[0046] In some embodiments, hematopoietic stem cells and / or progenitor cells are CD34 + In some embodiments, the hematopoietic stem cells and / or progenitor cells are derived from the subject's 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.

[0047] This disclosure provides, in some embodiments, genetically engineered hematopoietic stem cells and / or progenitor cells produced by the methods described herein.

[0048] In another embodiment, the present disclosure provides a method for treating hematopoietic disorders, comprising administering an effective amount of genetically engineered hematopoietic stem cells and / or progenitor cells or cell populations described herein to a subject in need thereof. In some embodiments, the hematopoietic disorder is a hematopoietic malignancy.

[0049] In some embodiments, the method further comprises administering an effective amount of a CD33-targeting agent to a target, the agent comprising an antigen-binding fragment that binds to CD33. In some embodiments, the CD33-targeting agent is an immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment that binds to CD33.

[0050] In some embodiments, the Disclosure provides genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein for use in the treatment of hematopoietic disorders, the treatment comprising administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or cell population to a subject in need thereof, and further comprising administering an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33 to the subject.

[0051] In some embodiments, the Disclosure provides a CD33-targeting agent for use in the treatment of hematopoietic disorders, comprising an antigen-binding fragment that binds to CD33, wherein the treatment comprises administering an effective amount of the CD33-targeting agent to a subject in need, and further comprising administering an effective amount of genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein to the subject.

[0052] A combination for use in the treatment of hematopoietic disorders, comprising a genetically engineered hematopoietic stem cell or progenitor cell or cell population as described herein, and a CD33-targeting agent containing an antigen-binding fragment that binds to CD33, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cell or progenitor cell or cell population and the CD33-binding agent to a patient in need.

[0053] In some embodiments, genetically engineered hematopoietic stem cells or progenitor cells or cell populations are administered in combination with a CD33-targeting agent. In some embodiments, genetically engineered hematopoietic stem cells or progenitor cells or cell populations are administered before the CD33-targeting agent. In some embodiments, the CD33-targeting agent is administered before the genetically engineered hematopoietic stem cells or progenitor cells or cell populations.

[0054] In some embodiments, the immune cells are T cells. In some embodiments, the immune cells, genetically engineered hematopoietic stem cells and / or progenitor cells, or both, are allogeneic. In some embodiments, the immune cells, genetically engineered hematopoietic stem cells 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 to human CD33.

[0055] In some embodiments, the method further comprises administering an effective dose of an EMR2-targeting agent, the agent comprising an antigen-binding fragment that binds to EMR2. In some embodiments, the EMR2-targeting agent is an immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment that binds to EMR2.

[0056] In some embodiments, the Disclosure provides genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein for use in the treatment of hematopoietic disorders, the treatment comprising administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or cell population to a subject in need thereof, and further comprising administering an effective amount of an EMR2-targeting agent comprising an antigen-binding fragment that binds to EMR2 to the subject.

[0057] In some embodiments, the Disclosure provides an EMR2-targeting agent for use in the treatment of hematopoietic disorders, comprising an antigen-binding fragment that binds to EMR2, wherein the treatment comprises administering an effective amount of the EMR2-targeting agent to a subject in need, and further comprising administering an effective amount of genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein to the subject.

[0058] A combination for use in the treatment of hematopoietic disorders, comprising genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein, and an EMR2-targeting agent containing an antigen-binding fragment that binds to EMR2, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or cell population and the EMR2-binding agent to a patient in need.

[0059] In some embodiments, genetically engineered hematopoietic stem cells or progenitor cells or cell populations are administered in combination with an EMR2-targeting agent. In some embodiments, genetically engineered hematopoietic stem cells or progenitor cells or cell populations are administered before the EMR2-targeting agent. In some embodiments, the EMR2-targeting agent is administered before the genetically engineered hematopoietic stem cells or progenitor cells or cell populations.

[0060] In some embodiments, the immune cells are T cells. In some embodiments, the immune cells, genetically engineered hematopoietic stem cells and / or progenitor cells, or both, are allogeneic. In some embodiments, the immune cells, genetically engineered hematopoietic stem cells 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 to human EMR2.

[0061] In some embodiments, the method further includes administering an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33, and an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen. In some embodiments, the CD33-targeting agent is an immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment that binds to CD33. In some embodiments, the CD33-targeting agent and the CD33-targeting agent are immune cells expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen. In some embodiments, the CD33-targeting agent and the CD33-targeting agent are immune cells. In some embodiments, the agent targeting CD33 and at least one further lineage-specific cell surface antigen is an immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment that binds to CD33 and at least one further lineage-specific cell surface antigen.

[0062] In some embodiments, the Disclosure provides genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein for use in the treatment of hematopoietic disorders, the treatment comprising administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or cell populations to a subject in need thereof, and further comprising administering an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33, and an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen.

[0063] In some embodiments, the Disclosure provides a CD33-targeting agent for use in the treatment of hematopoietic disorders, comprising an antigen-binding fragment that binds to CD33, and an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen, wherein the treatment comprises administering to a subject in need an effective dose of the CD33-targeting agent and an effective dose of the agent that targets at least one further lineage-specific cell surface antigen and comprises an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen, and further comprises administering to an effective dose of genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein.

[0064] A combination for use in the treatment of hematopoietic disorders comprising genetically engineered hematopoietic stem cells or progenitor cells or cell populations as described herein, a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33, and an effective amount of an agent targeting at least one further lineage-specific cell surface antigen comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen, wherein the treatment comprises administering to a patient in need an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or cell population, as well as the CD33-binding agent and the agent that binds to at least one further lineage-specific cell surface antigen.

[0065] In some embodiments, a drug that targets CD33 and includes an antigen-binding fragment that binds to CD33 is the same drug as a drug that targets at least one further lineage-specific cell surface antigen and includes an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen. In some embodiments, the drug is an immune cell that targets CD33 and at least one further lineage-specific cell surface antigen.

[0066] In some embodiments, genetically engineered hematopoietic stem cells or progenitor cells or cell populations are administered in combination with a CD33-targeting agent and a agent targeting at least one further lineage-specific cell surface antigen. In some embodiments, genetically engineered hematopoietic stem cells or progenitor cells or cell populations are administered before the CD33-targeting agent and the agent targeting at least one further lineage-specific cell surface antigen. In some embodiments, the CD33-targeting agent and the agent targeting at least one further lineage-specific cell surface antigen are administered before the genetically engineered hematopoietic stem cells or progenitor cells or cell populations. In some embodiments, the CD33-targeting agent is administered before the genetically engineered hematopoietic stem cells or progenitor cells or cell populations, and the agent targeting at least one further lineage-specific cell surface antigen is administered after the genetically engineered hematopoietic stem cells or progenitor cells or cell populations. In some embodiments, a drug targeting at least one further lineage-specific cell surface antigen is administered before the genetically engineered hematopoietic stem cells or progenitor cells or cell population, while a drug targeting CD33 is administered after the genetically engineered hematopoietic stem cells or progenitor cells or cell population.

[0067] In some embodiments, the immune cells are T cells. In some embodiments, the immune cells, genetically engineered hematopoietic stem cells and / or progenitor cells, or both, are allogeneic. In some embodiments, the immune cells, genetically engineered hematopoietic stem cells 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 to human CD33 and / or at least one further lineage-specific cell surface antigen. In some embodiments, the antigen-binding fragment in the chimeric receptor is a single-chain antibody fragment (scFv) that specifically binds to at least one further human lineage-specific cell surface antigen.

[0068] In some embodiments, at least one further lineage-specific cell surface antigen is EMR2.

[0069] In some embodiments, agents targeting CD33 and / or EMR2 and / or further lineage-specific cell surface antigens target modified, de-expressed, or deleted epitopes in genetically engineered hematopoietic stem cells or progenitor cells or cell populations administered in conjunction with the agent, as described herein.

[0070] In some embodiments, the subjects are human patients with Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, or multiple myeloma. In some embodiments, the subjects are human patients with leukemia, which is acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia. The characteristics of the compositions and methods described herein are also described in the embodiments listed below.

[0071] The characteristics of the compositions and methods described in this book are also described in the embodiments listed below. 1. Genetically engineered hematopoietic stem cells or progenitor cells comprising at least one nucleotide substitution in a gene encoding a lineage-specific antigen, wherein the nucleotide substitution is contained within a sequence encoding a splice element, resulting in alternative splicing of the transcript encoded by the gene, the alternative splicing causing a decrease in the expression level of the epitope encoded by the gene compared to a wild-type equivalent cell, and the epitope is targeted by an immunotherapy agent. 2. The splice element is a genetically engineered hematopoietic stem cell or progenitor cell according to any of the preceding embodiments, selected from the group consisting of a splice acceptor, a splice donor, a splice enhancer, and a splice silencer. 3. Genetically engineered hematopoietic stem cells or progenitor cells according to Embodiment 1 or 2, wherein alternative splicing causes the skipping of exons encoding epitopes. 4. Alternative splicing is performed on genetically engineered hematopoietic stem cells or progenitor cells as described in any of the preceding embodiments, which causes the epitope-coding exons to be extended. 5. A genetically engineered hematopoietic stem cell or progenitor cell according to any of the preceding embodiments, wherein the nucleotide substitution is from "C" to "T" or from "G" to "A". 6. A genetically engineered hematopoietic stem cell or progenitor cell according to any of the preceding embodiments, wherein the nucleotide substitution is from "A" to "G" or from "T" to "C". 7. The nucleotide substitution is performed using a CRISPR-based base editor system in genetically engineered hematopoietic stem cells or progenitor cells as described in any of the preceding embodiments. 8. The reduced expression level of the epitope is in cells differentiated from (e.g., terminally differentiated) hematopoietic stem cells or progenitor cells as described in any of the preceding embodiments, wherein the wild-type equivalent cells are cells differentiated from (e.g., terminally differentiated) wild-type hematopoietic stem cells or progenitor cells. 9. The genetically engineered hematopoietic stem cells or progenitor cells according to Embodiment 8, wherein the cells differentiated from the hematopoietic stem cells or progenitor cells are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 10. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification results in a reduced expression level of the epitope encoded by exon 2 of CD33 compared to a wild-type equivalent cell. 11. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification results in a reduced expression level of the epitope encoded by exon 2 of CD33, which is less than 20% of the level compared to a wild-type equivalent cell. 12. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site in exon 2 of CD33. 13. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the nucleotide sequence of the intron 1 / exon 2 junction of CD33. 14. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 1. 15. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 2. 16. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 3. 17. The modifications are made using a CRISPR-based base editor system in the genetically engineered hematopoietic stem cells or progenitor cells described in Embodiments 10-16. 18. Genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 10-17, wherein the modification results in a reduced expression level of the epitope encoded by exon 2 of CD33 (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the level in the wild-type equivalent cells) compared to wild-type equivalent cells. 19. The reduced expression level of the CD33 epitope is in cells differentiated from hematopoietic stem cells or progenitor cells (e.g., terminally differentiated cells), and the wild-type equivalent cells are cells differentiated from wild-type hematopoietic stem cells or progenitor cells (e.g., terminally differentiated cells), as described in Embodiments 10-18. 20. The genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 19, wherein the cells differentiated from the hematopoietic stem cell or progenitor cell are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 21. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification results in a reduced expression level of the epitope encoded by exon 13 of EMR2 compared to a wild-type equivalent cell. 22. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modified splice donor site induces early codon termination and the production of mutant or truncated EMR2 compared to a wild-type equivalent. 23. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in a splice acceptor or exon splicing enhancer site in exon 13 of EMR2. 24. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in the nucleotide sequence of the intron 12 / exon 13 junction of EMR2. 25. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is made by using a gRNA containing one of the nucleotide sequences of SEQ ID NO: 4 or 46-47. 26. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 21-25, wherein the modifications are made using a CRISPR-based base editor system. 27. Genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 21-25, wherein the modification results in a reduced expression level of the epitope encoded by exon 13 of EMR2 (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the level in the wild-type equivalent cells) compared to wild-type equivalent cells. 28. The genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 21-26, wherein the reduction in the expression level of the EMR2 epitope is in cells differentiated from (e.g., terminally differentiated) hematopoietic stem cells or progenitor cells, and the wild-type equivalent cells are cells differentiated from (e.g., terminally differentiated) wild-type hematopoietic stem cells or progenitor cells. 29. The genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 29, wherein the cells differentiated from the hematopoietic stem cell or progenitor cell are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 30. Genetically engineered hematopoietic stem cells or progenitor cells according to any of the prior embodiments, which are CD34+. 31. Genetically engineered hematopoietic stem cells or progenitor cells according to any of the preceding embodiments, derived from target bone marrow cells or peripheral blood mononuclear cells. 32. The subject is a human patient with a hematopoietic malignancy, and the subject is a genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 31. 33. The subject is a healthy human donor (e.g., an HLA-matched donor), and the subject is a genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 31. 34. Genetically engineered hematopoietic stem cells or progenitor cells comprising: the nucleotide substitution, wherein at least one nucleotide substitution in a gene encoding a lineage-specific antigen, the nucleotide substitution being contained within a sequence encoding a splice element, resulting in alternative splicing of the transcript encoded by the gene, the alternative splicing causing a decrease in the expression level of an epitope encoded by the gene compared to a wild-type equivalent cell, and the epitope being targeted by an immunotherapy agent; and the nucleotide substitution, wherein at least one nucleotide substitution in a gene encoding at least one further lineage-specific antigen, the nucleotide substitution being contained within a sequence encoding a splice element, resulting in alternative splicing of the transcript encoded by the gene, the alternative splicing causing a decrease in the expression level of an epitope encoded by the gene encoding at least one further lineage-specific antigen compared to a wild-type equivalent, and both epitopes being targeted by an immunotherapy agent(s). 35. The genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 34, wherein the splice element is selected from the group consisting of a splice acceptor, a splice donor, a splice enhancer, and a splice silencer. 36. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 34-35, wherein alternative splicing causes the skipping of exons encoding epitopes. 37. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 34-35, wherein alternative splicing causes the epitope-coding exon to be extended. 38. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 34-37, wherein the nucleotide substitution is from "C" to "T" or from "G" to "A". 39. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 34-37, wherein the nucleotide substitution is from "A" to "G" or from "T" to "C". 40. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 34-39, wherein the nucleotide substitution is made using a CRISPR-based base editor system. 41. The genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 34-40, wherein the reduction in the expression level of the epitope is in cells differentiated from (e.g., terminally differentiated) hematopoietic stem cells or progenitor cells, and the wild-type equivalent cells are cells differentiated from (e.g., terminally differentiated) wild-type hematopoietic stem cells or progenitor cells. 42. The genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 41, wherein the cells differentiated from the hematopoietic stem cell or progenitor cell are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 43. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of an endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site in exon 2 of CD33, and a modification of a modified element in the exon of an endogenous gene encoding at least one further lineage-specific cell surface antigen. 44. A genetically engineered hematopoietic stem cell or progenitor cell comprising: a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site in exon 2 of CD33; and a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in the splice donor site in exon 13 of EMR2. 45. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor or exon splicing enhancer site within exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site within exon 2 of CD33, resulting in a reduced expression level of the epitope encoded by exon 2 of CD33 compared to a wild-type equivalent cell. The genetically engineered hematopoietic stem cell or progenitor cell further comprising a modified splice donor site within exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in the splice donor site within exon 13 of EMR2, resulting in a reduced expression level of the epitope encoded by exon 13 of EMR2 compared to a wild-type equivalent cell, and / or inducing early codon termination and the production of mutant or truncated EMR2 compared to a wild-type equivalent cell. 46. ​​A genetically engineered hematopoietic stem cell or progenitor cell comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site in exon 2 of CD33, and is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 1, and further comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in the splice donor site in exon 13 of EMR2, and is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 4 or any one of 46-47, the genetically engineered hematopoietic stem cell or progenitor cell. 47. A genetically engineered hematopoietic stem cell or progenitor cell comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site in exon 2 of CD33, and is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 2, and further comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in the splice donor site in exon 13 of EMR2, and is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 4 or any one of 46-47, the genetically engineered hematopoietic stem cell or progenitor cell. 48. A genetically engineered hematopoietic stem cell or progenitor cell comprising a modified splice acceptor or exon splicing enhancer site in exon 2 of the endogenous CD33 gene, wherein the modification is a nucleotide substitution in the splice acceptor or exon splicing enhancer site in exon 2 of CD33, and is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 3, and further comprising a modified splice donor site in exon 13 of the endogenous EMR2 gene, wherein the modification is a nucleotide substitution in the splice donor site in exon 13 of EMR2, and is made by using a gRNA containing the nucleotide sequence of SEQ ID NO: 4 or any one of 46-47, the genetically engineered hematopoietic stem cell or progenitor cell. 49. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 43-48, wherein the modifications(s) are made using a CRISPR-based base editor system. 50. Genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 43-49, wherein the modification results in a reduced expression level of the epitope encoded by exon 2 of CD33 (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the level in the wild-type equivalent cells) and / or a reduced expression level of the epitope encoded by exon 13 of EMR2 (e.g., less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% of the level in the wild-type equivalent cells). 51. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 43-50, expressing less than 20% of the epitope encoded by exon 2 of CD33 as expressed by the wild-type equivalent, and / or less than 20% of the epitope encoded by exon 13 of EMR2 as expressed by the wild-type equivalent. 52. The reduced expression level of the CD33 and / or EMR2 epitope is in cells differentiated from the hematopoietic stem cells or progenitor cells (e.g., terminally differentiated), and the wild-type equivalent cells are cells differentiated from wild-type hematopoietic stem cells or progenitor cells (e.g., terminally differentiated), as described in Embodiments 43-51. 53. The genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 52, wherein the cells differentiated from the hematopoietic stem cell or progenitor cell are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 54. Genetically engineered hematopoietic stem cells or progenitor cells according to embodiments 34-53, which are CD34+. 55. Genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 34-53, derived from target bone marrow cells or peripheral blood mononuclear cells. 56. The subject is a human patient with a hematopoietic malignancy, and the subject is a genetically engineered hematopoietic stem cell or progenitor cell according to Embodiment 55. 57. The subject is a healthy human donor (e.g., an HLA-matched donor), and the subject is a genetically engineered hematopoietic stem cell or progenitor cell as described in Embodiment 55. 58. Genetically engineered hematopoietic stem cells or progenitor cells according to Embodiments 43 to 57, produced by a process comprising contacting the endogenous CD33 gene with a catalytically impaired CRISPR endonuclease fused to cytosine deaminase or adenosine deaminase (base editor), and contacting the endogenous EMR2 gene with the catalytically impaired CRISPR endonuclease fused to cytosine deaminase or adenosine deaminase (base editor). 59. Genetically engineered hematopoietic stem cells or progenitor cells as described in any of the preceding embodiments, which do not contain mutations in any of the predicted off-target sites. 60. A cell population comprising multiple genetically engineered hematopoietic stem cells or progenitor cells as described in any of the prior embodiments (e.g., including hematopoietic stem cells, hematopoietic progenitor cells, or a combination thereof). 61. A cell population comprising multiple genetically engineered hematopoietic stem cells or progenitor cells, each containing a deletion or modification of exon 2 of the endogenous CD33 gene and a deletion or modification of exon 13 of the endogenous EMR2 gene. 62. The cell population according to Embodiment 61, further comprising one or more cells containing one or more unengineered CD33 genes and / or unengineered EMR2 genes. 63. The cell population according to Embodiments 61-62, further comprising one or more cells that are homozygous wild-type for CD33 and / or homozygous wild-type for EMR2. 64. A cell population according to any one of embodiments 61 to 63, further comprising one or more cells that are heterozygous wild-type for CD33 and / or heterozygous wild-type for EMR2. 65. The cell population according to any one of embodiments 61 to 64, wherein the reduced expression levels of CD33 and / or EMR2 are in cells differentiated from hematopoietic stem cells or progenitor cells (e.g., terminally differentiated cells), and the wild-type equivalent cells are cells differentiated from wild-type hematopoietic stem cells or progenitor cells (e.g., terminally differentiated cells). 66. The cell population according to Embodiment 65, wherein the cells differentiated from the hematopoietic stem cells or progenitor cells are myeloblasts, monoblasts, monocytes, macrophages, or natural killer cells. 67. A cell population according to any one of embodiments 61 to 66, comprising hematopoietic stem cells and hematopoietic progenitor cells. 68. A pharmaceutical composition comprising genetically engineered hematopoietic stem cells or progenitor cells as described in any of Embodiments 1 to 59. 69. A pharmaceutical composition comprising a cell population as described in any of embodiments 60 to 67. 70. A method for producing genetically engineered hematopoietic stem cells or progenitor cells as described in any of Embodiments 1 to 33 or a cell population as described in any of Embodiments 60 to 67, (i) Prepare hematopoietic stem cells or progenitor cells (e.g., wild-type hematopoietic stem cells or progenitor cells), (ii) A method for producing genetically engineered hematopoietic stem cells and / or progenitor cells, comprising introducing into the cells (a) a guide RNA (gRNA) containing a targeting domain that targets a nucleotide sequence in the genome of the hematopoietic stem cell or progenitor cell containing a splice element, and (b) a catalytically impaired Cas protein fused to a DNA modifying enzyme fused to cytosine deaminase or adenosine deaminase (base editor). 71. A method for producing genetically engineered hematopoietic stem cells or progenitor cells as described in any of Embodiments 34 to 59 or a cell population as described in any of Embodiments 60 to 67, (i) Prepare hematopoietic stem cells or progenitor cells, (ii) Introducing into the cells (a) a first guide RNA (gRNA) containing a targeting domain that targets a first nucleotide sequence in the genome of the hematopoietic stem cell or progenitor cell containing a splice element, and (b) a first catalytically impaired Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor), (iii) A method for producing genetically engineered hematopoietic stem cells or progenitor cells, comprising introducing into the cells (a) a second guide RNA (gRNA) comprising a targeting domain that targets a second nucleotide sequence in the genome of the hematopoietic stem cell or progenitor cell comprising a splice element, and (b) a second catalytically impaired Cas9 endonuclease fused to a cytosine deaminase or adenosine deaminase (base editor). 72. The method according to Embodiments 70-71, wherein the method results in nucleotide substitutions in endogenous genes(s) encoding the series-specific cell surface antigens(s). 73. The method according to any one of embodiments 70 to 72, wherein the method targets a splice element in an endogenous gene(s), and the method results in alternative splicing of a transcript encoded by the gene(s). 74. The method according to any one of embodiments 70 to 73, wherein the alternative splicing causes the exon encoding the epitope to be skipped. 75. The method according to any one of embodiments 70 to 73, wherein the alternative splicing causes the exon encoding the epitope to be extended. 76. The method according to any one of embodiments 70 to 75, wherein the splice element(s) is a splice donor, a splice acceptor, a splice enhancer, or a splice silencer. 77. The method according to any one of embodiments 70 to 76, wherein the base editor(s) are cytosine base editors. 78. The method according to any one of embodiments 70 to 76, wherein the base editor(s) are adenine base editors. 79. The method according to any one of embodiments 70 to 78, wherein the endonuclease(s) is Cas9 nickasase. 80. The method according to embodiment 77, wherein the cytosine base editor (or multiple base editors) is BE4Max. 81. The method according to Embodiment 78, wherein the adenosine base editor (or multiple base editors) is ABE8e. 82. The method according to any one of embodiments 70 to 81, wherein the alternative splicing results in a decrease in the expression of the epitope encoded by exon 2 of CD33. 83. The method according to any one of embodiments 70 to 81, wherein the alternative splicing results in reduced expression of the epitope encoded by exon 13 of EMR2, and / or early codon termination and production of mutant or truncated EMR2, compared to equivalent wild-type cells. 84. A guide ribonucleic acid (gRNA) comprising any of the sequences described in Sequence IDs 1-4 and 46-47, or their reverse complement, or a sequence having at least 90% or 95% identity with any of the aforementioned, or a sequence having one or fewer, two or fewer, or three or fewer mutations with any of the aforementioned. 85. The gRNA according to Embodiment 84, comprising one or more chemical modifications (e.g., chemical modifications to nucleic acid bases, sugars, or skeletal portions). 86. A gRNA according to either embodiment 84 or 85 that binds to Cas9. 87. A kit or composition comprising a gRNA selected from the group consisting of gRNAs including sequence numbers 1-3 and combinations thereof, or a nucleic acid encoding said gRNA. 88. The kit according to Embodiment 87, further comprising a second gRNA, or a nucleic acid encoding the second gRNA, wherein the second gRNA targets a lineage-specific cell surface antigen other than CD33. 89. The kit or composition according to Embodiment 88, wherein the second gRNA targets EMR2. 90. The kit or composition according to any one of embodiments 89 and 90, wherein the second gRNA comprises one of sequence numbers 4 or 46-47. 91. A kit or composition according to any one of embodiments 88 to 90, further comprising a third gRNA, or a nucleic acid encoding the third gRNA. 92. The kit or composition according to Embodiment 91, wherein the third gRNA targets lineage-specific cell surface antigens other than CD33 and EMR2. 93. A kit or composition according to any one of embodiments 87 to 92, further comprising one or more catalytically impaired CRISPR endonucleases fused to a cytosine deaminase or adenosine deaminase (base editor). 94. Use of a CRISPR-based base editor system to reduce CD33 expression in hematopoietic stem cell or progenitor cell samples containing gRNAs with sequence numbers 1-3. 95. Use of a CRISPR-based base editor system to reduce EMR2 expression in hematopoietic stem cell or progenitor cell samples containing gRNAs SEQ ID NOs. 4 and 46-47. 96. Use of a CRISPR-based base editor system to reduce the expression of CD33 and / or EMRR2 in a sample of hematopoietic stem cells or progenitor cells containing gRNAs SEQ ID NOs: 1-4 and 46-47. 97. A method for producing genetically engineered hematopoietic stem cells or progenitor cells, (i) Prepare hematopoietic stem cells or progenitor cells, (ii) The method for producing genetically engineered hematopoietic stem cells or progenitor cells, comprising introducing into the cells (a) guide RNA (gRNA) containing sequence numbers 1 to 3, and (b) a nuclease (e.g., endonuclease) that binds to the gRNA fused to cytosine deaminase or adenosine deaminase (base editor). 98. A method for producing genetically engineered hematopoietic stem cells or progenitor cells, (i) Prepare hematopoietic stem cells or progenitor cells, (ii) The method for producing the genetically engineered hematopoietic stem cells or progenitor cells, comprising introducing into the cells (a) a guide RNA (gRNA) having a nucleotide sequence identical to at least 90% of sequence numbers 1-3, and (b) a Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor). 99. The method according to embodiments 97 and 98, wherein the gRNA sequence targets the intron 1 / exon 2 junction of CD33. 100. The method according to Embodiments 97 to 99, wherein the gRNA targets a nucleotide sequence including SEQ ID NO: 37. 101. The method according to any one of embodiments 97 to 100, wherein the method results in a nucleotide substitution in the sequence encoding the splice element of exon 2 of CD33, the nucleotide substitution resulting in alternative splicing of the gene-encoded transcript. 102. The method according to Embodiment 101, wherein the alternative splicing of the transcript results in a decrease in the expression of the CD33 exon 2 epitope. 103. The method according to any one of embodiments 97 to 102, wherein the base editor is a cytosine base editor and is BE4max. 104. The method according to any one of embodiments 97 to 102, wherein the base editor is an adenosine base editor, and is ABE8e. 105. The method according to any one of embodiments 97 to 104, which results in genetically engineered hematopoietic stem cells or progenitor cells having a reduced expression level of the CD33 exon 2 epitope compared to equivalent wild-type cells. 106. The method according to any one of embodiments 97 to 105, performed on a plurality of hematopoietic stem cells or progenitor cells. 107. The method according to any one of embodiments 97 to 106, wherein a cell population according to any one of embodiments 60 to 67 is produced. 108. A method for producing genetically engineered hematopoietic stem cells or progenitor cells, (i) Prepare hematopoietic stem cells or progenitor cells, (ii) The method for producing genetically engineered hematopoietic stem cells or progenitor cells, comprising introducing into the cells (a) a guide RNA (gRNA) containing sequence number 4 or 46-47, and (b) a nuclease (e.g., endonuclease) that binds to the gRNA fused to cytosine deaminase or adenosine deaminase (base editor). 109. A method for producing genetically engineered hematopoietic stem cells or progenitor cells, (i) Prepare hematopoietic stem cells or progenitor cells, (ii) The method for producing the genetically engineered hematopoietic stem cells or progenitor cells, comprising introducing into the cells (a) a guide RNA (gRNA) having a nucleotide sequence at least 90% identical to sequence number 4 or 46-47, and (b) a Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor). 110. The method according to embodiments 108 and 109, wherein the gRNA sequence targets the intron 12 / exon 13 junction of EMR2. 111. The method according to Embodiments 108-110, wherein the gRNA targets a nucleotide sequence including SEQ ID NO: 40. 112. The method according to any one of embodiments 108 to 111, wherein the method results in a nucleotide substitution in the sequence encoding the splice element of exon 13 of EMR2, the nucleotide substitution resulting in alternative splicing of the gene-encoded transcript. 113. The method according to Embodiment 112, wherein the alternative splicing of the transcript results in reduced expression of the exon 13 epitope of EMR2 and / or induces early codon termination and the production of mutant or truncated EMR2 compared to the wild-type equivalent. 114. The method according to any one of embodiments 108 to 112, wherein the base editor is a cytosine base editor and is BE4max. 115. The method according to any one of embodiments 108 to 112, wherein the base editor is an adenosine base editor, and is ABE8e. 116. The method according to any one of embodiments 108 to 115, which results in genetically engineered hematopoietic stem cells or progenitor cells having a reduced expression level of the EMR2 exon 13 epitope compared to equivalent wild-type cells. 117. The method according to any one of embodiments 108 to 116, performed on a plurality of hematopoietic stem cells or progenitor cells. 118. The method according to any one of embodiments 108 to 117, wherein a cell population according to any one of embodiments 60 to 67 is produced. 119. A method for producing genetically engineered hematopoietic stem cells or progenitor cells, (i) Prepare hematopoietic stem cells or progenitor cells (e.g., wild-type hematopoietic stem cells or progenitor cells), (ii) Introducing into the cells (a) guide RNA (gRNA) containing sequence numbers 1-3, and (b) a nuclease (e.g., endonuclease) (e.g., Cas9 endonuclease) that binds to the gRNA fused to cytosine deaminase or adenosine deaminase (base editor), (iii) A method for producing genetically engineered hematopoietic stem cells or progenitor cells, comprising introducing into the cells (a) a guide RNA (gRNA) that targets at least one further lineage-specific cell surface antigen, and (b) a nuclease (e.g., endonuclease) (e.g., Cas9 endonuclease) that binds to the gRNA fused to cytosine deaminase or adenosine deaminase (base editor). 120. A method for producing genetically engineered hematopoietic stem cells or progenitor cells, (i) Prepare genetically engineered hematopoietic stem cells and / or progenitor cells, (ii) Introducing into the cells (a) guide RNA (gRNA) containing at least 90% identical nucleotide sequences to SEQ ID NOs: 1-3, and (b) Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor), (iii) A method for producing genetically engineered hematopoietic stem cells and / or precursors, comprising introducing into the cells (a) a guide RNA (gRNA) that targets at least one further lineage-specific cell surface antigen, and (b) a Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor). 121. The method according to any one of Embodiments 119 to 120, which results in the genetically engineered hematopoietic stem cells or progenitor cells having a reduced expression level of the CD33 exon 2 epitope and / or a reduced expression level of the exon epitope of at least one further lineage-specific cell surface antigen compared to equivalent wild-type cells. 122. The method according to any one of embodiments 119 to 121, performed on a plurality of hematopoietic stem cells or progenitor cells. 123. The method or use of any of Embodiments 119 to 122, performed on a cell population comprising multiple hematopoietic stem cells and multiple hematopoietic progenitor cells. 124. The method according to any one of embodiments 119 to 123, wherein a cell population according to any one of embodiments 60 to 67 is produced. 125. The method according to any one of Embodiments 119 to 123, wherein the at least one further lineage-specific cell surface antigen is EMR2, and the method results in a decrease in the expression level of the epitope encoded by exon 13 of EMR2, and / or induces early codon termination and the production of mutant or truncated EMR2, compared to a wild-type equivalent. 126. The method according to embodiment 125, wherein the gRNA is sequence number 4 or 46-47. 127. The method according to any one of embodiments 97 to 126, wherein the nucleic acids of (a) and (b) are encoded in a vector introduced into the cell. 128. The method according to Embodiment 127, wherein the vector is a viral vector. 129. The method according to any one of embodiments 97 to 126, wherein the base editor is in the form of a protein, and (a) and (b) are introduced into the cell as a pre-formed ribonucleoprotein complex. 130. The method according to Embodiment 129, wherein the ribonucleoprotein complex is introduced into the cells via electroporation. 131. The method according to any one of embodiments 97 to 130, wherein the gRNA is a single-molecule guide RNA (sgRNA). 132. The method according to any one of embodiments 97 to 131, wherein the gRNA is a modified sgRNA. 133. The method according to any one of embodiments 97 to 132, wherein the gRNA is a chemically modified sgRNA. 134. The method according to any one of embodiments 97 to 133, wherein the hematopoietic stem cells or progenitor cells are CD34+. 135. The method according to any one of embodiments 97 to 133, wherein the hematopoietic stem cells or progenitor cells are derived from the target bone marrow cells or peripheral blood mononuclear cells (PBMCs). 136. The method according to Embodiment 135, wherein the subject has a hematopoietic disorder. 137. The method according to embodiment 135, wherein the subject is a healthy donor. 138. Genetically engineered hematopoietic stem cells or progenitor cells produced by the method or use described in any of Embodiments 97 to 137. 139. A cell population comprising multiple genetically engineered hematopoietic stem cells or progenitor cells as described in Embodiment 138 (for example, including hematopoietic stem cells, hematopoietic progenitor cells, or a combination thereof). 140. A method for treating a hematopoietic disorder, comprising administering to a subject in need thereof an effective amount of genetically engineered hematopoietic stem cells or progenitor cells as described in any of embodiments 1 to 43 and 138, or a cell population as described in any of embodiments 60 to 67 and 169. 141. Genetically engineered hematopoietic stem cells or progenitor cells according to any one of embodiments 1 to 20 and 138, or a cell population according to any one of embodiments 60 to 67 and 169, for use in the treatment of hematopoietic disorders, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or the cell population to a subject in need thereof, and further comprising administering an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33 to the subject thereof. 142. A combination for use in the treatment of hematopoietic disorders, comprising genetically engineered hematopoietic stem cells or progenitor cells as described in any of embodiments 1 to 20 and 138, or a cell population as described in any of embodiments 60 to 67 and 169, and a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or the cell population and the CD33-binding agent to a patient in need thereof. 143. Genetically engineered hematopoietic stem cells or progenitor cells according to any of embodiments 1-9, 21-29 and 138, or a cell population according to any of embodiments 60-67 and 169, for use in the treatment of hematopoietic disorders, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or the cell population to a subject in need thereof, and further comprising administering an effective amount of an EMR2-targeting agent comprising an antigen-binding fragment that binds to EMR2 to the subject thereof. 144. A combination for use in the treatment of hematopoietic disorders, comprising genetically engineered hematopoietic stem cells or progenitor cells as described in any of embodiments 1-9, 21-29, and 138, or a cell population as described in any of embodiments 60-67 and 169, and an EMR2-targeting agent comprising an antigen-binding fragment that binds to EMR2, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or the cell population and the EMR2-binding agent to a patient in need thereof. 145. Genetically engineered hematopoietic stem cells or progenitor cells according to any of embodiments 1-9, 43-59 and 138, or a cell population according to any of embodiments 60-67 and 169, for use in the treatment of hematopoietic disorders, wherein the treatment comprises administering an effective amount of the genetically engineered hematopoietic stem cells or progenitor cells or the cell population to a subject in need thereof, and further comprising administering to the subject an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33, and an effective amount of a CD33-targeting agent comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen. 146. A combination for use in the treatment of hematopoietic disorders, comprising: genetically engineered hematopoietic stem cells or progenitor cells as described in any of embodiments 1-9, 43-59 and 138, or a cell population as described in any of embodiments 60-67 and 169; a CD33-targeting agent comprising an antigen-binding fragment that binds to CD33; and a drug that targets at least one further lineage-specific cell surface antigen comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen, wherein the treatment comprises administering to a patient in need the genetically engineered hematopoietic stem cells or progenitor cells or the cell population, as well as an effective amount of the drug that binds to CD33 and the at least one further lineage-specific cell surface antigen. 147. The combination of the at least one further lineage-specific cell surface antigen described in Embodiment 145 and the genetically engineered hematopoietic stem cells or progenitor cells described in Embodiment 146, wherein the at least one further lineage-specific cell surface antigen is EMR2. 148. Genetically engineered hematopoietic stem cells or progenitor cells according to any one of embodiments 1 to 59 and 138, or a cell population according to any one of embodiments 60 to 67 and 169, for use in cancer immunotherapy. 149. The patient has hematopoietic disorders, and the patient has genetically engineered hematopoietic stem cells or progenitor cells according to any one of embodiments 1 to 59 and 138, or a cell population according to any one of embodiments 60 to 67 and 169, for use in cancer immunotherapy. 150. Genetically engineered hematopoietic stem cells or progenitor cells according to any one of embodiments 1 to 59 and 138, or a cell population according to any one of embodiments 60 to 67 and 169, for use in hematopoietic lipopulation in patients with hematopoietic disorders. 151. Genetically engineered hematopoietic stem cells or progenitor cells as described in any of embodiments 1 to 59 and 138, or a cell population as described in any of embodiments 60 to 67 and 169, for use in a method for treating hematopoietic disorders, wherein the genetically engineered hematopoietic stem cells or progenitor cells or the cell population described herein lipopure the patient. 152. Genetically engineered hematopoietic stem cells or progenitor cells according to any of embodiments 1-59 and 138, or cell populations according to any of embodiments 60-67 and 169, for use in immunotherapy to reduce the cytotoxic effect of agents targeting CD33 and / or at least one further lineage-specific cell surface antigen. 153. Genetically engineered hematopoietic stem cells or progenitor cells as described in any of Embodiments 1 to 59 and 138, or a cell population as described in any of Embodiments 60 to 67 and 169, for use in a method of immunotherapy using a drug that targets CD33 and / or at least one further lineage-specific cell surface antigen, wherein the genetically engineered hematopoietic stem cells or progenitor cells or the cell population described herein reduce the cytotoxic effect of the drug that targets CD33 and / or at least one further lineage-specific cell surface antigen. 154. The method, cells, drug, or combination according to any of Embodiments 140 to 153, wherein the genetically engineered hematopoietic stem cells or progenitor cells or the cell population are administered in combination with the drug that targets CD33 and / or at least one further lineage-specific cell surface antigen. 155. The method, cells, drugs, or combinations according to any of Embodiments 140 to 153, wherein the genetically engineered hematopoietic stem cells or progenitor cells or the cell population are administered prior to the drug that targets CD33 and / or at least one further lineage-specific cell surface antigen. 156. The method, cells, drugs, or combinations according to any of Embodiments 140-153, wherein the agent targeting CD33 and / or at least one further lineage-specific cell surface antigen is administered prior to the genetically engineered hematopoietic stem cells or progenitor cells or the cell population. 157. The hematopoietic disorder is a hematopoietic malignancy, as described by any of embodiments 140 to 156, by any of the methods, cells, drugs, uses, or combinations thereof. 158. The method, cells, drugs, use, or combination according to any of Embodiments 140 to 157, further comprising administering to the subject an effective amount of a drug that targets CD33 and comprises an antigen-binding fragment that binds to CD33, and / or an effective amount of a drug that targets at least one further lineage-specific cell surface antigen and comprises an antigen-binding fragment that binds to the at least one further lineage-specific cell surface antigen. 159. The method, cells, drugs, use, or combination according to Embodiment 158, wherein the drug targeting CD33 is an immune cell expressing a chimeric antigen receptor (CAR) comprising the antigen-binding fragment that binds to CD33, and the drug targeting at least one further lineage-specific cell surface antigen is an immune cell expressing a chimeric antigen receptor (CAR) comprising the antigen-binding fragment that binds to the at least one further lineage-specific cell surface antigen. 160. The methods, cells, drugs, uses, or combinations according to Embodiments 152-159, wherein the at least one further lineage-specific cell surface antigen is EMR2. 161. The method, cells, drugs, use, or combination described in Embodiment 159, wherein the immune cells are T cells. 162. The method, cells, drugs, use, or combination described in any of Embodiments 140 to 161, wherein the subject is a human patient having Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, or multiple myeloma. 163. The method, cells, drugs, uses, or combinations according to Embodiment 163, wherein the subject is a human patient having leukemia, which is acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia.

[0072] The following drawings are part of this specification and are included to further illustrate certain aspects of the present disclosure, which can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0073] [Figure 1] Exemplary illustrations of series-specific antigens for types 0, 1, 2, and 3 are presented. [Figure 2] This is a schematic diagram showing immune cells expressing a chimeric receptor that targets CD307, a type O lineage-specific cell surface antigen. Multiple myeloma (MM) cells and other CD307-expressing cells such as plasma cells are targeted by immune cells expressing the anti-CD307 chimeric receptor. [Figure 3] This is a schematic diagram showing immune cells expressing a chimeric receptor that targets CD33, a type 2 lineage-specific cell surface antigen. The image shows acute myeloid leukemia (AML) cells expressing 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 then be used to generate myeloid cells. [Figure 4A] The base editing scheme of CD33 is shown. Figure 4A shows details of the exon 1-3 region and possible splicing results. ag: splicing acceptor site (SA). gt: splicing donor site. Dotted lines represent possible splicing events. GO gentuzumab ozogamicin (antibody (Ab) icon) recognizes the epitope located in exon 2. [Figure 4B]The base editing mechanism of CD33 is shown. Figure 4B shows the intron 1 (lowercase) / exon 2 (uppercase) junction DNA sequence with exon 2 SA (red) and exon splicing enhancer region (yellow ESE) highlighted. [Figure 4C] The base editing mechanism of CD33 is shown. Figure 4C shows that the full-length CD33 mRNA (CD33FL) contains seven exons. Exon 2 encodes an Ig-like V-type domain. CD33Δ2 lacks exon 2 due to a common polymorphism (rs12459419) that results in a modified exon splicing enhancer (ESE) site by changing C to T. [Figure 5A] This demonstrates that ABE induces A>G conversion at the target site with up to 95% efficiency, with minimal indel formation and exon 2 skipping. Figure 5A shows the Sanger sequencing profile of edited cells compared to the wild-type genome sequence (top). Cytosine or adenine edits that mutate ESE or SA are indicated by arrows. [Figure 5B] ABE introduces A>G conversion at the target site with up to 95% efficiency, resulting in minimal indels and inducing exon 2 skipping. Figure 5B shows the results of HTS analysis by CRISPResso2 representing the editing results at the target site. Approximately 250 bp surrounding the target nucleotide was PCR-amplified from the extracted genomic DNA and sequenced using Illumina MiSeq. The results indicate that each target base acquired the intended mutation. [Figure 5C] ABE induces A>G conversion at the target site with up to 95% efficiency, resulting in minimal indel formation and exon 2 skipping. Figure 5C shows the evaluation of CD33 expression in edited cells 7 days after electroporation by FACS analysis using two different antibody clones, WM53 and P67.6, which recognize epitopes located within exon 2. After editing with BE4, approximately 30% of CD34+ cells express CD33, compared to less than 5% after editing with ABE. [Figure 5D]ABE induces exon 2 skipping with up to 95% efficiency in introducing A>G conversion at the target site, resulting in minimal indel formation. Figure 5D shows that editing of ESE or SA induces exon 2 skipping. Exon skipping in CD34+ edited cells was characterized by PCR against cDNA using a set of primers specific to CD33Δ2 (spanning exon junctions 1-3) or primers common to all isoforms (within exons 1, 5, and 7). PCR products were separated by polyacrylamide gel electrophoresis and visualized with SYBR-safe fluorescence. Under the gel, Sanger sequencing of the PCR products confirmed that exon 2 was absent in edited cells, while all other exons were intact. [Figure 6A] This study demonstrates that WT or CD33Δ2 monocytes differentiated in vitro exhibit normal phagocytic activity, and that CD34+CD33Δ2 cells possess resistance to GO cell cytotoxicity in vitro. Figure 6A shows that WT or CD33Δ2 monocytes differentiated in vitro exhibit comparable phagocytic activity when measured by the internal transport of E. coli bioparticles. The left side shows a representative FACs plot of internal transport of E. coli bioparticles by WT or CD33Δ2 monocytes differentiated in vitro. Treatment with cytochalasin D, an actin polymerization inhibitor, suppresses phagocytosis. The right side shows a graph quantifying phagocytosis. [Figure 6B] This study demonstrates that differentiated WT or CD33Δ2 monocytes in vitro exhibit normal phagocytic activity, and that CD34+CD33Δ2 cells possess resistance to GO cytotoxicity in vitro. Figure 6B shows that CD34+CD33Δ2 cells are resistant to GO cytotoxicity in vitro. Cells were incubated with GO for 48 hours, and cytotoxicity was analyzed by FACS. CD34+CD33Δ2 cells exhibit the same resistance to GO cytotoxicity as donors with the homozygous rs12459419 A14V SNP. [Figure 7A]This shows that CD34+CD33Δ2 cells engraft in vivo, reproduce a complete hematopoietic system, and exhibit resistance to gemtuzumab ozogamicin (GO). Figure 7A is a graph showing the frequency of human CD45+ cells in the bone marrow (BM) and spleen analyzed 16 weeks post-transplant, as well as the frequencies of myeloid progenitor cells (CD123) and lymphoid progenitor cells (CD10), as well as mature myeloid cells (CD14) and lymphoid cells (CD19), and T cells (CD3) within the human CD45 population. [Figure 7B] This demonstrates that CD34+CD33Δ2 engrafts in vivo, reproduces a complete hematopoietic system, and exhibits resistance to gemtuzumab ozogamicin (GO). Figure 7B shows representative images of H&E staining and anti-CD33 immunohistochemistry of BM in mice engrafted with CD34+WT or CD34+CD33Δ2. [Figure 7C] This study demonstrates that CD34+CD33Δ2 cells engraft in vivo, reproduce a complete hematopoietic system, and exhibit resistance to gemtuzumab ozogamicin (GO). Figure 7C shows that CD34+CD33Δ2 cells are GO-resistant in vivo. Peripheral blood (PB) of mice 12 weeks after transplantation was analyzed for the presence of CD33+CD14+ cells or CD33Δ2CD14+ cells. Mice were then injected with 2.5 ugr of GO, and blood was collected and bagged one week after GO treatment to evaluate the presence of myeloid cells in the PB and BM of humanized mice. Before GO treatment, CD34+WT transplanted mice and CD34+CD33Δ2 transplanted mice showed the same frequency of CD14+ cells in the PB (FACS plot above). One week after GO injection, CD33-CD14+ cells were detected in the PB and BM of mice engrafted with CD34+CD33Δ2 cells, but CD33+ and CD14+ cells were eradicated in the PB and BM of CD34+WT engrafted mice. [Figure 7D]This study demonstrates that CD34+CD33Δ2 engrafts in vivo, replicates a complete hematopoietic system, and exhibits resistance to gemtuzumab ozogamicin (GO). Figure 7D shows a graph of CD33-on-target AG editing at the target site (A7) in WT (unedited) or edited cells engrafted from bone marrow samples 16 weeks after transplantation. 16 weeks after transplantation, the CD33 locus was amplified from mouse bone marrow-derived genomic DNA. The amplicons were sequenced by HTS, and A-to-G editing at position A7 was quantified. [Figure 8A] The results of the off-target analysis are shown. Figure 8A is a table summarizing the top 19 off-target gene loci identified. [Figure 8B] The results of the off-target analysis are shown. Figure 8B is a graph evaluating the editing from A to G at position A7 of the top 19 off-target gene loci identified in human WT (unedited) or edited cells engrafted from bone marrow 16 weeks after transplantation. [Figure 8C] The results of the off-target analysis are shown. Figure 8C is a graph of the evaluation of indels at the top 19 off-target gene loci identified in human WT (unedited) or edited cells engrafted from bone marrow 16 weeks after transplantation. [Figure 9A] The base editing mechanisms of CD33 and EMR2 are shown, demonstrating that ABE introduces A>G conversion at target sites leading to double editing in CD34+ cells. Figure 9A (top) shows details of EMR2, exon 13, and gtgagt:splicing donor site (SD). Figure 9A (bottom) shows the intron 12 (lowercase) / exon 13 (uppercase) junction DNA sequence, with exon 13 SD (red) and the bolded protospacer highlighted. [Figure 9B] The base editing schemes for CD33 and EMR2 are shown, demonstrating that ABE introduces A>G conversion at target sites, leading to double editing in CD34+ cells. Figure 9B shows the Sanger sequencing profiles of edited cells compared to the wild-type genome sequence (top). Arrows indicate adenine editing that mutates SA in CD33 exon 2 or SD in EMR2 exon 13. [Figure 9C] The base editing schemes for CD33 and EMR2 are shown, demonstrating that ABE introduces A>G conversion at target sites, leading to double editing in CD34+ cells. Figure 9C shows FACS analysis of WT and edited cells one week after nucleofection. [Figure 10A] A schematic diagram of an exemplary chimeric receptor containing an antigen-binding fragment that targets CD33 is shown. Figure 10A: A typical chimeric receptor that targets CD33, containing an anti-CD33 scFv, a hinge domain, a transmembrane domain, a costimulatory domain, and a signaling domain. [Figure 10B] A schematic diagram of an exemplary chimeric receptor containing an antigen-binding fragment that targets CD33 is shown. Figure 10B: A CD33-targeting chimeric receptor containing an anti-CD33 scFv, a hinge domain derived from CD8, a transmembrane domain derived from CD8, and intracellular domains derived from CD28 and CD3ζ. [Figure 10C] A schematic diagram of an exemplary chimeric receptor containing an antigen-binding fragment that targets CD33 is shown. Figure 10C: A CD33-targeting chimeric receptor containing an anti-CD33 scFv, a hinge domain derived from CD8, a transmembrane domain derived from CD8, and intracellular domains derived from ICOS (or CD27, 4-1BB, or OX-40) and CD3ζ. [Figure 10D] A schematic diagram of an exemplary chimeric receptor containing an antigen-binding fragment that targets CD33 is shown. Figure 10D: A CD33-targeting chimeric receptor containing an anti-CD33 scFv, a hinge domain derived from CD8, a transmembrane domain derived from CD8, and intracellular domains derived from OX40, CD28, and CD3ζ. [Figure 11] This is a schematic diagram of an immunotoxin. [Figure 12A]Figure 12A: Western blot using a primary antibody that recognizes CD3ζ. The table shows the estimated molecular weight of each chimeric receptor tested. [Figure 12B] Figure 12B: Expression of anti-CD33 chimeric receptor in K562 cells transduced with an empty vector or a vector encoding the anti-CD33 chimeric receptor. Flow cytometry analysis showing an increase in the cell population positively stained for the anti-CD33 chimeric receptor. [Figure 13A] This shows that the anti-CD33 chimeric receptor binds to CD33. Figure 13A: Ponceau-stained protein gel. Lanes 1, 3, 5: CD33 molecules. Lanes 2, 4, 6: CD33 molecules + APC conjugate. [Figure 13B] This shows that the anti-CD33 chimeric receptor binds to CD33. Figure 13B: Western blot using a primary antibody that recognizes CD3ζ. Lanes 1, 3, and 5 contain the CD33 molecule and the co-incubated chimeric receptor, while lanes 2, 4, and 6 contain the CD33-APC conjugate and the co-incubated chimeric receptor. [Figure 13C] This shows that the anti-CD33 chimeric receptor binds to CD33. Figure 13C: Flow cytometry analysis showing an increase in the cell population expressing the anti-CD33 chimeric receptor and binding to CD33. [Figure 14] The images show the cytotoxicity of K562 cells by NK92 cells expressing the indicated chimeric receptor. A: CART1 and CART2 compared to an empty HIVzsG vector. B: CART3 compared to an empty HIVzsG vector. [Figure 15]This graph shows the cytotoxicity (represented as cytotoxicity rate on the y-axis) of CD33-deficient K562 cells by NK92 cells expressing the indicated chimeric receptor. A: Unsorted population of K562 cells pretreated with CD33-targeting CRISPR / Cas reagent. B: Single clone of CD33-deficient K562 cells. The columns, from left to right, correspond to an empty HIVzsG vector, CART1, CART2, and CART3. [Figure 16] Flow cytometry analysis of primary T cell populations is shown. A: Cell sorting based on the expression of T cell markers C4+, CD8+, or both CD4+CD8+. B: Relative expression of CD33 in the primary T cell population shown. [Figure 17] This image shows the cytotoxicity of K562 cells by primary T cells expressing the chimeric receptor shown. A: CD4+ T cells. B: CD4+ / CD8+ (CD4 / 8) and CD8+ (CD8). [Modes for carrying out the invention]

[0074] Cancer immunotherapy that targets antigens present on the surface of cancer cells is particularly challenging when the target antigens are also present on the surface of normal, non-cancer cells that are necessary for, or critically involved in, the development and / or survival of the target. Targeting these antigens may result in adverse effects on the target because the cytotoxic effects of immunotherapy are directed not only at cancer cells but also at these other cells.

[0075] The methods, nucleic acids, and cells described in this book enable the targeting of antigens (e.g., type 1 or type 2 antigens) present not only in cancer cells but also in cells important for the development and / or survival of the target. This method involves (1) reducing the number of cells possessing the target lineage-specific cell surface antigen using a drug that targets such antigen, and (2) replacing normal cells (e.g., non-cancer cells) that present the antigen and are therefore susceptible to drug administration with hematopoietic cells lacking the lineage-specific cell surface antigen. The methods described in this book can maintain surveillance of target cells, including cancer cells expressing the lineage-specific cell surface antigen of interest, and can also maintain a population of non-cancer cells expressing lineage-specific antigens that may be important for the development and / or survival of the target.

[0076] Therefore, this book describes the combined use of immune cells expressing chimeric receptors containing antigen-binding fragments that target lineage-specific cell surface antigens (e.g., CD33) with hematopoietic cells such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) that lack lineage-specific cell surface antigens, for the treatment of hematopoietic malignancies. This book also provides chimeric receptors, nucleic acids encoding them, vectors containing them, and immune cells (e.g., T cells) that express such chimeric receptors.

[0077] This disclosure also provides genetically engineered hematopoietic cells lacking lineage-specific antigens, such as those described herein, and methods for producing them (e.g., genome editing methods).

[0078] This document also describes the combined use of immune cells expressing chimeric receptors containing antigen-binding fragments targeting lineage-specific cell surface antigens (e.g., CD33) and at least one further lineage-specific cell surface antigen (e.g., EMR2) for treating hematopoietic malignancies, with hematopoietic cells such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) lacking lineage-specific cell surface antigens (maybe more than one). This document also provides chimeric receptors, nucleic acids encoding them, vectors containing them, and immune cells (e.g., T cells) expressing such chimeric receptors. This disclosure also provides genetically engineered hematopoietic cells lacking lineage-specific antigens, such as those described herein, and methods for producing them (e.g., genome editing methods).

[0079] This book also describes methods for genome editing of hematopoietic cells using CRISPR-based base editor systems. The use of CRISPR-based base editor systems enables high editing efficiency of HSCs / HSPCs using CRISPR-based cytosine and adenine base editors (CBE and ABE). CBE and ABE are Cas9 nickase fused to cytidine deaminase or adenosine deaminase, respectively, enabling precise base substitution in the target region without generating double-spin roots (DSBs). Base editors are considered safe editing tools because they avoid DSBs and eliminate unwanted indels, translocations, or rearrangements caused by DSBs.

[0080] This book demonstrates the highly effective use of base editors, particularly the adenosine base editor ABE8e, for modifying hematopoietic cells by specifically altering the nucleotide sequence of splice elements. Modification of splice elements results in alternative splicing of gene-encoded transcripts and further leads to a decrease in the expression level of gene-encoded epitopes, such as those encoded by exons of a gene. This book demonstrates the use of base editors and gRNAs to modify splice acceptor or exon enhancer sites within exon 2 of CD33. In particular, genome editing using the ABE8e base editor and a guide RNA specifically designed to target junction DNA sequences showed efficiency of up to 95% without indels.

[0081] This book also demonstrates the use of a base editor to modify the splice donor site in exon 13 of EMR2.

[0082] Furthermore, Cas9 nickase fused to cytidine deaminase or adenosine deaminase as described in this book can be used in mRNA or protein form. The latter form yields fewer off-target effects than the former and has been successfully used to edit HSCs, which engrafted in vivo and conferred resistance to GO to CD34 cells.

[0083] See, for example, Examples 1-5.

[0084] definition The terms “subject,” “individual,” and “patient” are used synonymously and refer to vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, human primates, non-human primates, or species such as mice, cattle, horses, dogs, or cats. In the context of this disclosure, the term “subject” also includes tissues and cells that can be cultured in vitro or ex vivo, or manipulated in vivo. The term “subject” may be used synonymously with the term “organism.”

[0085] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used synonymously. These refer to nucleotides in polymeric form of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Examples of polynucleotides include, but are not limited to, coding or non-coding regions of genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), small 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 in a polynucleotide may be further modified. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can also be modified after polymerization, for example, by conjugation with labeling agents.

[0086] The term "hybridization" refers to a reaction in which one or more polynucleotides react to form a stabilized complex via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur in Watson-Crick base pairings, Hoogstein bonds, or any other sequence-specific manner. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridized strand, or any combination thereof. Hybridization reactions can constitute a step in a broader process, such as the initiation of PCR or enzymatic cleavage of polynucleotides. A sequence that can hybridize with a given sequence is called its "complement."

[0087] The term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and which enables the expression of mRNA, protein, polypeptide, or peptide by a host cell when the vector is brought into contact with the cell under conditions sufficient to express mRNA, protein, polypeptide, or peptide in the cell. The vectors of this disclosure are not naturally occurring as a whole. Parts of the vectors may be naturally occurring. The non-naturally occurring recombinant expression vectors of this disclosure may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, may be synthesized or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides.

[0088] As used in this book, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by physical or chemical means.

[0089] The terms “antibody,” “antibody fragment,” “antibody fragment,” “functional fragment of an antibody,” or “antigen-binding moiety” are used synonymously to mean one or more fragments or portions of an antibody that possess the ability to specifically bind to a particular antigen (Holliger et al., Nat. Biotech. (2005) 23(9):1126). The antibody in this disclosure may be an antibody and / or a fragment thereof. Antibody fragments include Fab, F(ab')2, scFv, disulfide-bonded Fv, Fc, or variants and / or mixtures. The antibody may be a chimeric antibody, a humanized antibody, a single-chain antibody, or a bispecific antibody. All antibody isotypes, including IgA, IgD, IgE, IgG, and IgM, are included in this disclosure. Preferred IgG subtypes include IgG1, IgG2, IgG3, and IgG4. The variable region of the light or heavy chain of an antibody consists of a framework region interrupted by three hypervariable regions called complementarity-determining regions (CDRs). The CDR of the antibody or antigen-binding moiety in this case may be of non-human or human origin. The framework of the antibody or antigen-binding moiety in this case may be human, humanized, non-human (e.g., a mouse framework modified to reduce antigenicity in humans), or synthetic (e.g., a consensus sequence).

[0090] The antibody or antigen-binding portion in this case is approximately 10 -7 Less than M, approximately 10 -8 Less than M, approximately 10 -9 Less than M, approximately 10 -10 Less than M, approximately 10 -11 Less than M, or about 10 -12 Dissociation constant less than M (K D ) can be specifically bound to the antibody. The affinity of the antibody according to this disclosure can be easily determined using conventional techniques (see, for example, Scatchard et al., Ann. NYAcad. Sci. (1949) 51:660, and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents).

[0091] The terms “chimeric receptor,” “chimeric antigen receptor,” or alternatively “CAR” are used synonymously throughout and refer to recombinant polypeptide constructs comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to here as the “intracellular signaling domain”) containing a functional signaling domain derived from a stimulating molecule as defined below. Lee et al., Clin. Cancer Res. (2012) 18(10):2780, Jensen et al., Immunol Rev. (2014) 257(1):127. In one embodiment, the stimulating molecule is a zeta chain associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. The co-stimulatory molecule may be 4-1BB (i.e., CD137), CD27 and / or CD28, or fragments of these molecules. In another embodiment, CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulating molecule. CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulating molecule. Alternatively, CAR comprises a chimeric fusion protein comprising an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulating molecule. The antigen-recognition portion of CAR, encoded by a nucleic acid sequence, may contain any series-specific antigen-binding antibody fragment.The antibody fragment may include one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or any combination thereof.

[0092] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity via a defined signaling pathway, either by generating second messengers or by acting as an effector in response to such messengers.

[0093] The terms “zeta” or alternatively “zeta chain,” “CD3 zeta,” or “TCR zeta” are defined as the protein provided as GenBank accession numbers NP_932170, NP_000725, or XP_011508447, or as homogeneous residues derived from non-human species such as mice, rodents, monkeys, and apes, while the “zeta-stimulating domain” or alternatively “CD3 zeta-stimulating domain” or “TCR zeta-stimulating domain” are defined as amino acid residues derived from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signals necessary for T cell activation.

[0094] The terms "genetically engineered" or "genetically modified" refer to cells being manipulated by genetic engineering, such as genome editing. That is, the cells contain heterologous sequences that are not naturally present in the cells. Typically, these heterologous sequences are introduced via vector systems or other means for introducing nucleic acid molecules into cells, including liposomes. The heterologous nucleic acid molecules may be integrated into the cell's genome or exist extrachromosomally, for example, in the form of plasmids. The term also includes embodiments of introducing genetically engineered, isolated CAR polypeptides into cells.

[0095] The term "self-derived" refers to any material that originates from the same individual as material that is later reintroduced into the same individual.

[0096] The term "homogenetic" refers to any material originating from different animals belonging to the same species as the individual into which the material is introduced. Two or more individuals are said to be homogenous if their genes at one or more loci are not identical.

[0097] The term "cell lineage" refers to a group of cells that share a common ancestor and develop from the same type of identifiable cell into specific identifiable / functional cells. The cell lineages used in this book include, but are not limited to, those of the respiratory, prostate, pancreas, mammary gland, kidney, intestine, nervous system, skeletal system, blood vessels, liver, hematopoiesis, muscle, or heart.

[0098] When used in relation to the gene expression or function of lineage-specific antigens, the term "inhibition" refers to a reduction in the level of gene expression or function of the lineage-specific antigen, and inhibition is a result of interference with gene expression or function. Inhibition may be complete, in which case there is no detectable expression or function, or it may be partial. Partial inhibition can range from near-complete inhibition to almost no inhibition. By eliminating specific target cells, CAR T cells can effectively inhibit the overall expression of a particular cell lineage.

[0099] Cells such as hematopoietic cells that are "deficient in lineage-specific antigens" refer to cells whose expression levels of lineage-specific antigens are substantially reduced compared to naturally occurring equivalents, such as endogenous hematopoietic cells of the same type, or cells that do not express lineage-specific antigens, i.e., cells that cannot be detected by standard assays such as FACS. In some cases, the expression level of lineage-specific antigens in "antigen-deficient" cells may be less than approximately 40% (e.g., 30%, 20%, 15%, 10%, 5%, or less) of the expression level of the same lineage-specific antigens in naturally occurring equivalents.

[0100] As used in this book, the term "splice element" includes splice acceptor sites, splice donor sites, splice enhancer sites, and splice silencer sites.

[0101] In this book, the term "approximately" refers to a range of ±5% of a given value. For example, an expression level of approximately 40% could include any expression level between 35% and 45%.

[0102] Drugs that target lineage-specific cell surface antigens Aspects of this disclosure provide agents that target, for example, lineage-specific cell surface antigens (or more) on target cancer cells (e.g., agents that target CD33, e.g., agents comprising an antigen-binding fragment that binds to CD33, and agents that target at least one further lineage-specific cell surface antigen, e.g., agents comprising an antigen-binding fragment that binds to at least one further lineage-specific cell surface antigen). Such agents may include an antigen-binding fragment that binds to and targets lineage-specific cell surface antigens (or more). In some cases, the antigen-binding fragment may be a single-chain antibody (scFv) that specifically binds to the lineage-specific antigen.

[0103] A. Lineage-specific cell surface antigens In this book, the terms “series-specific,” “series-specific cell surface antigen,” and “cell surface series-specific antigen” may be used synonymously and refer to any antigen associated with one or more populations of a cell lineage(s) that is sufficiently present on the cell surface. For example, an antigen may be present in one or more populations of a cell lineage(s) but absent (or at reduced levels) on the cell surface of other cell populations.

[0104] Generally, lineage-specific cell surface antigens can be classified based on several factors, including whether the antigen and / or the cell population presenting it are necessary for the survival and / or development of the host organism. Table 1 below provides an overview of exemplary types of lineage-specific antigens. See also Figure 1. [Table 1]

[0105] As shown in Table 1 and Figure 1, type O lineage-specific cell surface antigens are necessary for tissue homeostasis and survival, and cell types possessing type O lineage-specific cell surface antigens may also be necessary for the survival of the subject. Therefore, given the importance of type O lineage-specific cell surface antigens or cells possessing type O lineage-specific cell surface antigens in homeostasis and survival, inhibiting or eliminating such antigens and cells possessing such antigens may be detrimental to the survival of the subject, making it difficult to target this category of antigens using conventional CAR T cell immunotherapy. Hence, lineage-specific cell surface antigens (such as type O lineage-specific antigens) and / or cell types possessing such antigens may be necessary for survival, for example, by performing essential and non-overlapping functions in the subject, in which case this type of lineage-specific antigen may be unsuitable as a target for CAR T cell-based immunotherapy.

[0106] Unlike type O antigens, type I lineage-specific cell surface antigens and cells possessing type I lineage-specific cell surface antigens are not required for the homeostasis or survival of the target tissue. Targeting type I lineage-specific cell surface antigens is unlikely to result in adverse outcomes for the target. For example, CAR T cells engineered to target CD307, a type I antigen uniquely expressed in both normal plasma cells and multiple myeloma (MM) cells, lead to the elimination of both cell types (Figure 2) (Elkins et al., Mol Cancer Ther. (2012) 10:2222). However, because plasma cell lineages are consumed for the survival of the organism, CD307 and other type I lineage-specific antigens are suitable antigens for CAR T cell-based immunotherapy. Type I class lineage-specific antigens can be expressed in a wide variety of different tissues, including the ovaries, testes, prostate, breast, endometrium, and pancreas. In some embodiments, the drug targets a lineage-specific cell surface antigen that is a type I antigen.

[0107] Targeting type 2 antigens presents significant challenges compared to targeting type 1 antigens. Type 2 antigens are characterized by (1) the antigen being unnecessary for the organism's survival (i.e., not required for survival), and (2) the cell lineage possessing the antigen being essential for the organism's survival (i.e., a specific cell lineage is required for survival). For example, CD33 is a type 2 antigen expressed in both normal myeloid cells and acute myeloid leukemia (AML) cells (Dohner et al., (2015) NEJM 373:1136). As a result, CAR T cells engineered to target the CD33 antigen may lead to the death of both normal and AML cells, which may be incompatible with the target's survival (Figure 3). In some embodiments, the drug targets the cell surface of lineage-specific antigens that are type 2 antigens.

[0108] A wide variety of antigens can be targeted by the methods and compositions of this disclosure. Monoclonal antibodies against these antigens may be commercially purchased or produced using standard techniques, including, as described above, immunizing animals with the antigen of interest and then performing a standard somatic cell hybridization technique as described in Kohler and Milstein, Nature (1975) 256:495. Antibodies or nucleic acids encoding antibodies can be sequenced using any standard DNA or protein sequencing technique.

[0109] In some embodiments, the lineage-specific cell surface antigens targeted using the methods and cells described herein are lineage-specific cell surface antigens of leukocytes or subpopulations of leukocytes. In some embodiments, the lineage-specific cell surface antigens are antigens associated with myeloid cells. In some embodiments, the lineage-specific cell surface antigens are cell membrane differentiation antigens (CDs). Examples of CD antigens, but not limited to, are 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, and CD2. 6, CD27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD 42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD6 6a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD73, 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、CD 120a、CD120b、CD121a、CD121b、CD121a、CD121b、CD122、CD123、CD124、CD125 、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、C D137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、C D146、CD147、CD148、CD150、CD152、CD152、CD153、CD154、CD155、CD156a、CD 156b、CD156c、CD157、CD158b1、CD158b2、CD158d、CD158e1 / e2、CD158f、CD15 8g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD163 、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b 、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD17 9b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、 CD194、CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD202b、CD203 c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CDw210b、CD212、CD21 3a1, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD2 34, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256CD257, CD258, 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, CD29 7, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, 306, CD3 Includes 07a, CD307b, 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.

[0110] In some embodiments, the lineage-specific cell surface antigens are 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.

[0111] In some embodiments, the lineage-specific cell surface antigen is CD33.

[0112] Alternatively, or furthermore, the lineage-specific cell surface antigen may be a cancer antigen, for example, a lineage-specific cell surface antigen that is differentially present on cancer cells. In some embodiments, the cancer antigen is an antigen specific to a tissue or cell lineage. Examples of lineage-specific cell surface antigens associated with specific types of cancer include, but are not limited to, CD20, CD22 (non-Hodgkin lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (acute myeloid leukemia (AML)), CD10 (gp100) (common (pre-B) acute lymphocytic leukemia and malignant melanoma), 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 (gynecologic cancers, cholangiocarcinoma and pancreatic ductal adenocarcinoma), and prostate-specific membrane antigens.

[0113] In some embodiments, the lineage-specific cell surface antigen is CD33 and is associated with AML cells. In some embodiments, the lineage-specific cell surface antigen is EMR2 and is associated with AML cells.

[0114] B. Antigen-binding fragments Any antibody or its antigen-binding fragment (e.g., one that binds to CD33 or at least one further lineage-specific cell surface antigen, e.g., EMR2) can be used to construct agents that target the lineage-specific cell surface antigens described herein. Such antibodies or antigen-binding fragments can be prepared by conventional methods, for example, using hybridoma technology or recombinant technology.

[0115] For example, antibodies specific to a target lineage-specific antigen can be produced by conventional hybridoma techniques. Using cell surface lineage-specific antigens that can be coupled to carrier proteins such as KLH, host animals can be immunized to produce antibodies that bind to the complex. The immunization pathways and schedules for host animals generally follow established conventional techniques for antibody stimulation and production, as further described herein. General techniques for the production of mouse antibodies, humanized antibodies, and human antibodies are known in the art and described herein. It is intended that antibody-producing cells of any mammalian subject, including humans, or derived therefrom, can be manipulated to serve as a basis for producing mammalian hybridoma cell lines, including humans. Typically, a certain amount of immunogen, including those described herein, is inoculated intraperitoneally, intramuscularly, intraorally, subcutaneously, intraplantarally, and / or intradermally in a host animal.

[0116] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique described in Kohler and Milstein Nature (1975) 256:495-497 or a modified version described by Buck et al., In Vitro (1982) 18:377-381. Hybridization may utilize available myeloma strains, including but not limited to X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, Calif., USA. Generally, this technique involves fusing myeloma cells and lymphoid cells using a fusion agent such as polyethylene glycol or by electrical means 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 eliminate unhybridized parent cells. All culture media described in this book can be used to culture hybridomas that secrete monoclonal antibodies, with or without serum supplementation. As an alternative to cell fusion techniques, EBV immortalized B cells may be used to produce the TCR-like monoclonal antibodies described in this book. The hybridomas are cultured on a large scale, subcloned as needed, and the supernatant is assayed for antiimmunogen activity using conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[0117] Hybridomas usable as antibody sources include all derivatives and progeny cells of parental 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 procedures. Monoclonal antibodies can be isolated from culture media or body fluids by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if necessary. If undesirable activity is present, it can be removed, for example, by passing the preparation through an adsorbent made of an immunogen bound to a solid phase, thereby eluting or releasing the desired antibody from the immunogen. Immunizing a host animal with a fragment containing a target amino acid sequence conjugated to an immunogenic protein in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or a soy trypsin inhibitor, using a target antigen or a bifunctional agent or derivatizer, such as maleimidobenzoylsulfosuccinimide (conjugation via a cysteine ​​residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR (where R and R1 are different alkyl groups), can result in a population of antibodies (e.g., monoclonal antibodies).

[0118] If necessary, the antibody of interest (e.g., one produced by a hybridoma) may be sequenced, and the polynucleotide sequence may then be cloned into a vector for expression or proliferation. The sequence encoding the antibody of interest may be maintained in the vector within host cells, and the host cells may then be expanded cultured and frozen for future use. Alternatively, the polynucleotide sequence may be used for genetic engineering to "humanize" the antibody or to improve its affinity (affinity maturation) or other characteristics. For example, the constant region may be engineered to more closely resemble the human constant region to avoid an immune response if the antibody is to be used in human clinical trials and treatments. It may be desirable to genetically engineer the antibody sequence to obtain high affinity for lineage-specific antigens. It will be obvious to those skilled in the art that modifications to one or more polynucleotides in an antibody may maintain its binding specificity to the target antigen.

[0119] In other embodiments, fully human antibodies can be obtained using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Humanized antibodies or human antibodies can also be generated using transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response. Examples of such techniques include Xenomouse™ from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In alternative methods, antibodies can be recombinantly produced by phage display or yeast techniques. See, for example, U.S. Patents 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winter et al., Annu. Rev. Immunol. (1994) 12:433-455. Alternatively, human antibodies and antibody fragments may be produced in vitro from an immunoglobulin variable (V) domain gene repertoire derived from non-immunized donors using phage display technology (McCafferty et al., Nature (1990) 348:552-553).

[0120] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by standard methods. For example, the F(ab')2 fragment can be produced by pepsin digestion of the antibody molecule, and the Fab fragment can be generated by reducing the disulfide crosslinks of the F(ab')2 fragment.

[0121] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombination techniques. For instance, using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of a monoclonal antibody), DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in one or more expression vectors, which are then transfected into host cells that otherwise do not produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, thereby achieving the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication WO87 / 04462. Next, the DNA may be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (Morrison et al., Proc.Nat.Acad.Sci.(1984)81:6851), or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies, that have binding specificity to a target antigen can be prepared.

[0122] Techniques developed for the production of "chimeric antibodies" are well known in this field. See, for example, Morrison et al., Proc. Natl. Acad. Sci. (1984) 81, 6851, Neuberger et al., Nature (1984) 312, 604, and Takeda et al., Nature (1984) 314:452.

[0123] Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. (1989) 86:10029-10033. In one example, the variable regions of the VH and VL of a parental non-human antibody are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Next, the same molecular modeling analysis is used to identify framework amino acid residues predicted to be important for the formation of the correct CDR structure. In parallel, the parental VH and VL sequences are used as search queries to identify human VH and VL chains with amino acid sequences homologous to those of the parental non-human antibody from any antibody gene database. Next, human VH and VL acceptor genes are selected.

[0124] The CDR region within the selected human acceptor gene can be replaced with a CDR region derived from the parental non-human antibody or its functional variant. If necessary, the corresponding residue in the human acceptor gene can be substituted using residues within the parental chain framework region that are predicted to be important for interaction with the CDR region (see description above).

[0125] Single-chain antibodies can be prepared via recombination techniques, which involve linking a nucleotide sequence encoding a heavy-chain variable region with a nucleotide sequence encoding a light-chain variable region. Preferably, a mobile linker is incorporated between the two variable regions. Alternatively, phage or yeast scFv libraries can be produced by applying techniques reported for the production of single-chain antibodies (U.S. Patents 4,946,778 and 4,704,692), and scFv clones specific to lineage-specific antigens can be identified from the library according to a standard procedure. Positive clones may be subjected to further screening to identify those that bind to lineage-specific cell surface antigens.

[0126] In some cases, the target lineage-specific cell surface antigen is CD33, and the antigen-binding fragment specifically binds to CD33, such as human CD33. Exemplary amino acid and nucleic acid sequences of the heavy chain and light chain variable regions of anti-human CD33 antibodies are shown below. CDR sequences are shown in bold, and amino acid sequences are underlined.

[0127] Amino acid sequence of the anti-CD33 heavy chain variable region (SEQ ID NO: 5) [ka]

[0128] Nucleic acid sequence of the anti-CD33 heavy chain variable region (SEQ ID NO: 6) CAGGTGCAGCTGCAGCAGCCCGGCGCCGAGGTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGATCAAGCAGACCCCCGGCCAGGGCCTGGAGTGGGTGGGCGTGATCTACCCCGGCAACGACGACATCAGC TACAACCAGAAGTTCCAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCACCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGAGGTGAGGCTGAGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC

[0129] Amino acid sequence of the anti-CD33 light chain variable region (SEQ ID NO: 7) [ka]

[0130] Nucleic acid sequence of the anti-CD33 heavy chain variable region (SEQ ID NO: 8) GAGATCGTGCTGACCCAGAGCCCCGGCAGCCTGGCCGTGAGCCCCGGCGAGAGGGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCAGCCAGAAGAACTACCTGGCCTGGTACCAGCAGATCCCCGGCCAGAGCCCCAGGCTGCTGATCTACTGGG CCAGCACCAGGGAGAGCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTACTGCCACCAGTACCTGAGCAGCAGGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGG

[0131] Anti-CD33 antibody-binding fragments for use in constructing CD33-targeting agents described in this document may contain the same heavy and / or light chain CDR regions as those of SEQ ID NO: 5 and SEQ ID NO: 7. Such antibodies may contain amino acid residue changes in one or more of the framework regions. In some cases, the anti-CD33 antibody fragment may contain a heavy chain variable region that shares at least 70% sequence identity with SEQ ID NO: 5 (e.g., 75%, 80%, 85%, 90%, 95%, or more) and / or a light chain variable region that shares at least 70% sequence identity with SEQ ID NO: 7 (e.g., 75%, 80%, 85%, 90%, 95%, or more).

[0132] The "percent identicality" of the two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc.Natl.Acad.Sci.USA(1990)87:2264-68, modified as described in Karlin and Altschul Proc.Natl.Acad.Sci.USA(1993)90:5873-77. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J.Mol.Biol.(1990)215:403-10. Performing a BLAST protein search using the XBLAST program with a score of 50 and a word length of 3 will yield amino acid sequences homologous to the protein molecule disclosed herein. If a gap exists between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. (1997) 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used.

[0133] C. Immune cells expressing chimeric receptors In some embodiments, the drugs that target lineage-specific cell surface antigens as described herein are immune cells expressing chimeric receptors containing antigen-binding fragments (e.g., single-chain antibodies) capable of binding to lineage-specific antigens (e.g., CD33, EMR2). Recognition of target cells (e.g., cancer cells) with lineage-specific antigens on their cell surface by the antigen-binding fragment of the chimeric receptor transmits an activation signal to the signaling domain(s) of the chimeric receptor (e.g., a co-stimulatory signaling domain and / or cytoplasmic signaling domain), thereby activating the effector function in immune cells expressing the chimeric receptor.

[0134] As used in this text, a chimeric receptor refers to a non-native molecule that can be 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 domains derived from different molecules. In addition to the antigen-binding fragments described herein, a chimeric receptor may further include one or more of the following: a hinge domain, a transmembrane domain, at least one costimulatory domain, and a cytoplasmic signaling domain. In some embodiments, a chimeric receptor includes, from N-terminus to C-terminus, an antigen-binding fragment that binds to a cell surface lineage-specific antigen, a hinge domain, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, a chimeric receptor further includes at least one costimulatory domain.

[0135] In some embodiments, the chimeric receptors described herein include a hinge domain that may be located between the antigen-binding fragment and the transmembrane domain. A hinge domain is an amino acid segment commonly found between two domains of a protein that can allow for protein mobility and the movement of one or both domains toward each other. Any amino acid sequence that results in such mobility and movement of the antigen-binding fragment toward another domain of the chimeric receptor can be used.

[0136] The hinge domain may contain approximately 10 to 200 amino acids, for example, 15 to 150 amino acids, 20 to 100 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain may be approximately 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 acid lengths.

[0137] In some embodiments, the hinge domain is a naturally occurring hinge domain of a protein. Any hinge domain of any protein known in the Art to contain a hinge domain is suitable for use in the chimeric receptor described herein. In some embodiments, the hinge domain is at least a portion of a naturally occurring hinge domain of a protein that confers mobility to the chimeric receptor. In some embodiments, the hinge domain is of CD8α or CD28α. In some embodiments, the hinge domain is a portion of a CD8α hinge domain, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of a CD8α or CD28α hinge domain.

[0138] The hinge domains 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 domain is a hinge domain that binds the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain is of the antibody and includes the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain includes the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain includes the hinge domain of the antibody and 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 includes the hinge region of the IgG1 antibody and the CH2 and CH3 constant regions. In some embodiments, the hinge region includes the hinge region of the IgG1 antibody and the constant CH3 region.

[0139] Within the scope of this disclosure are also chimeric receptors that include a hinge domain which is a peptide not found in nature. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor is (Gly x Ser) n A peptide linker such as a linker, where x and n are independently integers from 3 to 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or more.

[0140] Further peptide linkers that may be used in the hinge domain of the chimeric receptors described in this book are known in the art. See, for example, Wriggers et al. Current Trends in Peptide Science (2005) 80(6):736-746 and PCT Publication WO2012 / 088461.

[0141] In some embodiments, the chimeric receptors described herein may include a transmembrane domain. The transmembrane domain for use in the chimeric receptor may be any form known in the art. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable in the cell membrane, preferably the eukaryotic cell membrane. Transmembrane domains suitable for use in the chimeric receptors used herein can be obtained from naturally occurring proteins. Alternatively, the transmembrane domain may be a synthetic, naturally occurring protein segment, such as a thermodynamically stable hydrophobic protein segment in the cell membrane.

[0142] Transmembrane domains are classified based on their topology, including the number of times the transmembrane domain crosses the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, while multi-pass membrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). In some embodiments, the transmembrane domain is a single-pass transmembrane domain. In some embodiments, the transmembrane domain is a single-pass transmembrane domain with the N-terminus of the chimeric receptor facing the extracellular side of the cell and the C-terminus of the chimeric receptor facing the intracellular side of the cell. In some embodiments, the transmembrane domain is derived from a single-pass transmembrane protein. In some embodiments, the transmembrane domain is of CD8α. In some embodiments, the transmembrane domain is of CD28. In some embodiments, the transmembrane domain is of ICOS.

[0143] In some embodiments, the chimeric receptors described herein include one or more co-stimulatory signaling domains. As used herein, the term “co-stimulatory signaling domain” refers to at least a portion of a protein that mediates intracellular signaling to induce immune responses, such as effector function. The co-stimulatory signaling domains of the chimeric receptors described herein may be cytoplasmic signaling domains derived from co-stimulatory proteins that transmit signals and modulate responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

[0144] In some embodiments, the chimeric receptor comprises multiple (at least two, three, four, or more) co-stimulatory signaling domains. In some embodiments, the chimeric receptor comprises multiple co-stimulatory signaling domains obtained from different co-stimulatory proteins. In some embodiments, the chimeric receptor does not contain any co-stimulatory signaling domains.

[0145] Generally, many immune cells require co-stimulation in addition to antigen-specific signaling to promote cell proliferation, differentiation, and survival, and to activate cellular effector functions. Activation of co-stimulatory signaling domains within host cells (e.g., immune cells) can induce cells to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. Any co-stimulatory signaling domain of a co-stimulatory protein may be suitable for use in the chimeric receptors described herein. The type(s) of co-stimulatory signaling domains is selected based on factors such as the type of immune cell expressing the chimeric receptor (e.g., primary T cells, T cell lines, NK cell lines) and the desired immune effector function (e.g., cytotoxicity). Examples of co-stimulatory signaling domains for use in chimeric receptors include, but are not limited to, cytoplasmic signaling domains of co-stimulatory proteins, including 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 co-stimulatory domain is derived from 4-1BB, CD28, or ICOS. In some embodiments, the co-stimulatory domain is derived from CD28, and the chimeric receptor includes a second co-stimulatory domain derived from 4-1BB or ICOS.

[0146] In some embodiments, the co-stimulatory domain is a fusion domain comprising multiple co-stimulatory domains or a portion of multiple co-stimulatory domains. In some embodiments, the co-stimulatory domain is a fusion of co-stimulatory domains derived from CD28 and ICOS.

[0147] In some embodiments, the chimeric receptors described herein include a cytoplasmic signaling domain. Any cytoplasmic signaling domain may be used in the chimeric receptors described herein. Generally, cytoplasmic signaling domains stimulate cellular responses, such as inducing cellular effector functions (e.g., cytotoxicity), by relaying signals, such as the interaction between an extracellular ligand-binding domain and its ligand.

[0148] As will be apparent to those skilled in the art, the factor involved in T cell activation is the phosphorylation of the immunoreceptor-activating tyrosine motif (ITAM) in the cytoplasmic signaling domain. Any ITAM-containing domain known in the art can be used to construct the chimeric receptors described herein. Generally, ITAM motifs may include those obtained by separating two repeats of the amino acid sequence YxxL / I by 6-8 amino acids (where each x is independently any amino acid), resulting in the conserved motif YxxL / Ix(6-8)YxxL / I. In some embodiments, the cytoplasmic signaling domain is derived from CD3ζ.

[0149] Exemplary chimeric receptors are shown in Tables 2 and 3 below. [Table 2]

[0150] The following are nucleic acid sequences of exemplary components for constructing a chimeric receptor.

[0151] CD28 intracellular signaling domain - DNA - human (SEQ ID NO: 15) ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0152] ICOS intracellular signaling domain - DNA - human (SEQ ID NO: 16) CTATCAATTTTTGATCCTCCTCCTTTTAAAGTAACTCTTACAGGAGGATATTGCATATTATGAATCACAACTTTGTTGCCAGCTGAAGTTCTGGTTACCCATAGGATTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTCGACCTAAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCG CGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGGACAAGAGACGTGGCCGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAACCCTCAGGAAGGCCTTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGGAGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACAAGGACCACTACGACGCCCTTCCACATGCAGGCCTGCCCCCTCGC

[0153] CD28 / ICAS ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGC TATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0154] In some embodiments, the nucleic acid sequence encodes an antigen-binding fragment comprising a heavy chain variable region bound to CD33 and having the same CDR as the CDR of SEQ ID NO: 5, and a light chain variable region having the same CDR as the CDR of SEQ ID NO: 7. In some embodiments, the antigen-binding fragment comprises the heavy chain variable region presented in SEQ ID NO: 5 and the light chain variable region presented in SEQ ID NO: 7. In some embodiments, the chimeric receptor further comprises at least a transmembrane domain and a cytoplasmic signaling domain. In some embodiments, the chimeric receptor further comprises a hinge domain and / or a co-stimulatory signaling domain.

[0155] Table 3 presents exemplary chimeric receptors described in this book. The exemplary constructs have an antigen-binding fragment, a transmembrane domain, and a cytoplasmic signaling domain, from N-terminus to C-terminus. In some examples, the chimeric receptor further includes a hinge domain located between the antigen-binding fragment and the transmembrane domain. In some examples, the chimeric receptor further includes one or more costimulatory domains, which may be located between the transmembrane domain and the cytoplasmic signaling domain. [Table 3]

[0156] The amino acid sequences of the exemplary chimeric receptors listed in Table 3 above are shown below.

[0157] CART1 amino acid sequence (SEQ ID NO: 18) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTK LEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLR YFDVWGQGTTVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0158] CART2 amino acid sequence (SEQ ID NO: 19) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLE IKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFD VWGQGTTVTVSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0159] CART3 amino acid sequence (SEQ ID NO: 20) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0160] CART8 amino acid sequence (SEQ ID NO: 21) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLTKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0161] CART4 dual amino acid sequence (SEQ ID NO: 22) MWLQSLLLLGTVACSISIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF GGGTKLEIGSTSGSGKPGSGEGSTKGLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI YYCAKHYYYGGSYAMDYWGQGTSVTVSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFII FWVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0162] CART5 dual amino acid sequence (SEQ ID NO: 23) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTIS SVQPEDLAIYYCHQYLSSRTFGQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVI YPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0163] CART6 amino acid sequence (SEQ ID NO: 24) MWLQSLLLLGTVACSISIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIGST SGSGKPGSGEGSTKGLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0164] CART7 amino acid sequence (SEQ ID NO: 25) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTFG QGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVY YCAREVRLRYFDVWGQGTTVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0165] The nucleic acid sequences of the exemplary chimeric receptors listed in Table 3 above are shown below.

[0166] CART1 nucleic acid sequence (SEQ ID NO: 26)

[0167] CART2 nucleic acid sequence (SEQ ID NO: 27)

[0168] CART3 nucleic acid sequence (SEQ ID NO: 28)

[0169] CART4dual nucleic acid sequence (SEQ ID NO: 29)

[0170] CART5 dual nucleic acid sequence (SEQ ID NO: 30)

[0171] CART6 nucleic acid sequence (SEQ ID NO: 31)

[0172] CART7 nucleic acid sequence (SEQ ID NO: 32)

[0173] Furthermore, this book also envisions immune cells expressing chimeric receptors that target EMR2 in addition to those that target CD33 in AML patients. This can be achieved through two different approaches: 1) separately generating immune cells expressing anti-CD33 chimeric receptors and immune cells expressing anti-EMR2 chimeric receptors and injecting both types of immune cells separately into the patient, or 2) simultaneously generating immune cells that target both CD33 and EMR2 (Kakarla et al., Cancer (2014) 2:151).

[0174] Any of the chimeric receptors described in this book can be prepared by standard methods such as recombinant techniques. The methods for preparing chimeric receptors in this book involve generating nucleic acids encoding polypeptides that each of the domains of the chimeric receptor includes an antigen-binding fragment and, optionally, a hinge domain, a transmembrane domain, at least one costimulatory signaling domain, and a cytoplasmic signaling domain. In some embodiments, the nucleic acids encoding each of the components of the chimeric receptor are combined using recombinant techniques.

[0175] Each sequence of a component of a chimeric receptor can be obtained from any of the various sources known in the art by a standardized technique, such as PCR amplification. In some embodiments, one or more sequences of components of the chimeric receptor are obtained from human cells. Alternatively, one or more sequences of components of the chimeric receptor can be synthesized. Each sequence of a component (e.g., a domain) can be linked directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker) to form a nucleic acid sequence encoding the chimeric receptor using methods such as PCR amplification or ligation. Alternatively, the nucleic acid encoding the chimeric receptor may be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0176] Mutations of one or more residues in one or more of the components of the chimeric receptor (e.g., antigen-binding fragment) before or after joining the arrays of each of the components. In some embodiments, one or more mutations in the components of the chimeric receptor can be made to modulate (increase or decrease) the affinity of the component for the target (e.g., the antigen-binding fragment for the target antigen) and / or to modulate the activity of the component.

[0177] Any of the chimeric receptors described herein can be introduced into and expressed in suitable immune cells via 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 T cells that express CD8 (CD8 + ) or CD8 and CD4 (CD8 + / CD4 + ). In some embodiments, the T cells are T cells of established T cell lines, such as 293T cells or Jurkat cells.

[0178] Primary T cells can be obtained from any source, such as tissues such as peripheral blood mononuclear cells (PBMCs), bone marrow, spleen, lymph nodes, thymus, or tumor tissue. Suitable sources for obtaining the desired type of immune cells 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, e.g., 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 the subject to whom the immune cells expressing the chimeric receptor will later be administered. Immune cells administered to the same subject from which the cells were obtained are called autologous cells, while immune cells obtained from a subject other than the subject to whom the cells are administered are called allogeneic cells.

[0179] The desired host cell type can be cultured in a cell population obtained by co-incubating the cells with stimulating molecules. For example, anti-CD3 antibodies and anti-CD28 antibodies can be used for the expansion culture of T cells.

[0180] To construct immune cells expressing any of the chimeric receptor constructs described in this book, expression vectors for stable or transient expression of the chimeric receptor constructs can be constructed via the conventional methods described in this book and introduced into immune host cells. For example, the nucleic acid encoding the chimeric receptor may be cloned into a suitable expression vector, such as a viral vector operably linked to a suitable promoter. The nucleic acid and vector may be contacted with restriction enzymes under suitable conditions to create complementary ends on each molecule, which can pair with each other and be ligated. Alternatively, a synthetic nucleic acid linker may be ligated to the ends of the nucleic acid encoding the chimeric receptor. The synthetic linker may contain nucleic acid sequences corresponding to specific restriction sites 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.

[0181] Various promoters can be used for the expression of the chimeric receptors described in this book, and these promoters include, but are not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney's mouse leukemia virus (MMLV) LTR, myeloproliferative sarcoma virus (MPSV) LTR, splenic fociform virus (SFFV) LTR, simian virus 40 (SV40) early promoter, herpes simplex virus tk virus promoter, and elongation factor 1-alpha (EF1-α) promoter with or without the EF1-α intron. Further promoters for the expression of chimeric receptors include any promoter that is constitutively active in immune cells. Alternatively, any controllable promoter may be used so that its expression can be regulated within immune cells.

[0182] Furthermore, the vector may include, 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 pre-initial gene of human CMV for high levels of transcription; transcription termination signals 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 polyomatous replication origin and ColE1 for proper episomal replication; an internal ribosome binding site (IRES); a multipurpose multiplexing site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNAs; a "suicide switch" or "suicide gene" that, when activated, causes death of the vector-retaining cell (e.g., inducible caspases such as HSV thymidine kinase and iCasp9); and a reporter gene for evaluating the expression of a chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of vector preparation for the expression of chimeric receptors can be found, for example, in US2014 / 0106449, which is incorporated in its entirety by reference in this book.

[0183] In some embodiments, the chimeric receptor construct or the nucleic acid encoding the chimeric receptor is a DNA molecule. In some embodiments, the chimeric receptor construct or the nucleic acid encoding the chimeric receptor is a DNA vector that can electroporate immune cells (see, for example, Till et al., Blood (2012) 119(17):3940-3950). In some embodiments, the nucleic acid encoding the chimeric receptor is an RNA molecule that can electroporate immune cells.

[0184] Any vectors containing nucleic acid sequences encoding chimeric receptor constructs described herein are within the scope of this disclosure. Such vectors can be delivered into host cells, such as host immune cells, by preferred methods. Methods for delivering vectors to immune cells are well known in the art and may include: electroporation of DNA, RNA, or transposons; transfection reagents such as liposomes or nanoparticles for delivering 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 the expression of chimeric receptors are delivered to host cells by viral transduction. Exemplary methods of delivery using viruses include recombinant retroviruses (e.g., PCT Publications WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93 / 10218, WO91 / 02805; U.S. Patents 5,219,740 and 4,777,127; UK Patent No. 2,200,651). This includes, but is not limited to, alphavirus-based vectors (see also European Patent No. 0345242), alphavirus-based vectors, and adeno-associated virus (AAV) vectors (see, for example, PCT Publications WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655). In some embodiments, the vector for the expression of the chimeric receptor is a retrovirus. In some embodiments, the vector for the expression of the chimeric receptor is a lentivirus. In some embodiments, the vector for the expression of the chimeric receptor is an adeno-associated virus.

[0185] In cases where a vector encoding a chimeric receptor is introduced into host cells by the use of a viral vector, viral particles capable of infecting immune cells and containing the vector can be produced by any method known in the art, which can be found, for example, in PCT applications WO1991 / 002805A2, WO1998 / 009271A1, and U.S. Patent No. 6,194,191. The viral particles may be recovered from the cell culture supernatant and isolated and / or purified before contacting the viral particles with immune cells.

[0186] Methods for preparing host cells expressing any of the chimeric receptors described herein may include activating and / or expanding the culture of immune cells ex vivo. Activation of host cells means stimulating the host cells into an activated state, in which state the cells may be able to perform effector functions (e.g., cytotoxicity). The method for activating host cells depends on the type of host cell used for the expression of the chimeric receptor. Expanding the culture of host cells may include any method that results in an increase in the number of cells expressing the chimeric receptor, e.g., enabling the proliferation of host cells or stimulating the proliferation of host cells. The method for stimulating the expansion of host cell culture depends on the type of host cell used for the expression of the chimeric receptor and will be apparent to those skilled in the art. In some embodiments, host cells expressing any of the chimeric receptors described herein are activated and / or expanded in ex vivo before administration to a subject.

[0187] In some embodiments, the drug targeting a lineage-specific cell surface antigen(s) is an antibody-drug conjugate (ADC). As will be apparent to those skilled in the art, the term “antibody-drug conjugate” can be used synonymously with “immunotoxin” and refers to a fusion molecule comprising an antibody (or its antigen-binding fragment) conjugated to a toxin or drug molecule. When the antibody binds to the corresponding antigen, it enables the delivery of the toxin or drug molecule to a cell presenting the antigen on its cell surface (e.g., a target cell), thereby causing the death of the target cell.

[0188] In some embodiments, the drug 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 EMR2.

[0189] In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain CDR as the heavy chain variable region presented by SEQ ID NO: 5 and the same light chain CDRS as the light chain variable region presented by SEQ ID NO: 7. In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain variable region as presented by SEQ ID NO: 5 and the same light chain variable region as presented by SEQ ID NO: 7.

[0190] Toxins or drugs suitable for use in antibody-drug conjugates are well known in the art and will be obvious to those skilled in the art. See, for example, 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, and Elgundi et al., Advanced Drug Delivery Reviews (2017) 122:2-19.

[0191] In some embodiments, the antibody-drug conjugate may further include a linker (e.g., a peptide linker such as a cleavable linker) that conjugates the antibody and drug molecules. Examples of antibody-drug conjugates, but are not limited to, brentuximab vedotin, glenvatumumab vedotin / CDX-011, depatuxizumab mahodotin / ABT-414, and PSMA. ADC, Polatuzumab Vedotin / RG7596 / DCDS4501A, Denintuzumab Vedotin / SGN-CD19A, AGS-16C3F, CDX-014, RG7841 / DLYE5953A, RG7882 / DMUC406A, RG7986 / DCDS0780A, SGN-LIV1A, Enfortumab Vedotin / ASG-22ME, AG-15ME, AGS67E, Terisotuzumab Vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, Tisotuzumab Vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, Pinatuzumab Vedotin / RG7593 / DCDT2980S, Rifastuzumab Vedotin / RG7599 / DNIB0600A, Indusatuzumab Vedotin / MLN-0264 / TAK-264, Bundutuzumab Vedotin / RG7450 / DSTP3086S, Sofituzumab Vedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC 5T4, Trastuzumab emtansine / T-DM1, Milbetuximab sorabutansine / IMGN853, Coltuximab labutansine / SAR3419, Naratuximab emtansine / IMGN529, Induximab labutansine / BT-062, Anetumab labutansine / BAY 94-9343, SAR408701, SAR428926, AMG 224, PCA062, HKT288, LY3076226, SAR566658, lorbotuzumab meltansine / IMGN901, cantuzumab meltansine / SB-408075, cantuzumab labutancine / IMGN242, laprituximab emtansine / IMGN289, IMGN388, vibatuzumab meltansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP628, Vadasutuximab talirin / SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, Robalpituzumab tecillin / 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, CMB-401, Trastuzumab duocalmazine / SYD985, BMS-936561 / MDX-1203, Includes sacituzumab govitecan / IMMU-132, rabetsuzumab govitecan / IMMU-130, DS-8201a, U3-1402, milatuzumab doxorubicin / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, lupalzumab amadotin / BAY1129980, appletuzumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, and DSTA4637S / RG7861. In one example, the antibody-drug conjugate is gemtuzumab ozogamicin.

[0192] In some embodiments, when an antibody-drug conjugate binds to an epitope of a lineage-specific cell surface protein, internal movement of the antibody-drug conjugate is induced, and the drug (or toxin) may be released into the cell. In some embodiments, when an antibody-drug conjugate binds to an epitope of a lineage-specific cell surface protein, internal movement of the toxin or drug is induced, which allows the toxin or drug to kill cells expressing the lineage-specific protein (target cells). In some embodiments, when an antibody-drug conjugate binds to an epitope of a lineage-specific cell surface protein, internal movement of the toxin or drug is induced, which may control the activity of cells expressing the lineage-specific protein (target cells). The types of toxins or drugs used in the antibody-drug conjugates described herein are not limited to any particular type.

[0193] The ADCs described in this book may be used as follow-up treatments for patients who have received the combination therapies described in this book.

[0194] Hematopoietic cells lacking one or more lineage-specific cell surface antigens. This disclosure also provides hematopoietic cells, such as hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), that are genetically modified to be deficient in a lineage-specific cell surface antigen (e.g., CD33). In some embodiments, hematopoietic cells, such as hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs), are genetically modified to be deficient in a lineage-specific cell surface antigen (e.g., CD33) and at least one further lineage-specific cell surface antigen (e.g., EMR2).

[0195] In some embodiments, CRISPR-based base editor systems were used to genetically modify cells to reduce off-target effects and undesirable debilitating immunosuppressive side effects. In some embodiments, one CRISPR-based base editor system was used to modify cells to be deficient in a lineage-specific cell surface antigen, and a second CRISPR-based base editor system was used to modify cells to be deficient in at least one additional lineage-specific cell surface antigen. This paper demonstrates the successful use of CRISPR-based base editor systems to modify cells to be deficient in multiple lineage-specific cell surface antigens. In some embodiments, multiple CRISPR-based base editor systems may be used in the same cells to delete or inhibit multiple additional lineage-specific cell surface antigens.

[0196] This book demonstrates the use of a CRISPR-based base editor system to produce specific nucleotide substitutions in endogenous genes encoding lineage-specific cell surface antigens. In some embodiments, the nucleotide substitution is within a sequence encoding a splice element, and the nucleotide substitution results in alternative splicing of the transcript encoded by the gene. In some embodiments, the CRISPR-based base editor system targets a splice element in the endogenous gene, and the CRISPR-based base editor system results in alternative splicing of the transcript encoded by the gene. In some embodiments, alternative splicing causes the exon encoding the epitope to be skipped. In some embodiments, alternative splicing causes the exon encoding the epitope to be extended. In some embodiments, alternative splicing induces early codon termination. In some embodiments, the splice element is a splice donor, splice acceptor, splice enhancer, or splice acceptor. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitutions are "A" through "G". In some embodiments, the nucleotide substitutions are "T" through "C". In some embodiments, the epitope is targeted by a therapeutic or immunotherapeutic agent.

[0197] The CRISPR-based base editor systems described in this book include a catalytically impaired Cas protein fused to a DNA modifying enzyme, i.e., Cas9 nickase, which is fused to a cytosine deaminase or adenosine deaminase (base editor), and a single guide RNA. Each CRISPR-based base editor complex can bind to a lineage-specific antigen polynucleotide, allowing for the substitution of one or more nucleotides, thereby modifying the polynucleotide. Multiple CRISPR-based base editor systems can be used to modify multiple lineage-specific cell surface antigens.

[0198] In some embodiments, the CRISPR-based base editor system includes Cas nickase fused to a cytosine base editor. In some embodiments, the CRISPR-based base editor is a cytosine base editor. In some embodiments, the cytosine base editor is BE4max. In some embodiments, the CRISPR-based base editor system targets CD33, and the gRNA is selected from the group consisting of SEQ ID NOs: 1 and 2.

[0199] In some embodiments, the CRISPR-based base editor is an adenosine base editor. In some embodiments, the cytosine base editor is ABE8e. In some embodiments, the CRISPR-based base editor system targets CD33, and the gRNA is SEQ ID NO: 3.

[0200] In some embodiments, the splice acceptor or exon splicing enhancer site within exon 2 of CD33 is modified. In some embodiments, the nucleotide sequence of the intron 1 / exon 2 junction of CD33 is modified. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C". In some embodiments, the gRNA sequence hybridizes to a portion of the nucleotide sequence encoding CD33. In some embodiments, the gRNA sequence targets the splice acceptor or exon splicing enhancer site within exon 2 of the nucleotide sequence encoding CD33. In some embodiments, the gRNA sequence targets rs12459419, which is an SNP of CD33. In some embodiments, the gRNA sequence targets the intron 1 / exon 2 junction of CD33. In some embodiments, the gRNA sequence targets the nucleotide sequence containing SEQ ID NO: 37.

[0201] In some embodiments, the CRISPR-based base editor is an adenosine base editor. In some embodiments, the cytosine base editor is ABE8e. In some embodiments, the CRISPR-based base editor system targets EMR2 and the gRNA is SEQ ID NO: 4. In some embodiments, the CRISPR-based base editor system targets EMR2 and the gRNA is SEQ ID NO: 46. In some embodiments, the CRISPR-based base editor system targets EMR2 and the gRNA is SEQ ID NO: 47.

[0202] In some embodiments, the splice donor site within exon 13 of EMR2 is modified. In some embodiments, the nucleotide sequence of the intron 12 / exon 13 junction of EMR2 is modified. In some embodiments, the nucleotide substitution is from "C" to "T". In some embodiments, the nucleotide substitution is from "G" to "A". In some embodiments, the nucleotide substitution is from "A" to "G". In some embodiments, the nucleotide substitution is from "T" to "C". In some embodiments, the gRNA sequence hybridizes to a portion of the nucleotide sequence encoding EMR2. In some embodiments, the gRNA sequence targets the splice donor site within exon 13 of the nucleotide sequence encoding EMR2. In some embodiments, the gRNA sequence targets the intron 12 / exon 13 junction of EMR2. In some embodiments, the gRNA sequence targets the nucleotide sequence containing sequence number 40.

[0203] In some embodiments, all of the aforementioned CRISPR-based base editor systems can include base editor proteins and gRNAs in a ribonucleoprotein (RNP)-based delivery system.

[0204] In some embodiments, hematopoietic cells are HSCs, HPCs, or combinations thereof referred to herein as “HSPC” (“hematopoietic stem cells and / or progenitor cells”). In some embodiments, the cell population described herein comprises multiple hematopoietic stem cells; in some embodiments, the cell population described herein comprises multiple hematopoietic progenitor cells; and in some embodiments, the cell population described herein comprises multiple hematopoietic stem cells and multiple hematopoietic progenitor cells. HSCs can give rise to both myeloid progenitor cells and lymphoid progenitor cells, which further give rise to 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 for identification and / or isolation of HSCs. + ), and the absence of cell surface markers associated with leaning to a cell lineage. Therefore, in some embodiments, HSCs are CD34 + That is the case.

[0205] In some embodiments, HSCs are obtained from subjects such as mammalian subjects. In some embodiments, mammalian subjects are non-human primates, rodents (e.g., mice or rats), cattle, pigs, horses, or livestock. In some embodiments, HSCs are obtained from human patients, such as human patients with hematopoietic malignancies. In some embodiments, HSCs are obtained from healthy donors. In some embodiments, HSCs are obtained from subjects to whom immune cells expressing chimeric receptors are subsequently administered. HSCs administered to the same subject from which the cells were obtained are called autologous cells, while HSCs obtained from subjects other than the subject to which the cells are administered are called allogeneic cells.

[0206] HSCs can be obtained from any suitable source using conventional means known in the art. In some embodiments, HSCs are obtained from a sample of the subject, such as a bone marrow sample or a blood sample. Alternatively, or further, HSCs can be obtained from the umbilical cord. In some embodiments, HSCs are derived from bone marrow or peripheral blood mononuclear cells (PBMCs). Generally, bone marrow cells can be obtained from the iliac crest, femur, tibia, vertebra, rib, or other medullary cavity of the subject. Bone marrow can be collected from a patient and isolated by various separation and washing procedures known in the art. An exemplary procedure for isolating bone marrow cells includes the following steps: a) extracting a bone marrow sample; b) separating the bone marrow suspension into three fractions by centrifugation and collecting an intermediate fraction or buffy coat; c) centrifugating the buffy coat fraction from step (b) again in a separator, generally Ficoll®, to collect an intermediate fraction containing bone marrow cells; and d) washing the fraction collected from step (c) to recover reinfusionable bone marrow cells.

[0207] HSCs are normally present in the bone marrow, but they can be mobilized into the circulating blood by administering a mobilization agent to collect HSCs from peripheral blood. In some embodiments, the subjects from which HSCs are collected are administered a mobilization agent such as granulocyte colony-stimulating factor (G-CSF). The number of HSCs collected after mobilization using a mobilization agent is usually greater than the number of cells collected without the use of a mobilization agent. In some embodiments, the HSCs are peripheral blood HSCs.

[0208] In some embodiments, a sample is obtained from a subject, and then the desired cell type is enriched. For example, PBMC and / or CD34. + Hematopoietic cells can be isolated from blood, as described in this book. Cells can also be isolated from other cells, for example, by isolation and / or activation using antibodies that bind to epitopes on the cell surface of a desired cell type. Another method that can be used is negative selection, which uses antibodies against cell surface markers to selectively enrich specific cell types without activating the cells through receptor involvement.

[0209] Populations of HSCs can be expanded cultured before or after genetically engineering HSCs to eliminate lineage-specific cell surface antigens. Cells can be cultured under conditions including an expansion culture medium containing one or more cytokines, e.g., stem cell factor (SCF), Flt-3 ligand (Flt3L), thrombopoietin (TPO), interleukin-3 (IL-3), or interleukin-6 (IL-6). Cells may be expanded cultured for approximately 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 range required. In some embodiments, HSCs are cultured from a sample obtained from a subject to obtain a desired cell population (e.g., CD34). + / CD33 - After isolating the cells, they are cultured on a large scale before the genetic engineering procedure. In some embodiments, the HSCs are cultured on a large scale after the genetic engineering procedure, thereby selectively culturing cells that have undergone genetic modification and are deficient in lineage-specific cell surface antigens. In some embodiments, one cell ("clone") or several cells having the desired characteristics (e.g., phenotype or genotype) after genetic modification can be selected and cultured independently.

[0210] In some embodiments, hematopoietic cells are genetically engineered to be deficient in (e.g., not express) a lineage-specific cell surface antigen (e.g., CD33). In some embodiments, hematopoietic cells are genetically engineered to be deficient in (e.g., not express) a lineage-specific cell surface antigen (e.g., CD33) and at least one further lineage-specific cell surface antigen. In some embodiments, hematopoietic cells are genetically engineered to be deficient in the same lineage-specific cell surface antigen(s) that are targeted by a drug(s). As used in this text, a lineage-specific cell surface antigen(s) is considered deficient in hematopoietic cells if the expression of the lineage-specific cell surface antigen(s) in the hematopoietic cells is substantially reduced compared to naturally occurring hematopoietic cells of the same type (e.g., characterized by the presence of the same cell surface marker, such as CD34). In some embodiments, the expression of the lineage-specific cell surface antigen(s) is undetectable (e.g., does not express the lineage-specific cell surface antigen(s)). The expression level of lineage-specific cell surface antigens can be evaluated by any means known in the art. For example, the expression level of lineage-specific cell surface antigens can be evaluated by detecting the antigen with an antigen-specific antibody (e.g., flow cytometry, Western blotting).

[0211] In some embodiments, the expression of lineage-specific cell surface antigens (CPIs) in genetically engineered hematopoietic cells is compared to the expression of naturally occurring CSIs in hematopoietic cells (e.g., wild-type equivalents). In some embodiments, the genetic engineering results in a reduction of at least approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in the expression level of CSIs in naturally occurring hematopoietic cells. In other words, in some embodiments, genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of lineage-specific cell surface antigens (e.g., CD33) compared to naturally occurring hematopoietic cells (e.g., wild-type equivalents).

[0212] In some embodiments, the genetic engineering manipulation results in a reduction of at least approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in the expression level of wild-type lineage-specific cell surface antigen(s) (e.g., CD33) compared to the expression level of wild-type lineage-specific cell surface antigen(s) (e.g., CD33) in naturally occurring hematopoietic cells. In other words, in some embodiments, genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of wild-type lineage-specific cell surface antigens (e.g., CD33) compared to naturally occurring hematopoietic cells (e.g., wild-type equivalents).

[0213] In some embodiments, hematopoietic cells lack an entire endogenous gene encoding a lineage-specific cell surface antigen(s). In some embodiments, an entire endogenous gene encoding a lineage-specific cell surface antigen(s) is deleted. In some embodiments, hematopoietic cells contain a portion of an endogenous gene encoding a lineage-specific cell surface antigen(s). In some embodiments, hematopoietic cells express a portion of a lineage-specific cell surface antigen(s) (e.g., a truncated protein). In other embodiments, a portion of an endogenous gene encoding a lineage-specific cell surface antigen(s) is deleted. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the gene encoding a lineage-specific cell surface antigen(s) is deleted.

[0214] In some embodiments, the expression of epitopes encoded by exons of lineage-specific cell surface antigens (or more) in genetically engineered hematopoietic cells is compared to the expression of lineage-specific cell surface antigens (or more) in naturally occurring hematopoietic cells (e.g., wild-type equivalents). In some embodiments, the genetic engineering results in a reduction of at least approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in the expression level of lineage-specific cell surface antigens (or more) compared to the expression of lineage-specific cell surface antigens (or more) in naturally occurring hematopoietic cells. In other words, in some embodiments, genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the epitopes of lineage-specific cell surface antigens (e.g., CD33) compared to naturally occurring hematopoietic cells (e.g., wild-type equivalents).

[0215] In some embodiments, one or more nucleotide substitutions are made in an epitope encoded by an exon of an endogenous gene.

[0216] As will be understood by those skilled in the art, a portion of the nucleotide sequence encoding a lineage-specific cell surface antigen(s), or one or more non-coding sequences, may be deleted so that the antigen(s) are absent in hematopoietic cells (for example, the expression of the antigen(s) is substantially reduced).

[0217] In some embodiments, the lineage-specific cell surface antigen is CD33. The predicted structure of CD33 includes two immunoglobulin domains: an IgV domain and an IgC2 domain. In some embodiments, exon 2 of CD33 is deleted. In some embodiments, a modified splice acceptor or exon splicing enhancer within exon 2 of the endogenous CD33 gene is modified, and the modification results in a decrease in the expression level of the epitope encoded by exon 2 of CD33 compared to wild-type equivalent cells.

[0218] In some embodiments, at least one further lineage-specific cell surface antigen is EMR2. In some embodiments, exon 13 of EMR2 is deleted. In some embodiments, a modified splice donor within exon 13 of the endogenous EMR2 gene is modified, and the modification causes a decrease in the expression level of the epitope encoded by exon 13 of EMR2 compared to wild-type equivalent cells. In some embodiments, alternative splicing induces early codon termination and the production of mutant or truncated EMR2 compared to wild-type equivalent cells.

[0219] Any genetically engineered hematopoietic cells, such as HSCs, that lack or have modified cell surface lineage-specific antigens, can be prepared by standard methods or by methods described herein. In some embodiments, the genetic engineering is performed using genome editing. As used herein, “genome editing” refers to a method of modifying the genome of an organism, including any nucleotide sequence that codes for or does not code for proteins, in order to knock out the expression of a target gene.

[0220] In one aspect of this disclosure, tumor cells are replaced with a modified population of normal cells, using normal cells modified with lineage-specific antigens. Such modifications may include depletion or inhibition of any lineage-specific antigen using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based base editor system.

[0221] The CRISPR-Cas system has been successfully used to edit the genomes of a wide range of organisms, including but not limited to bacteria, humans, fruit flies, 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 See Research(2013)41(20):el88.

[0222] This disclosure utilizes a CRISPR-based base editor system that hybridizes with a target sequence in a lineage-specific antigen polynucleotide, wherein the CRISPR-based base editor system comprises a catalytically impaired Cas protein fused to a DNA modifying enzyme, i.e., Cas9 nickase, which is fused to a cytosine deaminase or adenosine deaminase (base editor), and a single guide RNA. The CRISPR-based base editor complex binds to the lineage-specific antigen polynucleotide, enabling the substitution of one or more nucleotides, thereby modifying the polynucleotide.

[0223] The CRISPR-based base editor system of this disclosure can bind to and / or cleave a region of interest in a cell surface lineage-specific antigen within a coding or non-coding region, in a gene, or adjacent to a gene, for example, in a leader sequence, trailer sequence, or intron, or in a non-transcribed region either upstream or downstream of a coding region. The guide RNA (gRNA) used in this disclosure may be designed so that the gRNA directs the binding of the base editor protein-gRNA complex to a predetermined cleavage site (target site) in the genome. The cleavage site may be selected to release a fragment containing a region of unknown sequence or a region containing SNPs, nucleotide insertions, nucleotide deletions, rearrangements, etc.

[0224] The guide RNA (gRNA) used in this disclosure may be designed so that the gRNA directs the binding of a base editor protein-gRNA complex to a predetermined target site in the genome. The target site may be a region including SNPs, nucleotide insertions, nucleotide deletions, rearrangements, etc.

[0225] The terms “gRNA,” “guide RNA,” and “CRISPR guide sequence” may be used synonymously throughout this text and refer to nucleic acids containing sequences that determine the specificity of the base editor proteins in a CRISPR-based base editor system. gRNA hybridizes (partially or fully) to a target nucleic acid sequence in the host cell’s genome. The gRNA or any portion of it that hybridizes to the target nucleic acid may be 15–25 nucleotides long, 18–22 nucleotides long, or 19–21 nucleotides long. In some embodiments, the gRNA sequence hybridizing to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some embodiments, the gRNA sequence hybridizing to the target nucleic acid is 10–30 or 15–25 nucleotides long.

[0226] In some embodiments, the gRNA includes a scaffold sequence in addition to a sequence that binds to the target nucleic acid. Expression of a gRNA encoding both a sequence complementary to the target nucleic acid and a scaffold sequence can have a dual function of both binding to (hybridizing) the target nucleic acid and recruiting an endonuclease to the target nucleic acid, resulting in site-specific CRISPR activity. In some embodiments, such a chimeric gRNA may be called a single guide RNA (sgRNA).

[0227] In some embodiments, the gRNA is modified, for example, chemically modified. The modified gRNA comprises at least one nucleotide having a modification to at least one chemical structure, which is a nucleic acid base, a sugar, and a phosphodiester bond or a backbone portion (e.g., a nucleotide phosphate). Exemplary gRNA modifications are obvious to those skilled in the art and can be found, for example, in Lee et al., Elife (2017) May 2;6 and U.S. Publication 2016 / 0289675. Further preferred modifications include phosphorothioate backbone modifications, 2'-O-Me modified sugars, 2'F modified sugars, substitution of ribose sugars with bicyclic nucleotide-cEt, 3' thioPACE (MSP), or any combination thereof. Suitable gRNA modifications are described, for example, in Rahdar et al. PNAS (2015) 112(51):E7110-E7117 and Hendel et al., Nat Biotechnol. (2015 Sep) 33(9):985-989, and their entire contents are incorporated into this book by reference.

[0228] In some embodiments, the gRNAs described herein are chemically modified. For example, a gRNA may contain one or more 2'-O modified nucleotides, such as 2'-O-methyl nucleotides. In some embodiments, a gRNA contains a 2'-O modified nucleotide, such as 2'-O-methyl nucleotide, at its 5' end. In some embodiments, a gRNA contains a 2'-O modified nucleotide, such as 2'-O-methyl nucleotide, at its 3' end. In some embodiments, a gRNA contains a 2'-O modified nucleotide, such as 2'-O-methyl nucleotide, at both its 5' and 3' ends. In some embodiments, a gRNA is 2'-O modified, such as 2'-O-methyl modified, at its 5' end, the second nucleotide from the 5' end, and the third nucleotide from the 5' end. In some embodiments, the gRNA is 2'-O modified, for example, 2'-O-methyl modified, at the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified, for example, 2'-O-methyl modified, at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified, for example, 2'-O-methyl 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 nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar.In some embodiments, the gRNA is modified with 2'-O modification, such as 2'-O-methyl modification, at the 5'-terminal nucleotide, the second nucleotide from the 5'-terminal, the third nucleotide from the 5'-terminal, the second nucleotide from the 3'-terminal, the third nucleotide from the 3'-terminal, and the fourth nucleotide from the 3'-terminal. In some embodiments, the 2'-O-methyl nucleotide includes a phosphate bond to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide includes a phosphorothioate bond to an adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide includes a thioPACE bond to an adjacent nucleotide.

[0229] In some embodiments, the gRNA may contain one or more 2'-O modified and 3'-phosphorus modified nucleotides, for example, 2'-O-methyl3'-phosphorothioate nucleotides. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide, for example, a 2'-O-methyl3'-phosphorothioate nucleotide, at the 5' end of the gRNA. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide, for example, a 2'-O-methyl3'-phosphorothioate nucleotide, at the 3' end of the gRNA. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide, for example, a 2'-O-methyl3'-phosphorothioate nucleotide, at both the 5' and 3' ends of the gRNA. In some embodiments, the gRNA contains a backbone in which one or more unbridged oxygen atoms are substituted with sulfur atoms. In some embodiments, the gRNA is modified with 2'-O and 3' phosphorus modifications, such as 2'-O-methyl3' phosphorothioate, at the 5' terminal nucleotide, the second nucleotide from the 5' terminal, and the third nucleotide from the 5' terminal. In some embodiments, the gRNA is modified with 2'-O and 3' phosphorus modifications, such as 2'-O-methyl3' phosphorothioate, at the 3' terminal nucleotide, the second nucleotide from the 3' terminal, and the third nucleotide from the 3' terminal. In some embodiments, the gRNA is modified with 2'-O and 3' phosphorus modifications, such as 2'-O-methyl3' phosphorothioate, at the 5' terminal nucleotide, the second nucleotide from the 5' terminal, the third nucleotide from the 5' terminal, the 3' terminal nucleotide, the second nucleotide from the 3' terminal, and the third nucleotide from the 3' terminal. In some embodiments, the gRNA is modified with 2'-O modification and 3'-phosphorus modification, such as 2'-O-methyl3'phosphorothioate modification, at the second nucleotide from the 3' end, the third nucleotide from the 3' end, and the fourth nucleotide from the 3' end.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' phosphorothioate modified, at the 5' terminal nucleotide, the second nucleotide from the 5' terminal, the third nucleotide from the 5' terminal, the second nucleotide from the 3' terminal, the third nucleotide from the 3' terminal, and the fourth nucleotide from the 3' terminal.

[0230] In some embodiments, the gRNA may contain one or more 2'-O modified and 3'-phosphorus modified nucleotides, for example, 2'-O-methyl3'thioPACE nucleotides. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide, for example, 2'-O-methyl3'thioPACE nucleotide at the 5' end of the gRNA. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide, for example, 2'-O-methyl3'thioPACE nucleotide at the 3' end of the gRNA. In some embodiments, the gRNA contains a 2'-O modified and 3'-phosphorus modified nucleotide, for example, 2'-O-methyl3'thioPACE nucleotide at both the 5' and 3' ends of the gRNA. In some embodiments, the gRNA contains a skeleton in which one or more non-bridged oxygen atoms are substituted with sulfur atoms, and one or more non-bridged oxygen atoms are substituted with acetate groups. In some embodiments, the gRNA is modified with 2'-O and 3' phosphorus, for example, 2'-O-methyl3'thioPACE, at the 5' terminal nucleotide, the second nucleotide from the 5' terminal, and the third nucleotide from the 5' terminal. In some embodiments, the gRNA is modified with 2'-O and 3' phosphorus, for example, 2'-O-methyl3'thioPACE, at the 3' terminal nucleotide, the second nucleotide from the 3' terminal, and the third nucleotide from the 3' terminal. In some embodiments, the gRNA is modified with 2'-O and 3' phosphorus, for example, 2'-O-methyl3'thioPACE, at the 5' terminal nucleotide, the second nucleotide from the 5' terminal, the third nucleotide from the 5' terminal, the 3' terminal nucleotide, the second nucleotide from the 3' terminal, and the third nucleotide from the 3' terminal. In some embodiments, the gRNA is modified with 2'-O modification and 3'-phosphorus modification, such as 2'-O-methyl3'thioPACE modification, 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 nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end 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-methyl3'thioPACE modified, at the nucleotide at the 5' end 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.

[0231] In some embodiments, the gRNA includes a chemically modified backbone. In some embodiments, the gRNA includes a phosphorothioate bond. In some embodiments, one or more non-crosslinked oxygen atoms are substituted with sulfur atoms. In some embodiments, the 5' nucleotide, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each include a phosphorothioate bond. In some embodiments, the 3' nucleotide, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each include a phosphorothioate bond. In some embodiments, the 5' nucleotide, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the 3' nucleotide, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each include a phosphorothioate bond. 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 phosphorothioate bond. In some embodiments, the nucleotide at the 5' end 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 phosphorothioate bond.

[0232] In some embodiments, the gRNA contains a thioPACE bond. In some embodiments, the gRNA contains a backbone in which one or more non-crosslinked oxygen atoms are substituted with sulfur atoms and one or more non-crosslinked oxygen atoms are substituted with acetate groups. In some embodiments, the 5' 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 contain a thioPACE bond. In some embodiments, the 3' 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 contain a thioPACE bond. In some embodiments, the 5' 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' 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 contain a thioPACE bond. 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 nucleotide at the 5' end 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.

[0233] Chemical modifications of gRNA are described, for example, in Hendel et al., Nature Biotech. (2015) 33(9), which is incorporated into this book by reference in its entirety.

[0234] As used in this book, “scaffold sequence,” also known as tracrRNA, refers to a nucleic acid sequence that recruits a Cas endonuclease to a target nucleic acid that has been bound (hybridized) to a complementary gRNA sequence. Any scaffold sequence containing at least one stem-loop structure and recruiting an endonuclease may be used in the genetic elements and vectors described herein. Exemplary scaffold sequences will be apparent 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, PCT application WO2014 / 093694, and PCT application WO2013 / 176772.

[0235] In some embodiments, the gRNA sequence does not include a scaffold sequence, and the scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further includes additional sequences that are complementary to a portion of the scaffold sequence and function to bind (hybridize) with the scaffold sequence to recruit an endonuclease to a target nucleic acid.

[0236] 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 teaching the complementarity between the gRNA sequence and the target polynucleotide sequence). A mismatch between the CRISPR guide sequence and the target nucleic acid near the 3' end has been shown to cause loss of 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).

[0237] The 3' end of the target nucleic acid is flanked by a protospacer-adjacent motif (PAM) that interacts with the endonuclease and may further contribute to the targeting of endonuclease activity to the target nucleic acid. The PAM sequence that flanks to the target nucleic acid is generally thought to be dependent on the endonuclease and its source. For example, in the case of Cas9 endonuclease from Streptococcus pyogenes, the PAM sequence is NGG. In the case of Cas9 endonuclease from Staphylococcus aureus, the PAM sequence is NNGRRT. In the case of Cas9 endonuclease from Neisseria meningitidis, the PAM sequence is NNNNGATT. In the case of Cas9 endonuclease from Streptococcus thermophilus, the PAM sequence is NNAGAA. In the case of Cas9 endonuclease from Treponema denticola, the PAM sequence is NAAAAC. In the case of Cpf1 nuclease, the PAM sequence is TTN.

[0238] In some embodiments, the genetic engineering of cells also includes introducing a CRISPR-based base editor protein into the cells. In some embodiments, the CRISPR-based base editor and the nucleic acid encoding the gRNA are provided in the same nucleic acid (e.g., a vector). In some embodiments, the CRISPR-based base editor protein and the nucleic acid encoding the gRNA are provided in different nucleic acids (e.g., different vectors). Alternatively, or further, the CRISPR-based base editor may be provided or introduced into cells in protein form. In some embodiments, the gRNA forms a complex with the CRISPR-based base editor in protein form.

[0239] In some embodiments, the Cas endonuclease is the 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 may be codon-optimized for expression in host cells. 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, which is called "nickase" or "Cas9n". The Cas9 niccasse endonuclease cleaves a single DNA strand of a target nucleic acid. See, for example, Dabrowska et al. Frontiers in Neuroscience (2018) 12(75). One or more mutations in the RuvC and HNH catalytic domains of the enzyme have been shown to improve Cas9 efficiency. See, for example, Sarai et al. Currently Pharma. Biotechnol. (2017) 18(13). In some embodiments, the Cas9 endonuclease is catalytically inactive Cas9. For example, dCas9 contains mutations in catalytically active residues (D10 and H840) and has no nuclease activity. Alternatively, or further, the Cas9 endonuclease may be fused to another protein or a portion thereof. In some embodiments, dCas9 is fused to a repressor domain such as the KRAB domain.In some embodiments, such dCas9 fusion proteins are used with constructs described herein for multiple gene repression (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 with constructs described herein for gene activation (e.g., CRISPR activation (CRISPRa)). In some embodiments, dCas9 is fused to an epigenetic regulatory domain such as a histone demethylase domain or a histone acetyltransferase domain. In some embodiments, dCas9 is fused to LSD1 or p300, or a portion thereof. In some embodiments, the dCas9 fusion is used for CRISPR-based epigenetic regulation. In some embodiments, dCas9 or Cas9 is fused to a Fok1 nuclease domain. In some embodiments, Cas9 or dCas9 fused to a Fok1 nuclease domain 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, the Cas9 / dCas9 protein fused to the fluorescent protein is used for labeling and / or visualizing genomic loci or for identifying cells expressing Cas endonucleases.

[0240] In some embodiments, the Cas endonuclease is modified to enhance the enzyme's specificity (e.g., to reduce off-target effects and maintain robust on-target cleavage). In some embodiments, the Cas endonuclease is a Cas9 variant with enhanced specificity (e.g., eSPCas9). See, for example, 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, for example, Kleinstiver et al. Nature (2016) 529:490-495.

[0241] Cas enzymes, such as Cas endonucleases, are known in the art, can be obtained from various sources, and / or can be engineered / modified to modulate the activity or specificity of one or more of the enzymes. In some embodiments, Cas enzymes are engineered / modified to recognize one or more PAM sequences. In some embodiments, Cas enzymes are engineered / modified to recognize one or more PAM sequences different from the PAM sequences that the Cas enzyme recognizes without engineering / modification. In some embodiments, Cas enzymes are engineered / modified to reduce the off-target activity of the enzyme.

[0242] In some embodiments, the nucleotide sequences encoding Cas endonucleases are further modified to alter the specificity of endonuclease activity (e.g., by reducing off-target cleavage, decreasing Cas endonuclease activity or lifespan in cells, increasing homologous recombination, and decreasing non-homologous end joining). See, for example, Komor et al. Cell (2017) 168:20-36. In some embodiments, the nucleotide sequences encoding Cas endonucleases are modified to alter the endonuclease's PAM recognition. For example, the Cas endonuclease SpCas9 recognizes the PAM sequence NGG, while relaxed variants of SpCas9 (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) containing one or more modifications of the endonuclease may recognize the PAM sequences NGA, NGAG, and NGCG. If a Cas endonuclease recognizes more potential PAM sequences compared to an unmodified Cas endonuclease, the PAM recognition of the modified Cas endonuclease is considered "relaxed." For example, the Cas endonuclease SaCas9 recognizes the PAM sequence NNGRRT, while a relaxed variant of SaCas9 containing one or more modifications to the endonuclease (e.g., KKH SaCas9) may 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 modifications to the endonuclease (e.g., RHA FnCas9) may recognize the PAM sequence YG. In another example, the Cas endonuclease is a Cpf1 endonuclease containing the substitution mutations S542R and K607R, which recognizes the PAM sequence TYCV. In one example, Cas endonuclease is a Cpf1 endonuclease containing the substitution mutations S542R, K607R, and N552R, which recognizes the PAM sequence TATV. See, for example, Gao et al. Nat. Biotechnol. (2017) 35(8):789-792.

[0243] In some embodiments, multiple (e.g., two, three, or more) Cas endonucleases are used. In some embodiments, at least one of the Cas endonucleases is a Cas9 enzyme. In some embodiments, at least one of the Cas endonucleases is a Cpf1 enzyme. In some embodiments, at least one of the Cas9 endonucleases is derived from Streptococcus pyogenes. In some embodiments, at least one of the Cas9 endonucleases is derived from Streptococcus pyogenes, and at least one Cas9 endonucleases is derived from an organism other than Streptococcus pyogenes.

[0244] In some embodiments, the endonuclease is a base editor. Base editor endonucleases generally contain a catalytically inactive Cas endonuclease fused to a functional domain. See, for example, Eid et al. Biochem. J. (2018) 475(11):1955-1964 and Rees et al. Nature Reviews Genetics (2018) 19:770-788.

[0245] CRISPR-based base editing systems generally include a Cas nickase or Cas fused to a deaminase that performs the editing, a gRNA that targets Cas to a specific gene locus, and a target base for editing within an editing window specified by the Cas protein.

[0246] Currently, there are two classes of base editors: cytosine (CBE) and adenine (ABE). CBE mediates the change from C to T (or G to A in the reverse strand). ABE performs the change from A to G (or T to C in the reverse strand). This accounts for only four of the twelve possible changes.

[0247] The first cytosine base editor was created by coupling cytidine deaminase with inactive dCas9 (Komor et al., Nature (2016) 533:420-424). These fusions convert cytosine to uracil without cleaving the DNA. Uracil is then converted back to thymine during DNA replication or repair. Fusing a uracil DNA glycosylase inhibitor (UGI) to dCas9 prevents base excision repair, which reverses the U mutation back to a C mutation. To increase base editing efficiency, Cas nickase was used instead of dCas9. The resulting editor BE3 makes the unmodified DNA strand appear "newly synthesized" to the cell by introducing a nick. Thus, the cell uses the U-containing strand as a template to repair the DNA and copy the base edit.

[0248] The fourth-generation base editor, BE4, reduces unwanted C→G or C→A conversions that can occur with previous BEs. These byproducts are likely due to removal by uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the UNG inhibitor UGI increases the purity of the base-edited product. APOBEC1-Cas9n and Cas9n-UGI linkers were extended to further improve product purity. These three improvements represent the fourth generation of base editors. Compared to BE3, BE4 results in a 2.3-fold reduction in C→G and C→A products, as well as a 2.3-fold reduction in indel formation.

[0249] Improving the efficiency of base editing for mammalian editing involves ensuring that the editor reaches the nucleus and is properly expressed. Improved nuclear localization signals and codon usage of BE4 have resulted in the creation of BE4max and AncBE4max with 4.2–6 times improved editing efficiency (Koblan et al., Nat Biotechnol (2018) 36:843-846).

[0250] Adenine base editors convert adenine to inosine, resulting in a change from A to G (Gaudelli et al., Nature (2017) 551:464-471). Since no known DNA adenine deaminase exists, further steps are needed to create an adenine base editor. The authors used directional evolution to create one from the RNA adenine deaminase TadA.

[0251] Adenine base editors have also been improved by enhancing nuclear localization and expression. In 2020, two papers were published describing further ABEs evolved from ABE7.10 with improved targeting flexibility and specificity. The first paper described the generation of ABE8e, which edits approximately 590 times faster than TadA of ABE7.10 without increasing off-target activity (Richter et al., Nat Biotechnol (2020) 38:883-891).

[0252] Furthermore, using ABE7.10 as a starting point, Gaudelli evolved the base editor into 40 new ABE8 variants (Gaudelli et al., Nat Biotechnol (2020) 38:892-900). Compared to ABE7.10, ABE8 showed 1.5 times higher editing efficiency at protospacer positions A5-A7, 3.2 times higher editing efficiency at NGG PAM positions A3-A4 and A8-A10, and 4.2 times higher editing efficiency in non-NGG PAM variants. ABE8 also exhibits improved base editing capabilities even at sites that were previously difficult to target. ABE8 is a promising tool for cell therapy applications as it can achieve 98-99% target modification in primary T cells.

[0253] In some embodiments, the catalytically inactive Cas endonuclease is dCas9. In some embodiments, the endonuclease comprises dCas9 fused to one or more uracilglycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises dCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas9 fused to ABE8e. 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 endonuclease comprises dCas9 fused to BE4max.

[0254] In some embodiments, the catalytically inactive Cas endonuclease is Cas9 nickase or Cas9n. In some embodiments, the endonuclease includes Cas9 nickase fused to one or more uracilglycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease includes Cas9 nickase fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the endonuclease includes Cas9 nickase fused to ABE8e. In some embodiments, the endonuclease includes Cas9 nickase fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)). In some embodiments, the endonuclease included Cas9 nickase fused to BE4max.

[0255] Examples of base editors include, but are not limited to, BE1, BE2, BE3, HF-BE3, BE4, BE4max, AncBE4max, 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, ABE7.10*, xABE, ABESa, ABE8e, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP. Further examples of base editors can be found, for example, in U.S. Publication No. 2018 / 0312825A1, U.S. Publication No. 2018 / 0312828A1, and PCT Publication No. WO2018 / 165629A1, which are incorporated into this book by reference in their entirety.

[0256] In some embodiments, the base editors are further modified to inhibit base excision repair at the target site and induce cellular mismatch repair. Any of the Cas endonucleases described herein may be fused to a Gam domain (bacteriophage Mu protein) to protect the Cas endonuclease from degradation and exonuclease activity. See, for example, Eid et al. Biochem. J. (2018) 475(11):1955-1964.

[0257] In some embodiments, the Cas endonucleases belong to class 2 type V of Cas endonucleases. Class 2 type V Cas endonucleases can be further categorized into type VA, type VB, type VC, and type VU. See, for example, Stella et al. Nature Structural & Molecular Biology (2017). In some embodiments, the Cas endonucleases are type VA Cas endonucleases, such as Cpf1 nuclease. In some embodiments, the Cas endonucleases are type VB Cas endonucleases, such as C2c1 endonucleases. See, for example, Shmakov et al. Mol Cell (2015) 60:385-397. In some embodiments, the Cas endonucleases are Mad7.

[0258] Alternatively, or further, Cas endonucleases are Cpf1 nucleases or their variants. As those skilled in the art will understand, Cas endonuclease Cpf1 nuclease is sometimes referred to as Cas12a. See, for example, Strohkendl et al. Mol. Cell (2018) 71:1-9. In some embodiments, host cells express Cpf1 nucleases derived from Provetella or Francisella species, Acidaminococcus species (AsCpf1), Lachnospiraceae bacterium (LpCpf1), or Eubacterium rectale. In some embodiments, the nucleotide sequence encoding the Cpf1 nuclease may be codon-optimized for expression in host cells. In some embodiments, the nucleotide sequence encoding the Cpf1 endonuclease is further modified to alter the activity of the protein.

[0259] A catalytically inactive variant of Cpf1 (Cas12a) is sometimes referred to as dCas12a. As described in this book, the catalytically inactive variant of Cpf1 may be fused to a functional domain to form a base editor. See, for example, Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas endonuclease is dCas12a. In some embodiments, the endonuclease comprises dCas12a fused to one or more uracilglycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises dCas12a fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas12a fused to ABE8e. In some embodiments, the endonuclease comprises dCas12a fused to a cytodin deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)). In some embodiments, the endonuclease comprises dCas12a fused to BE4max.

[0260] Alternatively, or even further, Cas endonucleases may be Cas14 endonucleases or their variants. Unlike Cas9 endonucleases, Cas14 endonucleases originate from archaea and tend to be smaller in size (e.g., 400-700 amino acids). Endonucleases do not require a PAM sequence. See, for example, Harrington et al. Science (2018).

[0261] Any of the Cas endonucleases described in this book can be regulated to control the level of Cas endonuclease expression and / or activity at a desired time. For example, it may be advantageous to increase the level of Cas endonuclease expression and / or activity at a specific stage of the cell cycle. The level of homology-directed repair has been shown to decrease during the G1 phase of the cell cycle; therefore, increasing the level of Cas endonuclease expression and / or activity during the S, G2, and / or M phases may increase homology-directed repair after Cas endonuclease editing. In some embodiments, the level of Cas endonuclease expression and / or activity increases during the S, G2, and / or M phases of the cell cycle. In one example, the Cas endonuclease was fused to the N-terminal region of human geminin. See, for example, Gutschner et al. Cell Rep. (2016) 14(6):1555-1566. In some embodiments, the expression and / or activity levels of Cas endonuclease decrease during the G1 phase. In one example, Cas endonuclease is modified to have decreased activity during the G1 phase. See, for example, Lomova et al. Stem Cells (2018).

[0262] Alternatively, or furthermore, any of the Cas endonucleases described herein may be fused to an epigenetic regulator (e.g., a chromatin-modifying enzyme, e.g., DNA methylase, histone deacetylase). See, for example, Kungulovski et al. Trends Genet. (2016) 32(2):101-113. Cas endonucleases fused to epigenetic regulators are sometimes called “epieffectors” and can enable transient and / or transient endonuclease activity. In some embodiments, the Cas endonuclease is dCas9 fused to a chromatin-modifying enzyme.

[0263] Furthermore, the compositions and methods described herein can be used in CRISPR prime editing methods. CRISPR-based prime editor systems may include Cas9 nickase fused to M-MLV reverse transcriptase (RT). Prime editor systems also utilize prime editing guide RNA (pegRNA). Prime editing allows for more nucleotide substitutions than CRISPR-based nucleotide editors, and by using this, it is possible to generate genetically engineered hematopoietic stem cells or progenitor cells described herein, in which the nucleotide substitutions are within sequences encoding splice elements, and the nucleotide substitutions result in alternative splicing of the gene-encoded transcript.

[0264] In further embodiments, homology-directed repair is used to generate genetically engineered hematopoietic stem cells or progenitor cells as described herein, in which the nucleotide substitutions are located within the sequence encoding the splice element, and the nucleotide substitutions result in alternative splicing of the gene-encoded transcript.

[0265] In some embodiments, the Disclosure provides compositions and methods for inhibiting lineage-specific cell surface antigens in hematopoietic cells using a CRISPR-based base editor system in which a guide RNA sequence hybridizes to a nucleotide sequence encoding a lineage-specific cell surface antigen. In some embodiments, the Disclosure provides compositions and methods for inhibiting multiple lineage-specific cell surface antigens in hematopoietic cells using a CRISPR-based base editor system in which a guide RNA sequence hybridizes to a nucleotide sequence encoding a lineage-specific cell surface antigen.

[0266] In some embodiments, the Disclosure provides compositions and methods for modifying lineage-specific cell surface antigens in hematopoietic cells using a CRISPR-based base editor system in which a guide RNA sequence hybridizes to a nucleotide sequence encoding a lineage-specific cell surface antigen. In some embodiments, the Disclosure provides compositions and methods for modifying multiple lineage-specific cell surface antigens in hematopoietic cells using a CRISPR-based base editor system in which a guide RNA sequence hybridizes to a nucleotide sequence encoding a lineage-specific cell surface antigen.

[0267] In some embodiments, the lineage-specific cell surface antigen is CD33, and the gRNA hybridizes to a portion of the nucleotide sequence encoding CD33. In some embodiments, the gRNA hybridizes to a sequence that flanks exon 2 of CD33 (Figure 4). Examples of gRNAs that target CD33 are shown in Table 4, but further gRNAs may be developed that hybridize to appropriate nucleotide sequences of CD33 and can be used in the methods described herein.

[0268] In some cases, the gRNAs for use in this disclosure may contain spacer sequences that are at least 90% (e.g., at least 93%, 95%, 96%, 97%, 98%, or 99%) identical to any of the exemplary guide RNA sequences in Table 4.

[0269] In some embodiments, the lineage-specific cell surface antigen is EMR2, and the gRNA hybridizes to a portion of the nucleotide sequence encoding EMR2. In some embodiments, the gRNA hybridizes to a sequence that flanks at exon 13 of the nucleotide sequence encoding EMR2 (Figure 9B). Examples of gRNAs targeting EMR2 are presented below, but further gRNAs may be developed that hybridize to appropriate nucleotide sequences of EMR2 and can be used in the methods described herein.

[0270] In some cases, the gRNAs for use in this disclosure may contain spacer sequences that are at least 90% (e.g., at least 93%, 95%, 96%, 97%, 98%, or 99%) identical to any of SEQ ID NOs. 4 and 46-47. [Table 4]

[0271] Furthermore, this disclosure provides compositions and methods for inhibiting a first lineage-specific cell surface antigen and at least one further lineage-specific cell surface antigen, i.e., a combination of the first lineage-specific antigen, a second lineage-specific antigen, a third lineage-specific antigen, a fourth lineage-specific antigen, and so on.

[0272] Furthermore, this disclosure provides compositions and methods for modifying a first lineage-specific cell surface antigen and at least one further lineage-specific cell surface antigen, i.e., a combination of the first lineage-specific antigen, a second lineage-specific antigen, a third lineage-specific antigen, a fourth lineage-specific antigen, and so on.

[0273] In some embodiments, the first series-specific antigen is CD33. In some embodiments, the second series-specific antigen is EMR2.

[0274] The "percent identity" of the two nucleic acids is determined using the algorithm of Karlin and Altschul Proc.Natl.Acad.Sci.USA(1990)87:2264-68, modified as described in Karlin and Altschul Proc.Natl.Acad.Sci.USA(1993)90:5873-77. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J.Mol.Biol.(1990)215:403-10. Performing a BLAST nucleotide search using the NBLAST program, score=100, and word length-12, nucleotide sequences homologous to the nucleic acid molecule of the present invention can be obtained. If a gap exists between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. (1997) 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used.

[0275] This book also provides a method for producing cells lacking or modified lineage-specific cell surface antigens, comprising preparing cells and introducing components of a CRISPR-based base editor system for genome editing into the cells. In some embodiments, nucleic acids containing guide RNA (gRNA) that hybridizes, or is predicted to hybridize, to a portion of the nucleotide sequence encoding the lineage-specific cell surface antigen are introduced into the cells. In some embodiments, the gRNA is introduced into the cells in a vector. In some embodiments, a CRISPR-based base editor is introduced into the cells. In some embodiments, the CRISPR-based base editor is introduced into the cells as a nucleic acid encoding the CRISPR-based base editor. In some embodiments, the gRNA and the nucleotide sequence encoding the CRISPR-based base editor are introduced into the cells in the same nucleic acid (e.g., the same vector). In some embodiments, the CRISPR-based base editor is introduced into the cells in the form of a protein. In some embodiments, the CRISPR-based base editor and gRNA are pre-formed in vitro and introduced into the cells as a complex. In some embodiments, the complex (e.g., a ribonucleoprotein complex) is introduced using electroporation.

[0276] This book also provides a method for producing cells deficient in or modified of multiple lineage-specific cell surface antigens, comprising preparing cells and introducing multiple CRISPR-based base editor systems for genome editing, namely, a CRISPR-based editor system for genome editing of lineage-specific cell surface antigens, and a CRISPR-based editor system for genome editing of at least one further lineage-specific cell surface antigen, for example, a first and a second CRISPR-based base editor system, into the cells. In some embodiments, components of the multiple CRISPR-based base editor systems are introduced into the cells. In some embodiments, a nucleic acid containing a guide RNA (gRNA) that hybridizes, or is predicted to hybridize, to a portion of a nucleotide sequence encoding at least one further lineage-specific cell surface antigen is introduced into the cells. In some embodiments, the gRNA is introduced into the cells in a vector. In some embodiments, the CRISPR-based base editor is introduced into the cells. In some embodiments, the CRISPR-based base editor is introduced into the cells as a nucleic acid encoding the CRISPR-based base editor. In some embodiments, the gRNA and the nucleotide sequence encoding the CRISPR-based base editor are introduced into the cell in the same nucleic acid (e.g., the same vector). In some embodiments, the CRISPR-based base editor is introduced into the cell in protein form. In some embodiments, the CRISPR-based base editor and gRNA are pre-formed in vitro and introduced into the cell as a complex. In some embodiments, the complex (e.g., a ribonucleoprotein complex) is introduced using electroporation.

[0277] In some embodiments, the first CRISPR-based base editor system is introduced into the cell in a different manner than the subsequent base editor system. In some embodiments, all CRISPR-based base editor systems are introduced into the cell using the same method.

[0278] This disclosure further provides engineered, non-naturally occurring vectors and vector systems capable of encoding one or more components of a CRISPR-based base editor system, the vector comprising (i) a (CRISPR)-Cas system guide RNA that hybridizes to a lineage-specific antigen sequence and (ii) a polynucleotide encoding a CRISPR-based base editor.

[0279] The vectors of this disclosure can drive 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 control function of the expression vector is usually provided by one or more regulatory elements. For example, commonly used promoters are derived from polyomas, 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.

[0280] The vectors of this disclosure can preferentially direct the expression of nucleic acids in specific cell types (e.g., tissue-specific regulatory elements are used to express nucleic acids). Such regulatory elements include promoters that may be tissue-specific or cell-specific. The term “tissue-specific” as applied to a promoter refers to a promoter that can direct the selective expression of a target nucleotide sequence for a particular type of tissue (e.g., seed) and for which the expression of the same target nucleotide sequence is relatively absent in different types of tissue. The term “cell-type specific” as applied to a promoter refers to a promoter that can direct the selective expression of a target nucleotide sequence for a particular type of cell and for which the expression of the same target nucleotide sequence is relatively absent in different types of cells within the same tissue. The term “cell-type specific” as applied to a promoter also means a promoter that can promote the selective expression of a target nucleotide sequence for a particular region within a single tissue. The cell-type specificity of a promoter can be evaluated using methods well known in the art, such as immunohistochemical staining.

[0281] Nucleic acids encoding CRISPR-based base editors can be introduced into mammalian cells or target tissues using conventional virus-based and non-virus-based gene transfer methods. Such methods can be used to administer nucleic acids encoding components of CRISPR-based base editor systems to cells or host organisms under culture.

[0282] Nonviral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acids, and nucleic acids compounded with a delivery vehicle. In one embodiment, the nonviral vector delivery system used is a pre-formed ribonucleoprotein complex (e.g., a complex containing a CRISPR-based base editor protein compounded with a targeting gRNA). The pre-formed ribonucleoprotein complex can then be introduced into cells via electroporation, particulate gun irradiation, or other physical delivery methods. In one embodiment, electroporation is used to introduce the pre-formed ribonucleoprotein complex into cells. See, for example, Example 1.

[0283] Viral vector delivery systems include DNA and RNA viruses that have either an episomal genome or an integrated genome after delivery to cells. Viral vectors may be administered directly to patients (in vivo) or used to manipulate cells in vitro or ex vivo, and the modified cells may be administered to patients. In one embodiment, the disclosure utilizes virus-based systems for gene transfer, including but not limited to retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus vectors. Furthermore, the disclosure provides vectors that can be integrated into a host genome, such as retroviruses or lentiviruses. Preferably, the vector used for expression in the CRISPR-based base editor system of the disclosure is a lentiviral vector.

[0284] In one embodiment, the disclosure provides introducing one or more vectors encoding a CRISPR-based base editor into a eukaryotic cell. The cell may be a cancer cell. Alternatively, the cell may be a hematopoietic cell, such as a hematopoietic stem cell. Examples of stem cells include pluripotent, multipotent, and unipotent stem cells. Examples of pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic cancer cells, and induced pluripotent stem cells (iPSCs). In a preferred embodiment, the disclosure provides introducing a CRISPR-based base editor into a hematopoietic stem cell.

[0285] The vectors of this disclosure are delivered to target eukaryotic cells. Modification of eukaryotic cells with a CRISPR-based base editor system can be performed under cell culture conditions, and this method includes isolating the eukaryotic cells from the target before modification. In some embodiments, this method further includes returning the eukaryotic cells and / or cells derived therefrom to the target.

[0286] Combination therapy As described in this book, drugs containing antigen-binding fragments that bind to lineage-specific cell surface antigens (e.g., CD33) can be administered to subjects in combination with hematopoietic cells lacking the lineage-specific cell surface antigen or its epitope, such as hematopoietic stem cells or progenitor cells produced using the CRISPR-based base editor system and gRNA described in this book, for example, gRNA containing the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0287] As described in this book, drugs containing antigen-binding fragments that bind to lineage-specific cell surface antigens (e.g., EMR2) can be administered to subjects in combination with hematopoietic cells lacking the lineage-specific cell surface antigen or its epitope, such as hematopoietic stem cells or progenitor cells produced using the CRISPR-based base editor system and gRNA described in this book, for example, gRNA containing the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 46, or SEQ ID NO: 47.

[0288] Furthermore, as described in this book, drugs containing antigen-binding fragments that bind to lineage-specific cell surface antigens (e.g., CD33) and drugs containing antigen-binding fragments that bind to at least one further lineage-specific cell surface antigen can be administered to subjects in combination with hematopoietic cells lacking the lineage-specific cell surface antigen or its epitope, for example, hematopoietic stem cells or progenitor cells produced using the CRISPR-based base editor system and gRNA described in this book, for example, the gRNA containing the following nucleotide sequence: (Sequence ID 1) (Sequence ID 2), or (Sequence ID 3), and (Sequence ID 4) (Sequence ID 46), or (Sequence ID 47).

[0289] As used in this book, “subject,” “individual,” and “patient” are used synonymously and refer to vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, human primates, non-human primates, or species such as mice, cattle, horses, dogs, or cats. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0290] In some embodiments, pharmaceutical compositions can be formed by mixing drugs and / or hematopoietic cells with a pharmaceutically acceptable carrier, which is also within the scope of this disclosure.

[0291] To carry out the methods described herein, an effective dose of an antigen-binding fragment containing a lineage-specific cell surface antigen(s) and an effective dose of hematopoietic cells may be administered simultaneously to a subject requiring treatment. As used herein, the term “effective dose” may be used synonymously with the term “therapeutic dose” and refers to the amount of a drug, cell population, or pharmaceutical composition (e.g., a composition containing a drug and / or hematopoietic cells) that, when administered to a subject requiring it, is sufficient to produce the desired activity. In the context of this disclosure, “effective dose” refers to the amount of a compound, cell population, or pharmaceutical composition that is sufficient to delay the manifestation, inhibit the progression, reduce, or alleviate at least one symptom of the disorder treated by the methods of this disclosure. Note that when a combination of active ingredients is administered, the effective dose of the combination may or may not include the amount of each ingredient that would have been effective if administered individually.

[0292] As will be recognized by those skilled in the art, the effective dose varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of parallel therapies (if any), the specific route of administration, and similar factors within the scope of the healthcare professional's knowledge and expertise. In some embodiments, the effective dose alleviates, reduces, remits, improves, reduces, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is human. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0293] As described in this book, hematopoietic cells and / or immune cells expressing chimeric receptors may be autologous to the subject; that is, the cells are obtained from a subject requiring treatment, genetically engineered to lack or modify the expression of cell surface lineage-specific antigens or chimeric receptor constructs, and then administered to the same subject. Administration of autologous cells to a subject may result in a reduction of host cell rejection compared to administration of non-autologous cells. Alternatively, the host cells may be allogeneic cells; that is, the cells are obtained from a first subject, genetically engineered to lack or modify the expression of cell surface lineage-specific antigens or chimeric receptor constructs, and then administered to a second subject that is the same species but different from the first subject. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient different from the donor.

[0294] In some embodiments, immune cells expressing any of the chimeric receptors described herein are administered to a subject in an amount effective in reducing the number of target cells (e.g., cancer cells) by at least 20%, for example, 50%, 80%, 100%, 2x, 5x, 10x, 20x, 50x, 100x, or more.

[0295] A typical amount of cells administered to a mammal (e.g., a human), i.e., immune cells or hematopoietic cells, may be in the range of, for example, 1 million to 100 billion cells, but amounts below or above this exemplary range are also within the scope of this disclosure. For example, a daily dose of cells may be approximately 1 million to 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range defined by any two of the aforementioned values), preferably approximately 10 million to 100 billion cells (e.g., approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 1 It could be 0 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the aforementioned values), more preferably approximately 100 million to approximately 50 billion cells (for example, approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells, or a range defined by any two of the aforementioned values).

[0296] 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 dose or number of immune cells expressing the chimeric receptor. One or more subsequent doses of the drug (e.g., immune cells expressing the chimeric receptor) may be provided 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 dose. Multiple doses of the drug, e.g., two, three, four, or more doses of the drug, may be administered to the subject weekly. The subject may receive multiple doses of the drug (e.g., immune cells expressing the chimeric receptor) in a week, then no drug administration for a week, followed by one or more further doses of the drug (e.g., multiple weekly doses of immune cells expressing the chimeric receptor). Immune cells expressing chimeric receptors may be administered every other day, three times a week, for two, three, four, five, six, seven, eight weeks, or longer.

[0297] In the context of this disclosure, to the extent that it relates to any of the disease conditions listed herein, terms such as “to treat” or “to treat” mean to alleviate or reduce at least one symptom associated with such condition, or to slow or reverse the progression of such condition. In the sense of this disclosure, the term “to treat” also means to prevent a disease, to delay its onset (i.e., the period prior to the clinical manifestation of the disease), and / or to reduce the risk of its onset or worsening. For example, in relation to cancer, the term “to treat” may mean to eliminate or reduce the tumor burden on a patient, or to prevent, delay, or inhibit metastasis.

[0298] In some embodiments, the drug(s) comprises an antigen-binding fragment that binds to lineage-specific cell surface antigen(s) and a population of hematopoietic cells that are deficient in or modified in lineage-specific cell surface antigen(s). Thus, in such a therapeutic method, the drug(s) recognize (bind to) target cells that express lineage-specific cell surface antigen(s) lipopulation of the cell type targeted by the drug(s). In some embodiments, the treatment of a patient may include the following steps: (1) administering to the patient a therapeutically effective dose of the drug(s) that targets lineage-specific cell surface antigen(s); and (2) injecting or reinjecting to the patient hematopoietic stem cells, either autologous or allogeneic, in which the expression of lineage-specific disease-associated antigen(s) is reduced or modified. In some embodiments, patient treatment may include the following steps: (1) administering a therapeutically effective dose to the patient of immune cells expressing a chimeric receptor, the immune cells comprising a nucleic acid sequence encoding a chimeric receptor that binds to a lineage-specific cell surface disease-associated antigen(s); and (2) injecting or reinjecting the patient with autologous or allogeneic hematopoietic cells (e.g., hematopoietic stem cells) in which the expression of the lineage-specific disease-associated antigen(s) is reduced or modified.

[0299] The effectiveness of a therapeutic method using a drug(s) containing antigen-binding fragments that bind to cell surface lineage-specific antigens(s) and a population of hematopoietic cells lacking or modified lineage-specific cell surface antigens(s) can be evaluated by any method known in the art and will be evident to a skilled medical professional. For example, the effectiveness of the therapy may be assessed by the survival of the subject or the cancer burden in the subject, tissue, or a sample thereof. In some embodiments, the effectiveness of the therapy is assessed by quantifying the number of cells belonging to a particular population or lineage of cells. In some embodiments, the effectiveness of the therapy is assessed by quantifying the number of cells presenting cell surface lineage-specific antigens.

[0300] In some embodiments, a drug containing an antigen-binding fragment that binds to a cell surface lineage-specific antigen is administered in combination with a population of hematopoietic cells.

[0301] In some embodiments, a drug(s) containing antigen-binding fragments that bind to lineage-specific cell surface antigens(s) (e.g., immune cells expressing the chimeric receptors described herein) is administered prior to the administration of hematopoietic cells. In some embodiments, a drug(s) containing antigen-binding fragments that bind to lineage-specific cell surface antigens(s) (e.g., immune cells expressing the chimeric receptors described herein) is administered at least approximately 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 earlier before the administration of hematopoietic cells. In some embodiments, a drug(s) containing antigen-binding fragments that bind to lineage-specific cell surface antigens(s) (e.g., immune cells expressing the chimeric receptors described herein) is administered to the subject multiple times prior to the administration of hematopoietic cells.

[0302] In some embodiments, hematopoietic cells are administered prior to the administration of a drug(s) containing antigen-binding fragments that bind to lineage-specific cell surface antigens(s) (e.g., immune cells expressing chimeric receptors as described herein). In some embodiments, the hematopoietic cell population is administered at least approximately 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 earlier, prior to the administration of a drug(s) containing antigen-binding fragments that bind to lineage-specific cell surface antigens(s).

[0303] In some embodiments, a drug(s) that target lineage-specific cell surface antigens (CPIs) and a hematopoietic cell population are administered substantially simultaneously. In some embodiments, a drug(s) that target lineage-specific cell surface antigens (CPIs) is administered, the patient is evaluated over a period of time, and then a hematopoietic cell population is administered. In some embodiments, a hematopoietic cell population is administered, the patient is evaluated over a period of time, and then a drug(s) that target lineage-specific cell surface antigens (CPIs) is administered.

[0304] The scope of this disclosure also includes multiple administrations (e.g., doses) of the drug and / or hematopoietic cell population. In some embodiments, the drug and / or hematopoietic cell population is administered to the subject once. In some embodiments, the drug and / or hematopoietic cell population is administered to the subject multiple times (e.g., at least two, three, four, five, or more times). In some embodiments, the drug and / or hematopoietic cell population is administered to the subject at regular intervals, for example, every six months.

[0305] In some embodiments, the subjects are human subjects with hematopoietic malignancies. As used in this text, hematopoietic malignancies refer to malignancies involving hematopoietic cells (e.g., blood cells including progenitor cells and stem cells). Examples of hematopoietic malignancies include, but are not limited to, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, or multiple myeloma. Examples of leukemia include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, and chronic lymphoblastic leukemia.

[0306] In some embodiments, leukemia is acute myeloid leukemia (AML). AML is characterized as a heterogeneous, clonal, neoplastic disease resulting from transformed cells that gradually acquire significant genetic alterations that disrupt major differentiation and growth regulatory pathways (Dohner et al., NEJM, (2015) 373:1136). The CD33 glycoprotein is expressed in the majority of myeloid leukemia cells as well as normal myeloid progenitor cells and monocyte progenitor cells, and has been 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 therapies have shown improved survival in some AML patients when combined with standard chemotherapy, but these effects have been accompanied by safety and efficacy concerns.

[0307] Other approaches aimed at targeting AML cells include generating T cells that express chimeric antigen receptors (CARs) that selectively target CD33 in AML. Buckley et al., Curr. Hematol. Malig. Rep. (2015) 2:65. However, data are limited, and it is uncertain how effective this approach may be in patient treatment (whether all targeted cells are eliminated). Furthermore, since myeloid cells are essential for life, depleting the target myeloid cells could have adverse effects on patient survival. This disclosure aims to address, at least in part, such problems associated with AML treatment.

[0308] Alternatively, or furthermore, 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, prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, gastrointestinal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, neoplasm in situ, kidney cancer, laryngeal cancer, liver cancer, fibroma, neuroblastoma, oral cancer (e.g., lips, tongue, mouth, and pharynx), retinoblastoma, rhabdomyosarcoma, renal cancer, respiratory cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary tract cancer, and other carcinomas and sarcomas.

[0309] Carcinoma is a cancer of epithelial origin. The carcinomas to which treatment by the methods of this disclosure is intended include acinar carcinoma, acinar carcinoma, follicular adenocarcinoma (also called adenoid cystic carcinoma, adenomyoepithelioma, cribriform carcinoma, and columnoma), adenomatous carcinoma, adenocarcinoma, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma (also called bronchiolar carcinoma, alveolar cell tumor, and pulmonary adenomatosis), basal cell carcinoma, basal cell carcinoma (also called basaloma or basiloma, and piloma carcinoma), basal carcinoma, basal squamous cell carcinoma, breast carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchial carcinoma, cerebral carcinoma, cholangiocarcinoma (also called cholangiocarcinoma and intrahepatic cholangiocarcinoma), choriocarcinoma, and colloid carcinoma. Carcinoma, comedone carcinoma, uterine body carcinoma, cribriform carcinoma, armory carcinoma, skin carcinoma, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, scirrhous carcinoma, embryonic carcinoma, cerebral carcinoma, upper eye carcinoma, epidermoid carcinoma, epithelioid adenoid carcinoma, ulcerative carcinoma, fibrocarcinoma, gelatinous carcinoma (gelatiniform carcinoma), gelatinous carcinoma (gelatinous Carcinoma), giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, piloma carcinoma, hematoid carcinoma, hepatocellular carcinoma (also called hepatocellular tumor, malignant hepatocellular tumor, and liver carcinoma), Huirthle cell carcinoma, hyaline carcinoma, adrenal carcinoma, infant embryonic carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ, Krompecher carcinoma, Kulchitzky cell carcinoma, lenticular carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, mammary carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma (carcinoma melanodes), melanotic carcinoma, mucinous carcinoma, mucinous cell carcinoma, mucosal epidermal carcinoma, mucinous carcinoma, mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, melanotic carcinoma This includes, but is not limited to, nigrum, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, pre-invasive carcinoma, prostate carcinoma, renal cell carcinoma of the kidney (also called renal adenocarcinoma and hypomephoroid carcinoma), replacement cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, potato-shaped carcinoma, elliptic cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, nodular carcinoma, verrucous carcinoma, and choriocarcinoma.In a preferred embodiment, the method of the present disclosure is used to treat subjects having breast cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, or kidney cancer.

[0310] Sarcomas are mesenchymal neoplasms that occur in bone and soft tissue. Various types of sarcomas are recognized, including liposarcoma (including myxoid liposarcoma and pleomorphic liposarcoma), leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (including Ewing's osteosarcoma, extraosseous (i.e., non-osseous) Ewing's sarcoma, and primitive neuroectodermal tumors [PNETs]), synovial sarcoma, angiosarcoma, and hemangiosarcoma. This includes arcomas, lymphangiosarcoma, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, tendonoid sarcoma (also known as invasive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangioectocytoma, malignant mesenchymal sarcoma, hydatidiform soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, connective tissue-forming small cell tumor, gastrointestinal stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma) - skeletal and extraskeletal, as well as chondrosarcoma.

[0311] In some embodiments, the cancer being treated may be a refractory cancer. “Refractory cancer,” as used herein, is a cancer that is resistant to the standard treatment prescribed. These cancers may initially appear to respond to treatment (and then recur), or they may be completely refractory to treatment. Typical standard treatments vary depending on the type of cancer and its stage in the subject. This may be chemotherapy, surgery, radiation, or a combination thereof. Those skilled in the art are aware of such standard treatments. Therefore, a subject receiving treatment for a refractory cancer according to this disclosure may have already been exposed to other treatments for that cancer. Alternatively, if the cancer is likely to be refractory (e.g., given an analysis of cancer cells or the subject's records), the subject may not have already been exposed to other treatments. Examples of refractory cancers include, but are not limited to, leukemia, melanoma, renal cell carcinoma, colon cancer, liver cancer, pancreatic cancer, non-Hodgkin lymphoma, and lung cancer.

[0312] Any of the chimeric receptor-expressing immune cells described in this book can be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier or excipient.

[0313] When used in connection with the compositions and / or cells of this disclosure, the term “pharmaceutically acceptable” refers to the molecular entities and other components of such compositions that are physiologically tolerable and do not typically cause adverse reactions when administered to mammals (e.g., humans). Preferably, as used herein, “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, more specifically in humans. “Acceptable” means that the carrier is compatible with the active component of the composition (e.g., nucleic acid, vector, cell, or therapeutic antibody) and that the composition does not adversely affect the subject to which it is administered. Any of the pharmaceutical compositions and / or cells used in the methods herein may contain a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or aqueous solution.

[0314] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphoric acid, citrate, 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; and other carbohydrates; metal complexes; and / or nonionic surfactants. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE. Hoover.

[0315] Kits for therapeutic use Within the scope of this disclosure are kits for using a drug that targets lineage-specific cell surface antigens in combination with a population of hematopoietic cells lacking one or more lineage-specific cell surface antigens. Such a kit may comprise one or more containers comprising a first pharmaceutical composition comprising any one drug (e.g., immune cells expressing the chimeric receptors described herein) containing antigen-binding fragments that bind to one or more lineage-specific cell surface antigens, and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising a population of hematopoietic cells (e.g., hematopoietic stem cells) lacking one or more lineage-specific cell surface antigens, and a pharmaceutically acceptable carrier.

[0316] In some embodiments, the kits described herein include gRNAs having sequences of SEQ ID NOs: 1–3. In some embodiments, the kits described herein include gRNAs having sequences of SEQ ID NOs: 4 and 46–47. In further embodiments, the kits may further include gRNAs having any of the sequences of SEQ ID NOs: 1–4 and 46–47. In some embodiments, the kits may further include reagents for a CRISPR-based base editor system, which include a catalytically impaired Cas protein fused to a DNA modifying enzyme, i.e., Cas9 nickase, fused to a cytosine deaminase or adenosine deaminase (base editor).

[0317] In some embodiments, the kit may include instructions for use of any of the methods described herein. The included instructions may include instructions for administering the first and second pharmaceutical compositions to a subject in order to achieve the intended activity in that subject. The kit may further include instructions for selecting a subject suitable for treatment based on the identification of whether the subject requires treatment. In some embodiments, the instructions include instructions for administering the first and second pharmaceutical compositions to a subject requiring treatment.

[0318] Instructions for use of the agents targeting cell surface lineage-specific antigens described herein, as well as the first and second pharmaceutical compositions, generally include information regarding the dosage, administration schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. Instructions supplied in the kits of this disclosure are typically those found on the label or in the accompanying leaflet. The label or accompanying leaflet indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate, a disease or disorder of interest.

[0319] The kits provided in this document are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, flasks, and flexible packaging. Packaging for use in combination with specific devices such as inhalers, nasal administration devices, or infusion devices is also intended. The kits may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). The container may have a sterile access port. At least one activator in the pharmaceutical composition is a chimeric receptor variant as described in this document.

[0320] The kit may optionally provide further components such as buffers and explanatory information. Typically, the kit includes a container and a label or accompanying document attached to or associated with the container. In one embodiment, the disclosure provides a product comprising the contents of the kit described above.

[0321] general technique Unless otherwise stated, the implementation of this disclosure will involve conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the skills of those skilled in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, 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 are fully described in the literature such as B. Perbal, A practical Guide To Molecular Cloning (1984), F.M. Ausubel et al. (eds.).

[0322] Those skilled in the art will likely be able to make the most of this disclosure based on the above description without further detail. Therefore, the following specific embodiments should be construed as merely illustrative and not as limiting the remainder of this disclosure. All publications referenced herein are incorporated by reference for the purposes or subjects referenced herein. [Examples]

[0323] Example 1: Efficient editing of HSC / HSPC using a base editor Materials and methods Human umbilical cord blood CD34+ stem cells were maintained in StemSpan SFEM II (STEMCELL Technologies Inc.) containing 1% penicillin streptomycin, along with the following human cytokines: 100 ng / mL TPO, 100 ng / mL SCF, 100 ng / mL IL6, and 100 ng / mL FLT3L and UM171 0.35 nM (Xcessbio, San Diego, CA, USA). All human cytokines were purchased from Biolegend (San Diego, CA, USA).

[0324] ABE protein and sgRNA were mixed in P3 buffer (Lonza, Amaxa P3 Primary Cell 4D-Nucleofector Kit) and incubated for 10 minutes. The cells were then washed with PBS, resuspended in P3 buffer, mixed with the Cas9 / sgRNA RNP complex, and electroporated using a 4D-Nucleofector. Following electroporation, the cells were cultured at 37°C until analysis or injection.

[0325] Editing of HSC / HSPC by ABE8e protein Using the Lonza 4D nucleofector and program DZ100, 1 million HSCs / HSPCs were nucleofected with 11ugr of ABE8e protein + 1.5ugr of chemically modified sgRNA (SEQ ID NO: 3).

[0326] Editing of HSCs / HSPCs with BE4max protein Using a Lonza 4D nucleofector and program DZ100, 500,000 HSCs / HSPCs were nucleofected with 8ugr of BE4max protein + 1.5ugr of chemically modified sgRNA (sequence number 1 or 2).

[0327] After electroporation, the cells were cultured at 37°C until analysis.

[0328] Editorial results and off-target evaluation: HTS analysis using CRISPResso2 Primers were designed to surround a 350 bp region around the protospacer sgRNA 208 or sgRNA SA. Each primer was adorned with an appropriate ILLUMINA adapter. The resulting PCR product was then re-amplified using ILLUMINA forward and reverse index primers to generate 250–300 bp single-ended Illumina sequencing. Each read was then aligned with a reference amplicon using CRISPResso2, and indel or base changes relative to the reference were identified within the window surrounding the protospacer. These were quantified by software. See Clement et al. Nature Biotechnology (2019) 37:224-26 and Huang et al. Nature Protocols (2021) 16(2):1089-1128.

[0329] PCR cDNA was prepared from 100 ng of cell-derived total RNA using the RNA to cDNA EcoDry mix (Takara). CD33 Δ2 A primer specific to (spanning exon junctions 1-3), CD33 FLPCR was performed for 30 cycles using primers specific to the isoform (spanning exon junctions 2-3 or exon 2) or primers common to all isoforms (within exons 1, 5, and 7). The PCR products were separated by polyacrylamide gel electrophoresis and analyzed by Sanger sequencing.

[0330] Flow cytometry Human CD34 was detected 7 days after electroporation using anti-CD33 antibody clones M53 and P67.6, which recognize epitopes located within exon 2. + Stem cells were analyzed.

[0331] statistics All statistical analysis was performed using Graphpad Prism 9.1.1. For continuous variables, an unpaired two-tailed t-test was performed. Differences in means were considered statistically significant if the p-value was <0.05; otherwise, they were considered not significant (ns; p>0.05).

[0332] result Three sgRNAs were designed to induce exon 2 skipping using a BE or ABE base editor (Figure 4). CD33 mRNA full length (CD33 FL ) contains seven exons, with exon 2 encoding the Ig-like V-type domain. CD33 Δ2 It lacks exon 2 due to a common polymorphism (rs12459419, A14VSNP) that results in a modified exon splicing enhancer (ESE) site where C is changed to T.

[0333] mRNA and ribonucleoprotein (RNP)-based delivery systems were tested, and their efficiency in primary cells was investigated. CD34 cells were electroporated with each of these gRNAs and either a base editor (BE) or adenine base editor (ABE) protein. Specifically, CD34 cells were electroporated with sgRNAs having ABE8e and SEQ ID NO: 3, BE4max and SEQ ID NO: 1, or BE4max and SEQ ID NO: 2 (Table 4).

[0334] As shown by Sanger sequencing, BE and ABE introduced either a C>T or A>G conversion to the target nucleotide (Figure 5A).

[0335] Furthermore, edited cells were analyzed using PCR and Illumina MiSeq. These results indicated that each target base acquired the intended mutation. Cells edited with SEQ ID NO: 3 and ABE8e showed only 4% wild-type reads. Cells edited with SEQ ID NO: 1 or 2 and BE4max showed approximately 9–12% wild-type reads (Figure 5B). Flow cytometry analysis of edited cells using two antibodies that recognize the epitope located within exon 2 of CD33 confirmed the absence of CD33 exon 2 in the edited cells (Figure 5C).

[0336] cDNA and CD33 Δ2 Edited cells were further analyzed for their editing results using PCR with primers specific to ESE (spanning exon junctions 1-3), primers specific to CD33FL (spanning exon junctions 2-3 or exon 2), or primers common to all isoforms (within exon 3, spanning exon junctions 3-5 or 4-5). The results showed that editing of ESE or SA induced exon 2 skipping without affecting other exons (Figure 5D).

[0337] Example 2: CD33 Δ2 The cells exhibit normal phagocytic ability in vitro and are resistant to GO. CD34 described in Example 1 + CD33 Δ2 The cells were further analyzed.

[0338] devour CD34 differentiated in vitro + CD33 WT and CD33 Δ2The ability of monocytes to phagocytose E coli biological particles in vitro was tested. Differentiated WT or CD33 monocytes were tested in vitro. Δ2 These monocytes exhibit comparable phagocytic ability when measured by the internal transport of E. coli bioparticles. See Figure 6A.

[0339] GO cytotoxicity Furthermore, CD34 + CD33 Δ2 The cells resisted GO cytotoxicity in vitro. Cells were incubated with GO for 48 hours and analyzed by FACS using Sytox Blue or 7AAD as the viability-determinating dye. CD34 + CD33 Δ2 This cell type exhibits the same resistance to GO cell cytotoxicity as donors with homozygous rs12459419(TT)A14V SNP (Figure 6B).

[0340] Example 3: CD34+CD33Δ2 can engraft multiple sequences in vivo over a long period and is resistant to CD33-targeted immunotherapy (GO). Materials and methods In vivo experiment This approach uses CD33 gene-edited stem cells (CD34) as a platform for CAR-T targeting and ADC delivery (GO) of the CD33 antigen. + CD33 Δ2 This may include transplanting CD34 cells that engraft and contribute to bone marrow hematopoiesis and lymphocyte regeneration. + CD33 Δ2 It is important to test the capabilities of the cells.

[0341] NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mouse or NOD.Cg-Prkdc scid Il2rg tm1WjlTg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ(NSG-SGM3) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were pre-treated with sublethal dose (100 cGy) total body irradiation (TBI). Mice that received sublethal irradiation were then treated with CD34+WT or CD34. + CD33 Δ2 The HSPC (cells edited using ABE8e and SEQ ID NO: 3 as described in Example 1) was injected via tail vein injection.

[0342] The temporal engraftment and lipopulation of the hematopoietic system (Figure 7A) were evaluated by analysis of peripheral blood (PB), spleen, and whole bone marrow (BM) using the following antibodies from Biolegend (San Diego, CA, USA) or BD Biosciences (San Jose, CA, USA): Ter119-PeCy5, Ly5-BV711, H2kd-BV711, hCD45-BV510, hCD3-Pacific Blue, hCD123-BV605, hCD33-APC, hCD14-APC / Cy7, hCD10-BUV395, hCD19-BV650, CD34-BV421, CD90-PeCy7, hCD38-BUV661, and hCD45RA-BUV737. Dead cells were eliminated using propidium iodide. Injected CD34 + Human-derived cells were gated with Ter119-, Ly5- / H2kd-human CD45+.

[0343] CD34 + CD33 Δ2 To demonstrate that cells possess GO resistance in vivo, PB cells in mice 12 weeks after transplantation were treated with CD33 + CD14 + Cells or CD33 Δ2 CD14 + The presence of cells was analyzed. Next, mice were injected with 2.5 ugr of GO, and blood was collected and bagged one week after treatment to evaluate the presence of PB and myeloid cells in the bone marrow of humanized mice. Before GO treatment, CD34 +WT Transplanted mice or CD34 + CD33Δ2 The transplanted mice had CD14 in PB. + This showed that the cell frequencies were the same (Figure 7D, FACS plot above). One week after GO injection, CD34 + CD33 Δ2 CD33 was found in the PB and BM of mice in which cells were engrafted. - CD14 + Although cells were detected, CD33 was not found in the PB and BM of CD34+WT engrafted mice. + Cells and CD14 + The cells had been eradicated.

[0344] Sixteen weeks after transplantation, the CD33 locus was amplified from mouse bone marrow-derived genomic DNA. The amplicon was sequenced by HTS, and the A-to-G editing at position A7 was quantified.

[0345] All experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee at Columbia University.

[0346] result Sixteen weeks after transplantation, human CD45+ cells, as well as myeloid progenitor cells (CD123) and lymphoid progenitor cells (CD10) within human CD45, and mature myeloid cells (CD14) and lymphoid cells (CD19), and T cells (CD3) were found in both the bone marrow and spleen (Figure 7B). All cells remained CD33-negative (Figure 7A).

[0347] CD34 after GO treatment + CD33 Δ2 CD33-CD14+ cells were detected in the peripheral blood and bone marrow of mice in which cells were engrafted, while CD33+ cells were not detected. + CD34 +WT The grafted mice have been eradicated. Therefore, CD33 WT The cells retain sensitivity to GO, but CD34 + CD33 Δ2The cells were insensitive. Figures 7B and 7C.

[0348] On-target editing at the target site (A7) in engrafted WT (unedited) or edited cells was analyzed from bone marrow samples 16 weeks after transplantation. 16 weeks after transplantation, the CD33 locus was amplified from mouse bone marrow-derived genomic DNA. Figure 7D.

[0349] Example 4: Off-target evaluation Materials and methods CIRCLEseq To identify off-target base editing, genomic DNA was extracted from an anonymized human donor-derived CD34+ enriched cell population using the QIAgen Gentra PureGene kit (catalog number 158445). Circle-seq was performed as previously reported (Tsai et al, Nature Methods (2017) 14:607-14). Briefly, purified genomic DNA was cut into 300 bp lengths using a Covaris S2 apparatus. Using the KAPA HTP Library Preparation Kit, PCR Free (KAPA Biosystems), the fragmented DNA was repaired at the ends, A-tails were added, and the DNA was ligated to uracil-containing stem-loop adapters. The DNA ligated to the adapters was treated with lambda exonuclease (NEB) and E. coli exonuclease I (NEB), followed by treatment with USER enzyme (NEB) and T4 polynucleotide kinase (NEB). Intramolecular circularization of DNA was performed using T4 DNA ligase (NEB), and the remaining linear DNA was degraded with Plasmid-Safe ATP-dependent DNase (Lucigen). In vitro cleavage was performed using 100 μl of 250 ng of Plasmid-Safe treated circularized DNA, 90 nM Cas9 nuclease (NEB), Cas9 nuclease buffer (NEB), and 90 nM synthetic chemically modified sgRNA (Synthego). An A-tail was added to the cleaved product, ligated with a hairpin adapter (NEB), treated with USER enzyme (NEB), and amplified by PCR using barcoded universal primers NEBNext Multiplex Oligos for Illumina (NEB) with Kapa HiFi Polymerase (KAPA Biosystems). The library was sequenced using 150 bp paired-end reads on an Illumina MiSeq instrument. CIRCLE-seq data analysis was performed using open-source CIRCLE-seq analysis software (https: / / github.com / tsailabSJ / circleseq) with default parameters.The human genome GRCh37 was used for alignment.

[0350] To evaluate off-target editing, the top 19 off-target sites identified by CIRCLE-seq were sequenced in human WT cells (unedited cells) or edited cells engrafted from bone marrow 16 weeks after transplantation.

[0351] For each off-target site, primers were designed to generate a 250–300 bp product containing an aligned off-target binding site for guide RNA and an added adapter for Illumina sequencing. After barcoding each sample by secondary PCR, the products were pooled and sequenced using a 300-cycle v2 Illumina MiSeq kit. The amplicons were sequenced by HTS to quantify the A-to-G editing at position A7.

[0352] To analyze the editing results in the obtained fastq files, each read was aligned with a reference amplicon using CRISPResso2, and indel or base changes were quantified.

[0353] CD34 edited using ABE8e and Sequence ID No. 3 as described in Example 1 + CD33 Δ2 The cells were further analyzed.

[0354] result Using CIRCLE-seq, the top 19 off-target gene loci were identified. Figure 8A.

[0355] As described above, sequencing was performed to evaluate A-to-G editing at the A7 position of the A7 target nucleotide in the top 19 off-target gene loci identified in engrafted human WT (unedited) or edited cells. As shown in Figure 8B, for most targets, A7 editing was similar in WT and edited cells.

[0356] As shown in Figure 8C, the percentage of target indels was similar in both WT cells and edited cells.

[0357] Example 5: Multiple base editing Two sgRNAs were designed. One induced exon skipping at CD33 using an ABE editor (SEQ ID NO: 3). The second induced exon 13 skipping at EMR2 using an ABE editor (SEQ ID NO: 4). Figures 4A and 9A.

[0358] Two RNPs (one for each target gene) were prepared separately using 5ugr AB8e+1.5ugr chemically modified sgRNA. After incubation, the RNPs were mixed with 500,000 HSCs / HSPCs and nucleofected using a Lonza 4D nucleofector and programmed DZ100.

[0359] Edited cells were analyzed for the introduction of nucleotide conversions. As shown by Sanger sequencing, the ABE base editor introduced A>G conversions to both loci, thereby editing the SA of the CD33 locus and the SD of the EMR2 locus (Figure 9B). Flow cytometry analysis of the edited cells confirmed that the binding of CD33 and EMR2 antibodies was suppressed by the editing in the edited cells (Figure 9C).

[0360] Example 6: Targeting of cell surface lineage-specific CD33 in acute myeloid leukemia (AML) This embodiment involves targeting of the CD33 antigen in AML. Specific steps of this embodiment are outlined in Table 5. [Table 5]

[0361] I. CD33-targeted chimeric antigen receptor (CAR) T-cell therapy A. Generation of anti-CD33 CAR structures The CD33-targeting chimeric antigen receptors described in this book may consist of the following components in 5' to 3' order: pHIV-Zsgreen lentiviral skeleton (www.addgene.org / 18121 / ), peptide signaling pathway, CD33 scFv, hinge, transmembrane domain of the CD28 molecule, intracellular domain of CD28, and signaling domain of the TCR-ζ molecule.

[0362] First, the peptide signal, anti-CD33 light chain (SEQ ID NO: 8), mobile linker, and anti-CD33 heavy chain (SEQ ID NO: 6) are cloned into the EcoRI site of pHIV-Zsgreen along with the optimal Kozak sequence.

[0363] The nucleic acid sequences of exemplary chimeric receptors that bind to CD33, which has the basic structure of light-chain-linker-heavy-chain-hinge-CD28 / ICOS-CD3ζ, are presented below.

[0364] Part 1: Light chain-linker-heavy chain (SEQ ID NO: 33): The Kozak start 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 separated by linkers. [ka]

[0365] Part 2: Hinge-CD28 / ICOS-CD3ζ NotI restriction enzyme recognition sites are shown in capital letters. Translation stop sites are shown in bold. BamHI restriction cleavage sites are shown underlined.

[0366] CD28 co-stimulatory domain (SEQ ID NO: 34) [ka]

[0367] ICOS co-stimulatory domain (SEQ ID NO: 35) [ka]

[0368] Fusion (hybrid) CD28 and ICOS costimulatory domain (SEQ ID NO: 36) [ka]

[0369] In the next step, the hinge region, CD28 domain (SEQ ID NO: 15), and cytoplasmic components of TCR-ζ are cloned into the NotI and BamHI sites of pHIV-Zsgreen (which already contains the peptide signaling molecule and CD33 scFv). Alternatively, the CD28 domain may be replaced with the ICOS domain (SEQ ID NO: 16).

[0370] In addition to the CD28 and ICOS domains, a fusion domain containing fragments of the CD28 and ICOS intracellular signaling domains is engineered (SEQ ID NO: 17) and used to generate further chimeric receptors. In such configurations, the chimeric receptor includes 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 (containing the TM region of CD28), and a signaling domain of the TCR-ζ molecule.

[0371] Examples of amino acid sequences of components that may be used to generate chimeric receptors are presented in this book, for example, the CD28 domain (SEQ ID NO: 10), the ICOS domain (SEQ ID NO: 11), the CD28 / ICOS hybrid domain (SEQ ID NO: 13), and TCR-ζ. Alternatively, chimeric receptors may be generated (Section B).

[0372] B. Alternative methods for generating anti-CD33 CAR constructs Schematic diagrams of exemplary chimeric receptors are shown in Figure 10, panels A-D. Chimeric receptors are generated using extracellular humanized scFv that recognize the CD33 antigen, linked to the extracellular CD8 hinge region, transmembrane domain, and cytoplasmic signaling domain, as well as the CD3 ζ signaling chain (Figure 10, panel B). DNA encoding the anti-CD33 chimeric receptor is generated using 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 10, panels C and D).

[0373] To generate anti-CD33 scFV sequences, the coding regions of the heavy and light chains (SEQ ID NOs: 6 and 8) of the variable region of the anti-CD33 antibody described above are amplified with specific primers and cloned into a pHIV-Zsgreen vector for intracellular expression. To evaluate the binding strength of scFv (single-chain variable fragment) to the target antigen, scFv is expressed in Hek293T cells. For this purpose, the vector (pHIV-Zsgreen containing the coding region) is transformed into E. coli Top10F bacteria to prepare plasmids. The expression vector encoding the resulting scFv antibody is introduced into Hek293T cells by transfection. After culturing the transfected cells for 5 days, the supernatant is removed and the antibody is purified.

[0374] The resulting antibodies can be humanized using framework substitution protocols known in the art. For example, see one such protocol provided by BioAtla (San Diego), which ligates a synthetic CDR coding fragment library derived from a template antibody to human framework region coding fragments from a human framework pool limited to germline sequences derived from functionally expressed antibodies (bioatla.com / applications / express-humanization / ).

[0375] Affinity maturation may be performed to improve antigen binding affinity. This can be achieved 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. Alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding in order to identify candidate hypervariable region residues suitable for modification. Furthermore, reported combinatorial libraries can also be used to improve antibody affinity (Rajpal et al., PNAS (2005) 102(24):8466). Alternatively, BioAtla has developed a platform for rapid and efficient antibody affinity maturation, which may be used for antibody optimization purposes (bioatla.com / applications / functional-maturation / ).

[0376] C. Assembly of CAR structures Next, anti-CD33 scFv is ligated to the extracellular CD8 hinge region, transmembrane domain, and cytoplasmic CD28 signaling domain, as well as the CD3 ζ signaling chain. Briefly, scFv is amplified as described above using primers specific to the anti-CD33 scFv sequence. The CD8 hinge and transmembrane domain (amino acids 135-205) are amplified using a plasmid (pUN1-CD8) (www.invivogen.com / puno-cd8a) containing the complete human CD8 coding sequence. The CD3 ζ fragment is amplified from the Invivogen plasmid pORF9-hCD247a (http: / / www.invivogen.com / PDF / pORF9-hCD247a_10E26v06.pdf) containing the complete human CD3 ζ coding sequence. Finally, CD28 (amino acids 153-220, corresponding to the TM and signaling domains of CD28) is amplified from cDNA generated using RNA collected from activated T cells by the Trizol method. A fragment containing anti-CD33-scFv-CD8-hinge+TM-CD28-CD3ζ is assembled using splice-overlap extension (SOE) PCR. The resulting PCR fragment is cloned into the pELPS lentiviral v...

Claims

1. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice acceptor site within the endogenous CD33 gene, wherein the modified splice acceptor site comprises an A-to-G nucleotide substitution located within a sequence targeted by a guide RNA comprising the sequence of Sequence ID No. 3, and the modified splice acceptor site causes a decrease in the expression level of the epitope encoded by exon 2 of CD33 compared to a wild-type equivalent cell.

2. Genetically engineered hematopoietic stem cells or progenitor cells comprising a modified splice donor site within the endogenous EMR2 gene, wherein the modified splice donor site comprises nucleotide substitutions located within a sequence targeted by a guide RNA including the sequence of SEQ ID NO: 47, SEQ ID NO: 46, or SEQ ID NO: 4, causing a decrease in the expression level of an epitope encoded by an exon of EMR2 compared to a wild-type equivalent cell.

3. (i) The modified splice donor site causes a decrease in the expression level of the epitope encoded by exon 13 of EMR2, and / or (ii) The nucleotide substitution is selected from the group consisting of C to T, G to A, A to G, and T to C. Genetically engineered hematopoietic stem cells or progenitor cells according to claim 2.

4. A genetically engineered hematopoietic stem cell or progenitor cell comprising a nucleotide substitution in a first endogenous gene encoding a lineage-specific antigen and a nucleotide substitution in a second endogenous gene encoding a lineage-specific antigen, The nucleotide substitution in the first endogenous gene is located within a splice element and causes a decrease in the expression level of the epitope encoded by the exon of the first endogenous gene compared to a wild-type equivalent cell, and the nucleotide substitution in the second endogenous gene is located within a splice element and causes a decrease in the expression level of the epitope encoded by the exon of the second endogenous gene compared to a wild-type equivalent cell. The first endogenous gene is the CD33 gene, and / or the second endogenous gene is the EMR2 gene. Genetically engineered hematopoietic stem cells or progenitor cells.

5. The genetically engineered hematopoietic stem cell or progenitor cell according to claim 4, wherein the first endogenous gene is the CD33 gene, the exon is exon 2 of the CD33 gene, and the second endogenous gene is the EMR2 gene, the exon is exon 13 of the EMR2 gene.

6. (i) The splice element in the first endogenous gene and / or the splice element in the second endogenous gene is selected from the group consisting of splice acceptors, splice donors, splice enhancers, and splice silencers. (ii) Alternative splicing causes the exons of the first endogenous gene and / or the exons of the second endogenous gene to be skipped or extended, (iii) The nucleotide substitution in the first endogenous gene and / or the nucleotide substitution in the second endogenous gene is selected from the group consisting of C to T, G to A, A to G, and T to C. Genetically engineered hematopoietic stem cells or progenitor cells according to claim 4.

7. The genetically engineered hematopoietic stem cells or progenitor cells, (i) CD34+, (ii) derived from the bone marrow cells or peripheral blood mononuclear cells of the subject, and / or (iii) No mutations in any of the predicted off-target sites, A genetically engineered hematopoietic stem cell or progenitor cell according to any one of claims 1 to 6.

8. The subject is a human patient with a hematopoietic malignancy or a healthy human donor, according to claim 7, wherein the subject is a genetically engineered hematopoietic stem cell or progenitor cell.

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

10. A method for producing genetically engineered hematopoietic stem cells or progenitor cells comprising at least one nucleotide substitution in the CD33 gene, wherein the method is: (i) Prepare hematopoietic stem cells or progenitor cells, (ii) Introducing into the cells (a) a guide RNA (gRNA) containing a targeting domain containing the sequence of Sequence ID No. 3, and (b) a catalytically impaired Cas9 endonuclease fused to adenosine deaminase (base editor). A method comprising, thereby producing the genetically engineered hematopoietic stem cells or progenitor cells.

11. A method for producing genetically engineered hematopoietic stem cells or progenitor cells comprising at least one nucleotide substitution in the EMR2 gene, wherein the method is: (i) Prepare hematopoietic stem cells or progenitor cells, (ii) Introducing into the cells (a) a guide RNA (gRNA) containing a splice element and a targeting domain that targets a nucleotide sequence in the genome of hematopoietic stem cells or progenitor cells located within the EMR2 gene, and (b) a catalytically impaired Cas9 endonuclease fused to an adenosine deaminase (base editor). A method comprising, thereby producing the genetically engineered hematopoietic stem cells or progenitor cells.

12. (A) The splice element is selected from the group consisting of a splice acceptor, a splice donor, a splice enhancer, and a splice silencer. (B) The at least one nucleotide substitution causes alternative splicing, which optionally causes the exon encoding the epitope of EMR2 to be skipped or extended, or (C) The at least one nucleotide substitution is selected from the group consisting of C to T, G to A, A to G, and T to C. The method according to claim 11.

13. The method according to claim 11, wherein the gRNA includes a sequence selected from the group consisting of SEQ ID NOs: 4 and 46-47.

14. A method for producing genetically engineered hematopoietic stem cells or progenitor cells comprising at least one nucleotide substitution in a first gene encoding a first lineage-specific antigen and at least one nucleotide substitution in a second gene encoding a second lineage-specific antigen, wherein the method is: (i) Prepare hematopoietic stem cells or progenitor cells, (ii) Introducing into the cells (a) a first guide RNA (gRNA) containing a splice element and a targeting domain that targets a first nucleotide sequence in the genome of the hematopoietic stem cell or progenitor cell located within the first gene, and (b) a first catalytically impaired Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor), (iii) Further introducing into the cells (c) a second guide RNA (gRNA) containing a splice element and a targeting domain that targets a second nucleotide sequence in the genome of the hematopoietic stem cell or progenitor cell located within the second gene, and (d) a second catalytically impaired Cas9 endonuclease fused to cytosine deaminase or adenosine deaminase (base editor). This includes, thereby producing the genetically engineered hematopoietic stem cells or progenitor cells, The first gene is CD33, and / or the second gene is the EMR2 gene. method.

15. (A) The splice element in the first gene and / or the splice element in the second gene is selected from the group consisting of splice acceptors, splice donors, splice enhancers, and splice silencers. (B) Alternative splicing causes the exons encoding the epitope of the first series-specific antigen and / or the epitope of the second series-specific antigen to be skipped or extended, (C) At least one nucleotide substitution in the first gene and / or at least one nucleotide substitution in the second gene is selected from the group consisting of C to T, G to A, A to G, and T to C. The method according to claim 14.

16. The method according to claim 14 or 15, wherein the first gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, and / or the second gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 4 and 46 to 47.

17. The method according to any one of claims 14 to 16, wherein the base editor of (ii)(b) and / or the base editor of (iii)(d) is a cytosine base editor.

18. The method according to any one of claims 14 to 16, wherein the base editor of (ii)(b) and / or the base editor of (iii)(d) is an adenosine base editor.

19. The first gRNA of (ii)(a) and the base editor of (ii)(b) are encoded in a vector introduced into the cell, wherein the vector is optionally a viral vector, and / or The second gRNA of (iii)(c) and the second base editor of (iii)(d) are encoded in a vector introduced into the cell, and optionally the vector is a viral vector. The method according to any one of claims 14 to 18.

20. The base editor of (ii)(b) is in protein form, the first gRNA of (ii)(a) and the base editor of (ii)(b) are introduced into the cell as a pre-formed ribonucleoprotein complex, the ribonucleoprotein complex is optionally introduced into the cell via electroporation, and / or The base editor of (iii)(d) is in protein form, the second gRNA of (iii)(c) and the base editor of (iii)(d) are introduced into the cell as a pre-formed ribonucleoprotein complex, and optionally the ribonucleoprotein complex is introduced into the cell via electroporation. The method according to any one of claims 14 to 18.

21. The hematopoietic stem cells or progenitor cells are CD34+, and / or The hematopoietic stem cells or progenitor cells are derived from the subject's bone marrow cells or peripheral blood mononuclear cells (PBMCs), and optionally the subject has a hematopoietic disorder. The method according to any one of claims 14 to 20.

22. Genetically engineered hematopoietic stem cells or progenitor cells produced by the method described in any one of claims 10 to 21.

23. A cell population for use in a method for treating hematopoietic disorders, wherein the method comprises administering an effective amount of the cell population to a subject in need thereof, the cell population comprising genetically engineered hematopoietic stem cells or progenitor cells as described in any one of claims 1 to 8 or 22, or the cell population as described in claim 9.

24. The aforementioned hematopoietic disorder is a malignant tumor of the hematopoietic system. The subjects are human patients with Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, or multiple myeloma, and optionally, the subjects are human patients with leukemia, specifically acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia. A cell population for use according to claim 23.

25. The method further comprises administering an effective amount of a drug that targets CD33 to the subject, wherein the drug comprises an antigen-binding fragment that binds to CD33, and / or The method further comprises administering an effective amount of an EMR2-targeting agent to the target, wherein the agent comprises an antigen-binding fragment that binds to EMR2, for use in the cell population according to claim 23 or 24.

26. The cell population for use according to claim 25, wherein the agent targeting CD33 and / or EMR2 is an immune cell expressing a chimeric antigen receptor (CAR) comprising an antigen-binding fragment, and optionally the immune cell is a T cell.

27. The cell population for use according to claim 26, wherein the immune cells, the genetically engineered hematopoietic stem cells or progenitor cells, or both are allogeneic or autologous.

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