WO2022228471A1 - 一种基因编辑的造血干细胞及其与car-t细胞的联合应用 - Google Patents

一种基因编辑的造血干细胞及其与car-t细胞的联合应用 Download PDF

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WO2022228471A1
WO2022228471A1 PCT/CN2022/089600 CN2022089600W WO2022228471A1 WO 2022228471 A1 WO2022228471 A1 WO 2022228471A1 CN 2022089600 W CN2022089600 W CN 2022089600W WO 2022228471 A1 WO2022228471 A1 WO 2022228471A1
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cells
cell
cell surface
surface protein
car
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French (fr)
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胡广
张佳元
王晓倩
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上海驯鹿生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention belongs to the technical field of tumor treatment, in particular to the combined application of gene-edited hematopoietic stem cell transplantation and chimeric antigen receptor T cells or antibody drugs, in particular to a hematopoietic stem cell or Progenitor cells and preparation methods thereof, and application of combined CAR-T cells or antibody drugs in the treatment of tumors.
  • Hematopoietic stem cells are a kind of hematopoietic tissue stem cells with self-renewal and multi-directional differentiation potential. They produce hematopoietic progenitor cells of various lines (such as lymphoid progenitor cells and myeloid progenitor cells) through directed differentiation, and then proliferate and differentiate into various hematopoietic progenitor cells. mature functional blood cells, thereby maintaining hematopoietic homeostasis throughout the body.
  • various lines such as lymphoid progenitor cells and myeloid progenitor cells
  • hematopoietic stem cell transplantation In the field of disease treatment, by replacing the patient's HSC with healthy autologous or allogeneic HSC, the method of rebuilding the patient's hematopoietic function and immune function to treat the disease is called hematopoietic stem cell transplantation. After more than 60 years of development and application, more than 1 million patients have received and benefited from hematopoietic stem cell transplantation. As the first regenerative method to enter clinical practice, hematopoietic stem cell transplantation has had a profound impact on the development of cell therapy-related clinical medicine.
  • GVHD chronic GVHD
  • 5%-20% the incidence of fatal GVHD
  • Severe GVHD is potentially life-threatening and requires immediate treatment if it occurs.
  • early engraftment is critical for post-transplantation and long-term immune reconstitution.
  • Pre-transplantation damage to the recipient's bone marrow microenvironment will produce a "bystander effect" on the transplanted donor hematopoietic stem cells, resulting in poor engraftment and reduced self-renewal capacity of the donor's hematopoietic stem cells, resulting in poor transplantation efficacy.
  • Chimeric antigen receptor T-cell (CAR-T) therapy is an immunotherapy technology that achieves tumor-targeted killing through genetic modification of T cells. By expressing receptors that can recognize specific antigens on the surface of T cells, the edited CAR-T cells can be targeted to eliminate tumor cells. Compared with traditional transplantation technology, the commonly used CAR-T products are all autologous, and this part of CAR-T cells will not cause the occurrence of GVHD. A number of clinical trials have confirmed that it has a good effect on patients with relapsed/refractory hematological tumors.
  • CAR-T cells targeting CD33 antigen will kill normal myeloid progenitor cells and myeloid cells in the treatment of acute myeloid leukemia, while CAR-T cells targeting CD19 antigen cannot differentiate between malignant B cells and normal B cells. Therefore, patients on these treatments must endure immunodeficiency due to myeloid developmental disorders (CD33) or long-term B cell depletion (CD19), and quality of life is greatly affected.
  • CAR-T cells do not persist in the body as long as fully chimeric donor cells in transplantation, and once the killing effect of CAR-T cells on tumor cells is lost, the primary disease may come back.
  • CAR-T therapy for patients with relapse after transplantation has good efficacy and safety. Therefore, the combined application of CAR-T therapy in each stage of transplantation can achieve complementarity or synergy, optimize the therapeutic effect, and expand the scope of applicability. It is expected to become a new therapeutic strategy worthy of promotion.
  • CD33 is a member of the sialic acid-binding immunoglobulin-like lectin family, which is expressed in all myeloid cells that differentiate from myeloid progenitor cells (CMP), so CD33 is also AML (acute myelocytic leukemia) Classic targets in diagnosis and therapy.
  • CMP myeloid progenitor cells
  • AML acute myelocytic leukemia
  • researchers use the CRISPR-Cas9 system to knock out the CD33 gene in human-derived hematopoietic stem cells. After CD33 knockout, the hematopoietic stem cells can differentiate into myeloid cells and function without being recognized by anti-CD33 CAR-T cells. and killing.
  • the purpose of the present invention is to provide a hematopoietic stem or progenitor cell protected from the action of chimeric antigen receptor T cells and its application.
  • the patient's autologous hematopoietic stem cells are modified, and the target antigen is modified by the gene editing technology, so that the modified hematopoietic stem cells are not killed by CAR-T cells or antibody drugs, and then the hematopoietic stem cells are transplanted back to the patient.
  • the target antigen is modified by the gene editing technology
  • the present invention provides the following technical solutions.
  • a cell having a cell surface protein wherein the cell surface protein is engineered to have epitope alterations that enable the cell to resist CAR-T or antibodies Drug killing.
  • the cells are hematopoietic stem cells or differentiated cells thereof.
  • the cells are lymphoid progenitors, myeloid progenitors, lymphoid cells, or myeloid cells.
  • the cell surface protein is a lymphoid cell-specific cell surface protein or a myeloid cell-specific cell surface protein.
  • the epitope alteration of the cell is due to the presence of single nucleotide mutations, splice site mutations and/or nucleotide fragment substitutions, insertions and/or deletions in the gene encoding the cell surface protein cause.
  • the cell surface protein is expressed with an exon deletion.
  • the cell surface protein is CD33, CD19 or BCMA.
  • the cell surface protein is CD33, which has deleted the amino acid segment encoded by the second exon of the CD33 gene.
  • the cell surface protein is CD33, and one or any combination of the following features are present in the CD33 gene of the cell: 1) the rs12459419 site is a nucleotide T; 2) the second exon The 3'ss site before the sequence is AA or CT; and 3) TTTCT in the second exon is replaced by AATCC.
  • this article provides the use of the cells described above in combination with CAR-T or antibody drugs in the preparation of antitumor drugs.
  • the CAR-T or antibody drug can target a cell surface protein of a tumor cell to kill the tumor cell, and the cell surface protein expressed by the cell is engineered to have an antigenic epitope change, The change of the antigenic epitope enables the cells to be killed by the CAR-T or antibody drugs.
  • the tumor is a hematological tumor, preferably AML.
  • a method for preparing a cell wherein the cell surface protein of the cell has an antigenic epitope change capable of being killed by anti-CAR-T or antibody-based drugs, the method comprising a gene encoding the cell surface protein Gene editing.
  • the gene editing comprises introducing single nucleotide mutations, splice site mutations and/or nucleotide fragment substitutions, insertions and/or deletions into the coding gene.
  • the gene editing is performed by introducing the CRISPR gene editing system into the cell.
  • the cells are hematopoietic stem cells or differentiated cells thereof.
  • the cells are lymphoid progenitors, myeloid progenitors, lymphoid cells, or myeloid cells.
  • the cell surface protein is a lymphoid cell-specific cell surface protein or a myeloid cell-specific cell surface protein.
  • the cell surface protein is expressed with an exon deletion.
  • the cell surface protein is CD33, CD19 or BCMA.
  • the cell surface protein is CD33
  • the gene editing comprises any one or any combination of: 1) mutating nucleotide C at rs12459419 to T; 2) mutating the second exon The nucleotide AG at the 3'ss site before the sequence was mutated to AA or CT; and 3) the nucleotide TTTCT in the second exon was mutated to AATCC.
  • the CRISPR gene editing system that mutates nucleotides C to T at rs12459419 includes an sgRNA with a target sequence of SEQ ID NO: 10, 11, 12, 14, or 15.
  • the CRISPR gene editing system for mutating nucleotide C at rs12459419 to nucleotide T comprises a sgRNA with a target sequence of SEQ ID NO: 10, 11 or 12 and a ssDNA comprising SEQ ID NO: 21;
  • the target sequence is the sgRNA of SEQ ID NO: 14 and the ssDNA comprising SEQ ID NO: 23; or the target sequence is the sgRNA of SEQ ID NO: 15 and the ssDNA comprising SEQ ID NO: 24.
  • the CRISPR gene editing system that mutates the nucleotide AG at the 3'ss site preceding the second exon sequence to AA comprises an sgRNA with a target sequence of SEQ ID NO: 13 or 16.
  • the CRISPR gene editing system that mutates the nucleotide AG at the 3'ss site before the second exon sequence to AA includes an sgRNA with a target sequence of SEQ ID NO: 13 or 16 and a single base cell A pyrimidine editor, the single base cytosine editor is preferably A3A-CBE3.
  • a CRISPR gene editing system that simultaneously mutates nucleotides C to T at rs12459419 and AG to CT at the 3'ss site preceding the second exon sequence comprises a target sequence of SEQ ID NO: 10 or 14 sgRNA.
  • a CRISPR gene editing system that simultaneously mutates nucleotides C to T at rs12459419 and AG to CT at the 3'ss site preceding the second exon sequence comprises a target sequence of SEQ The sgRNA of ID NO: 10 and the ssDNA comprising SEQ ID NO: 22 or the sgRNA of the target sequence of SEQ ID NO: 14 and the ssDNA comprising SEQ ID NO: 27.
  • a method of removing or reducing tumor cells in a subject comprising administering to the subject a CAR-T or antibody-based drug capable of killing the tumor cells and administering to the subject
  • the subject is supplemented with normal cells, wherein the CAR-T or antibody-based drug targets a cell surface protein of the tumor cell, and the cell surface protein expressed by the normal cell is engineered to have an antigenic epitope change, the The change of the antigenic epitope enables the normal cells to be killed by the CAR-T or antibody drugs.
  • the tumor cells are blood tumor cells and the normal cells are blood cells or hematopoietic stem or progenitor cells capable of differentiating into blood cells.
  • the cell surface protein is a lymphoid cell-specific cell surface protein or a myeloid cell-specific cell surface protein.
  • the epitope alteration in the normal cell is due to the presence of single nucleotide mutations, splice site mutations and/or nucleotide fragment substitutions, insertions and/or in the gene encoding the cell surface protein caused by deletion.
  • the cell surface protein of the normal cell is expressed with an exon deletion.
  • the cell surface protein is CD33, CD19 or BCMA.
  • the cell surface protein is CD33, which has deleted the amino acid segment encoded by the second exon of the CD33 gene.
  • the cell surface protein is CD33, and one or any combination of the following features is present in the gene encoding it: 1) the rs12459419 site is a nucleotide T; 2) the second exon sequence is preceded by The 3'ss site is AA or CT; and 3) TTTCT in the second exon is replaced by AATCC.
  • the tumor cells are AML cells.
  • this paper provides a pharmaceutical composition or drug combination comprising a CAR-T or antibody-based drug and normal cells, wherein the CAR-T or antibody-based drug can target cell surface proteins of tumor cells to kill the For tumor cells, the cell surface proteins expressed by the normal cells are modified to have antigenic epitope changes, and the antigenic epitope changes enable the normal cells to be killed by the CAR-T or antibody drugs.
  • the tumor cells are blood tumor cells and the normal cells are blood cells or hematopoietic stem or progenitor cells capable of differentiating into blood cells.
  • the cell surface protein is a lymphoid cell-specific cell surface protein or a myeloid cell-specific cell surface protein.
  • the epitope alteration of the normal cell is due to the presence of single nucleotide mutations, splice site mutations and/or nucleotide fragment substitutions, insertions in the nucleotide sequence encoding the cell surface protein and/or deletions.
  • the cell surface protein of the normal cell is expressed with an exon deletion.
  • the cell surface protein is CD33, CD19 or BCMA.
  • the cell surface protein is CD33, which has deleted the amino acid segment encoded by the second exon of the CD33 gene.
  • the cell surface protein is CD33, and one or any combination of the following features is present in the gene encoding it: 1) the rs12459419 site is a nucleotide T; 2) the second exon sequence is preceded by The 3'ss site is AA or CT; and 3) TTTCT in the second exon is replaced by AATCC.
  • the tumor cells are AML cells.
  • the engineered cells with altered epitopes provided herein can be used for tumor therapy in combination with CAR-T cells or antibody-based drugs.
  • Cells with altered epitopes are not killed by CAR-T or antibody drugs, and can be injected into patients to alleviate the side effects of CAR-T products or antibody drugs in tumor treatment.
  • Figure 1 is a graph of the analysis results of CD33 exon2 SNP genotypes between different cell lines in Example 1.
  • Figure 2 is a gel electrophoresis image of CD33 exon2 variable shear expression of different cell lines in Example 1.
  • 3A is a schematic diagram of the design of probe primers across CD33 exon2 in the RT-qPCR experiment in Example 1.
  • 3B is a graph of RT-qPCR results for detecting the deletion of CD33 exon2 in different cell lines in Example 1.
  • FIG. 4A is a flow cytometry result comparison of the expression of CD33 exon2 and total protein of different cell lines in Example 1.
  • FIG. 4A is a flow cytometry result comparison of the expression of CD33 exon2 and total protein of different cell lines in Example 1.
  • 4B is a statistical graph of the average fluorescence intensity of CD33 exon2 and total protein of different cell lines in Example 1.
  • Figure 5 is a histogram of the GFP positive rate and cell viability rate of different cell lines detected by FACS flow cytometry in Example 2, in which Figure I is the GFP positive rate, and Figure II is the cell viability rate.
  • 6A is a schematic diagram of the mutation status of the G base after the CD33 exon2 3'ss AG site is mutated by using the single-base cytosine editor A3A-CBE3 in Example 2.
  • FIG. 6B is a histogram obtained by using EditR to analyze the 3'ss single-base mutation efficiency in Example 2.
  • FIG. 6C is a comparison diagram of the ratio of E13/E2 in K562 cell line detected by RT-qPCR in Example 2.
  • FIG. 6C is a comparison diagram of the ratio of E13/E2 in K562 cell line detected by RT-qPCR in Example 2.
  • FIG. 6D is a comparison diagram of the ratio of E13/E2 in HL60 cell line detected by RT-qPCR in Example 2.
  • FIG. 6D is a comparison diagram of the ratio of E13/E2 in HL60 cell line detected by RT-qPCR in Example 2.
  • FIG. 7 is an analysis diagram of the mutation status obtained after mutating the SNP site and the 3'ss site by means of RNP combined with ssDNA electroporation into cells in Example 3.
  • FIG. 7 is an analysis diagram of the mutation status obtained after mutating the SNP site and the 3'ss site by means of RNP combined with ssDNA electroporation into cells in Example 3.
  • 8A is a graph showing the RT-qPCR results of E13/E2 after mutating the SNP site and the 3'ss site in Example 3.
  • FIG. 8B is a graph showing the results of flow-through antibody staining after mutating the SNP site and the 3'ss site in Example 3.
  • FIG. 8B is a graph showing the results of flow-through antibody staining after mutating the SNP site and the 3'ss site in Example 3.
  • Figure 9 is an analysis diagram of the mutation status of PE+ and PE- cells by Tyto flow sorting and the cells obtained after sorting in Example 4, wherein Figure I is a Tyto flow sorting diagram, and Figure II is a mutation status analysis diagram.
  • FIG. 10A is a sequence comparison diagram of CD33 and CD33 highly similar DNA in Example 5.
  • FIG. 10A is a sequence comparison diagram of CD33 and CD33 highly similar DNA in Example 5.
  • FIG. 10B is a schematic diagram of sg4-mediated editing in CD33 and off-target regions in Example 5.
  • FIG. 10B is a schematic diagram of sg4-mediated editing in CD33 and off-target regions in Example 5.
  • 11A is an analysis diagram of the sequence obtained after sg4-14 and sg4-15 mediate the mutation of the CD33 exon2 SNP site in Example 5.
  • FIG. 11B is a graph showing the results of flow cytometry detection of the expression level of the V region (P67.6) encoded by CD33 exon2 in cells after mutation in Example 5.
  • FIG. 11B is a graph showing the results of flow cytometry detection of the expression level of the V region (P67.6) encoded by CD33 exon2 in cells after mutation in Example 5.
  • FIG. 12A is a sequence analysis diagram of Sg3 and sg10-mediated AATCC mutation in Example 6.
  • FIG. 12A is a sequence analysis diagram of Sg3 and sg10-mediated AATCC mutation in Example 6.
  • FIG. 12B is a graph showing the results of flow cytometry detection of the expression level of the V region (P67.6) encoded by CD33 exon2 in cells after mutation in Example 6.
  • FIG. 12B is a graph showing the results of flow cytometry detection of the expression level of the V region (P67.6) encoded by CD33 exon2 in cells after mutation in Example 6.
  • Figure 13 is a graph showing the proportion of CAR-positive cells in CD33 CART cells detected by flow cytometry in Example 7.
  • Figure 14A is a flow cytometry result graph obtained after co-incubating CD33 CAR-T cells and edited NB4 cells for 0 hours in Example 7.
  • 14B is a graph of the detection results obtained after 0 hours of simultaneous flow staining of CD33 P67.6 and HIM-3-4 antibodies in Example 7.
  • Figure 14C is a graph of the flow cytometry results obtained after co-incubating CD33 CAR-T cells and edited NB4 cells for 96 hours in Example 7.
  • Figure 14D is a graph of the detection results obtained in Example 7 after simultaneous flow staining of CD33 P67.6 and HIM-3-4 antibodies for 96 hours.
  • Figure 14E is a graph of the proportion of target cells after mutating at 0, 22, 44, 68 and 96 hours after co-incubation of different cell lines with CD33 CAR-T cells in Example 7.
  • 15A is a sequence analysis diagram of cells obtained after electroporation of the RNP complexes of sg10 and cas9 in HSC cells in Example 8.
  • FIG. 15A is a sequence analysis diagram of cells obtained after electroporation of the RNP complexes of sg10 and cas9 in HSC cells in Example 8.
  • 15B is a graph of the detection results obtained after simultaneous flow staining of CD33 P67.6 and HIM-3-4 antibodies in Example 8.
  • 15C is a graph showing the proportion of CD14 positive cells detected on the 1st, 5th, 9th, and 14th day of differentiation by adding the medium for inducing the in vitro myeloid mononuclear differentiation of HSCs in Example 8.
  • 15D is a graph showing the content of CD14-positive cells at the 1st, 5th, 9th, and 14th day of differentiation in Example 8.
  • Figure 16 is a schematic representation of the deletion of the second exon of CD33 caused by a single nucleotide change.
  • Cell surface protein refers to a protein located at least partially on the cell surface after expression, which is usually a membrane protein with a partial amino acid sequence embedded in the cell membrane, and can also be an extracellular component attached to the cell surface.
  • cells derived from different tissues or organs they usually express their specific cell surface proteins, namely tissue-specific or cell-specific proteins. These cell surface proteins can also increase or decrease in expression as cells differentiate or mature.
  • the focus herein is on blood cell-specific cell surface proteins, such as proteins expressed primarily on the surface of lymphoid cells (eg, T cells or B cells) or myeloid cells (eg, various granulocytes, monocytes, etc.)
  • lymphoid cells eg, T cells or B cells
  • myeloid cells eg, various granulocytes, monocytes, etc.
  • cell surface markers such as CD33, CD19, CD21, BCMA, CD22, CD24, etc.
  • tumor cells the expression of some cell surface proteins is up-regulated, or some tumor-specific surface proteins are expressed, which can serve as targets for drug binding or action.
  • a “chimeric antibody receptor (CAR)” also known as a chimeric T cell receptor, a chimeric immune receptor, is an engineered membrane protein receptor molecule that binds the desired specificity Confer to immune effector cells, eg, the ability to bind to cell surface proteins such as tumor antigens.
  • Chimeric antigen receptors generally consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen binding domain is an scFv sequence responsible for recognizing and binding a specific antigen.
  • Intracellular signaling domains usually include immunoreceptor tyrosine activation motifs (ITAMs), such as the signaling domains derived from CD3 ⁇ molecules, which are responsible for activating immune effector cells for killing.
  • ITAMs immunoreceptor tyrosine activation motifs
  • the chimeric antigen receptor may also include a signal peptide at the amino terminus responsible for intracellular localization of the nascent protein, and a hinge region between the antigen binding domain and the transmembrane domain.
  • intracellular signaling domains can also include costimulatory domains derived from, for example, 4-1BB or CD28 molecules.
  • CAR-expressing T cells are abbreviated as CAR-T.
  • CAR-T uses the CAR expressed on its cell surface to recognize target cells, and after being activated by the target cells, it can kill the target cells accordingly.
  • the general process of using CAR-T cells to treat subjects is: collecting peripheral blood mononuclear cells (PBMCs) from subjects, isolating and culturing T cells, and introducing CAR codes through lentiviral transduction nucleic acid sequence, continue to culture and collect CAR+ cells, and infuse the CAR+ cells back into the subject.
  • PBMCs peripheral blood mononuclear cells
  • NK cells can be used to replace T cells for this process. Therefore, when referring to CAR-T, CAR-expressing NK cells may also be covered as appropriate.
  • an “antibody-based drug” as used herein refers to an antibody or antibody-drug conjugate (ADC) capable of binding to a cell surface protein resulting in a change in the activity of the cell expressing the cell surface protein.
  • ADC antibody or antibody-drug conjugate
  • the binding of antibody molecules to cell surface proteins may lead to inhibition of cell activity, growth arrest or death, or due to the binding of antibodies to cell surface proteins, the ligands of the cell surface proteins cannot bind normally, thereby causing cellular Activity is inhibited, growth arrests or death.
  • the cytotoxic agent when an antibody molecule is combined with a cytotoxic agent through a degradable linker, the cytotoxic agent can be targeted to cells expressing the antigen molecule by specifically recognizing the antibody to its corresponding antigen molecule, resulting in cell killing.
  • An “epitope” as used herein refers to the recognition and binding site of the corresponding antibody.
  • An epitope can be a contiguous amino acid sequence, or a three-dimensional structure formed by discontinuous amino acids, which is usually located on the surface of a protein molecule.
  • Antibody molecules bind to antigenic epitopes through the antigen-binding site formed by their heavy and light chain variable regions.
  • the antigen-binding domain in a CAR molecule typically includes an antigen-binding site in the form of an scFv that recognizes an antigenic epitope on a cell surface protein on a target cell.
  • An epitope change is at least an insertion, deletion and/or substitution of one or more amino acids that make up the epitope.
  • the epitope change is a contiguous amino acid deletion in a cell surface protein, eg, due to exon deletion due to alternative splicing of the pre-mRNA molecule.
  • the antibody molecule that recognizes the epitope will lose or weaken the binding ability.
  • the corresponding light chain variable region and heavy chain from the antibody molecule are included.
  • the CAR-T of the variable region (for single-domain antibodies, only the variable region of the heavy chain) also loses or weakens its target cell killing ability, that is, the target cell acquires the property of "anti-CAR-T killing".
  • this epitope change is only to obtain the anti-CAR-T killing ability for a specific CAR-T, without affecting the normal function of the cell surface protein.
  • Single nucleotide mutation refers to a single nucleotide change, including insertion, deletion or substitution, in an edited gene of a cell surface protein. This single nucleotide change results in a change in the amino acid sequence of the expressed cell surface protein, or if the single amino acid change occurs in a sequence involved in controlling RNA splicing (e.g., exonic splicing enhancers, intron splicing enhancers, 3'ss or 5'ss), resulting in splicing errors resulting in amino acid sequence changes. All of these amino acid sequence changes can change the antigenic epitope, so that the cells expressing the cell surface protein can obtain anti-CAR-T killing ability.
  • RNA splicing e.g., exonic splicing enhancers, intron splicing enhancers, 3'ss or 5'ss
  • a "splice site mutation” as used herein refers to a mutation associated with the splicing of a precursor RNA (pre-RNA). Mutation sites can, for example, be located in exon splicing enhancers, intron splicing enhancers, 3'ss or 5'ss, branch points, and other sequences so long as they result in changes in the amino acid sequence of the resulting protein. In some cases, the splice site mutation can result in the coding sequence of at least one exon not being present in the synthesized protein. In a specific embodiment, when a SNP site (rs12459419) in the second exon of the CD33 gene is T, the second exon can be deleted (see Figure 16).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR gene editing system used in this technology includes Cas nuclease and guide RNA (single-guide RNA, sgRNA), and optionally ssDNA as a repair template.
  • the sgRNA can be combined with Cas nuclease, and the other part of the sequence can be complementary to part of the target gene sequence. With the help of sgRNA recognition, the Cas nuclease can form a single-stranded or double-stranded nick at a specific site of the target gene.
  • HDR homologous recombination repair
  • NHEJ non-homologous end joining
  • CRISPR gene editing system such as CBE, ABE, and their various improved variants.
  • the fusion protein formed by ammonia enzyme forms nucleotide-directed changes at specific sites under the guidance of sgRNA.
  • Such base editors can be considered as improved CRISPR gene editing techniques, and they are also covered when this article refers to CRISPR gene editing.
  • DNA homologous recombination, site-specific cleavage by endonucleases eg, ZFNs and TALENs
  • endonucleases eg, ZFNs and TALENs
  • Normal cells herein refers to non-tumor cells, especially non-tumor cells that are killed during CAR-T (or antibody-based drug) therapy. Because CAR-T relies on its antigen-binding domain to recognize antigenic epitopes on tumor cells to kill tumor cells, but these epitopes are often expressed or present on non-tumor cells, causing them to also be killed by CAR-T, resulting in "on" -target, off-tumor” problem. These normal cells are killed, resulting in impairment of the subject's immune system or other functions. In medical practice, the above problems can be alleviated by supplementing the subject with these normal cells.
  • these normal cells may also carry antigenic epitopes recognized by CAR-T, in order to prevent being killed by CAR-T, these epitopes can be modified by the method provided in this article to avoid CAR-T killing. Therefore, in terms of preventing CAR-T killing, these normal cells are mainly epitope-modified cells. These normal cells can come from the subject itself or from other donors. These normal cells can be differentiated or mature cells, or stem cells, progenitor cells or precursor cells, such as hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • “Pharmaceutical composition” refers to a pharmaceutical formulation comprising at least one pharmaceutically active ingredient.
  • Pharmaceutical formulations may also generally include other ingredients such as pharmaceutically acceptable excipients, buffers, and the like.
  • a “pharmaceutical combination” as used herein means that at least two pharmaceutically active ingredients are in a separate state, ie in different pharmaceutical formulations, but which are suitable for sequential or simultaneous administration to a subject in order to produce superiority to the drug in the subject Therapeutic effect of active ingredients administered alone. This therapeutic effect can be reflected in the synergistic effect of active pharmaceutical ingredients, or it can reduce the side effects of one active pharmaceutical ingredient from another active ingredient.
  • the active pharmaceutical ingredients in a "pharmaceutical combination” may be present in the same pharmaceutical pack or separately in different pharmaceutical packs.
  • this paper provides a new therapeutic strategy for tumors, especially hematological tumors such as AML.
  • normal cells such as HSC transplantation
  • Tumor-specific antigens are selected as targets, but tumor-associated antigens can also be selected as targets.
  • Hematopoietic stem or progenitor cells provided herein that are protected from chimeric antigen receptor T cell killing can include a gene editing system for mutating nucleotide sequences encoding cell surface proteins.
  • the cell surface proteins include cell surface proteins targeted by CAR-T cells.
  • the antigenic surface protein includes any one of CD33 antibody recognition epitope, CD19 antibody recognition epitope or BCMA antibody recognition epitope.
  • the CD33 antibody of the hematopoietic stem cell or progenitor cell recognizes the SNP (single nucleotide polymorphism site, rs12459419) in the second exon region (exon2) corresponding to the epitope site
  • SNP single nucleotide polymorphism site
  • rs12459419 single nucleotide polymorphism site, rs12459419
  • the genotype is shown as a mutated T genotype; and/or, the TTTCT site in the second exon region corresponding to the CD33 antibody recognition epitope of the hematopoietic stem cell is shown as a mutation of AATCC.
  • CD33 is used as the target antigen, and commercialized AML cells are used for SNP and CD33 variable splicing experiments.
  • the mutation that can effectively cause the CD33 V structure to not bind to the antibody is screened; Later, it was found that there is a SNP site in CD33 exon2, and after mutating the SNP from C to T, exon 2 is deleted, which leads to the non-expression of the CD33 V domain, but after mutation, there is also a risk that the CD33 C domain will be affected; Therefore, the present invention unexpectedly found in further research that the introduction of a new AATCC mutagenic site can not only block the expression of the CD33 V domain, but also does not affect the expression of the CD33 C domain, and the resulting cells are not only resistant to Anti-CD33 CAR -T kills, and does not affect the normal in vitro differentiation of cells.
  • the SNP site and/or the TTTCT site in the second exon region of CD33 of mutant hematopoietic stem cells can effectively tolerate the killing of anti-CD33 CAR-T, and the cells can proceed normally.
  • the traditional knockout will cause the loss of gene function.
  • some genes are mutated by means of gene editing, resulting in changes in the conformation of the antigenic epitope. Or the failure of the antibody drug to target it has less impact on hematopoietic stem cells, and during the co-incubation process with CAR-T cells, the gene mutation provided by the present invention has stronger intracellular tolerance and higher survival rate. .
  • the gene editing methods and design ideas provided in the present invention can also be extended to other target antigens, such as lymphoid and myeloid cells (B cells, T cells, NK cells, monocytes, granulocytes, etc.)
  • B cells, T cells, NK cells, monocytes, granulocytes, etc. The specific expression of antigens greatly broadens the selection of tumor targets in cellular immunotherapy.
  • CAR-T cell therapy By combining hematopoietic stem cell transplantation and CAR-T cell therapy, it provides a new therapeutic idea for tumor immunotherapy.
  • the present invention can edit the relevant antigens of hematopoietic stem cells through base editing or knockout synergistic homologous recombination, and screen out the mutation sites of antigens such as CD19 and BCMA, which can differentiate into The B cells or plasma cells are resistant to targeting by anti-CD19-CAR-T cells (or anti-CD19 antibodies) or anti-BCMA-CAR-T cells (or anti-BCMA antibodies), while possessing normal cellular functions.
  • the 3'ss site of the CD33 gene of the hematopoietic stem cells expresses a mutation from AG to AA or CT.
  • the gene editing method of mutating the nucleotide sequence encoding CD33 can be used to change the antibody recognition epitope of CD19 or the antibody recognition epitope of BCMA.
  • the experimental method or design idea based on it is similar to CD33, but different The difference is that the sgRNA used in the gene editing system is different, and accordingly, the targeted site changes accordingly.
  • the gene editing system includes a CRISPR gene editing system.
  • the CRISPR gene editing system may include Cas enzymes and sgRNAs (or their expression vectors).
  • the sgRNA includes sgRNA for mutated CD33 antibody to recognize epitope site, sgRNA for mutated CD19 antibody to recognize epitope site or sgRNA for mutated BCMA antibody to recognize epitope site.
  • the sgRNA used to mutate the nucleotide sequence encoding the second exon region of CD33 includes: SEQ ID NO: 10 (sg4), SEQ ID NO: 11 (sg4-14), SEQ ID NO: 12 (sg4-15), any of the nucleotide sequences shown in SEQ ID NO: 18 (sg3) or SEQ ID NO: 20 (sg10).
  • the sg4 When using sg4 to mutate the SNP site, the sg4 is mismatched to the CD33 homologous sequence, resulting in off-target. Therefore, in the present invention, the 14th (sg4-14) or 15th (sg4-15) position on sg4 is further replaced. ) bases to reduce the possibility of sg4 off-targeting to regions of homology to CD33.
  • Both sg4-14 and sg4-15 can effectively mediate the mutation of SNP site C to T on CD33 exon2, and exon2 is deleted in the mutated cells; in the sg3 and sg10-mediated mutation group, TTTCT on the CD33 region is mutated to AATCC, and in the homologous region of CD33, the sequencing peak was single and there was no difference with the electroporated cas9 group, indicating that sg3 and sg10 would not off-target to CD33 homologous sequence to produce non-specific cleavage.
  • the sgRNA is any one of SEQ ID NO: 11 (Sg4-14), SEQ ID NO: 12 (Sg4-15) or SEQ ID NO: 20 (Sg10).
  • the sgRNA is SEQ ID NO: 20, and the TTTCT site in the second exon region corresponding to the CD33 epitope site of the prepared hematopoietic stem cell is a mutation of AATCC.
  • the antibody recognition epitope against CD19 or the antibody recognition epitope of BCMA is also designed for the corresponding sgRNA to achieve gene mutation.
  • the gene editing system further includes a repair template ssDNA.
  • the repair template ssDNA includes the nucleotide sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26.
  • the present invention also provides a method for preparing hematopoietic stem or progenitor cells as described above, the method comprising transferring a gene editing system into the hematopoietic stem or progenitor cells to be edited.
  • the transfer method includes any one of transduction, transfection or electroporation.
  • the method described in the present invention can be: preparing an RNP complex of cas9 and sgRNA, incubating, then mixing the RNP complex with an optional repair template ssDNA, adding the hematopoietic stem cell suspension to be edited, electroporation, After the electroporation is completed, culture medium is used to obtain hematopoietic stem cells transformed by gene editing.
  • the present invention also provides the application of the hematopoietic stem cells or progenitor cells in the preparation of medicines for treating tumors.
  • the present invention also provides the combination of the hematopoietic stem or progenitor cells and CAR-T cells in the preparation of drugs for the treatment of tumors, the development of antibodies or antibody-like molecules combined with small molecules, and the development of immune cell therapy drugs expressing antibody-like molecules or CAR molecules applications in .
  • hematopoietic stem cells modified by CD33 epitope combined with Anti-CD33 CAR-T hematopoietic stem cells modified by CD19 epitope combined with Anti-CD19 CAR-T
  • hematopoietic stem cells modified by BCMA epitope combined with Anti-BCMA CAR-T T its application in the treatment research of tumor cell immunity is of great significance for alleviating the side effects of CAR-T products in tumor treatment and improving the quality of life of patients.
  • the present invention also provides pharmaceutical compositions comprising hematopoietic stem or progenitor cells as described.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the present invention also provides a method of treatment comprising administering the above-mentioned pharmaceutical composition and CAR-T to a subject.
  • the subject is an animal or human suffering from tumor.
  • the invention provides the design of different sgRNAs.
  • the present invention further excludes sgRNAs that are prone to off-target and low-efficiency, and the prepared cells not only express the CD33 V domain, but also It does not affect the expression of CD33 C domain.
  • the present invention is a feasible method based on extensive basic research, creating "tumor-specific" antigens by gene editing mutant hematopoietic stem cells to facilitate cellular immunotherapy, and solving the problem that CAR-T cells kill tumor cells while also targeting tumor cells.
  • the "on-target, off-tumor” problem arising from healthy cells that normally express target antigens in patients provides a new therapeutic idea for tumor immunotherapy.
  • genomic DNA was extracted using PureLink TM Genomic DNA Mini Kit (for mutant cells, genomic DNA was generally collected 72 hours after electroporation).
  • PureLink TM Genomic DNA Mini Kit for mutant cells, genomic DNA was generally collected 72 hours after electroporation.
  • AmpliTaq Gold TM 360 Master Mix PCR was used to amplify the DNA fragments covering the target region. The PCR products were sequenced, and the base sequence map was obtained by Sanger sequencing, and the sequence genotypes were compared.
  • RNA was extracted using PrimeScript TM RT reagent Kit, and TB Premix DimerEraser TM kit reverse-transcribes RNA to obtain cDNA.
  • cDNA design a probe primer (E13) that spans CD33 exon2, which can specifically amplify the exon2-deleted sequence (m); the probe primer (E2) on exon2 can specifically amplify exon2 normally expressed Sequence (M), ct values for RT-qPCR were calculated and derived.
  • the m/M ct ratio reflects the degree of alternative splicing of CD33 exon2. The higher the ratio, the higher the missing expression of exon2 and the higher the occurrence of alternative splicing.
  • the LONZA electroporation instrument was used, and the electroporation procedures and electroporation kits recommended by the LONZA official website were used.
  • the specific electroporation conditions used for each cell were shown in Table 3.
  • the desired cells were centrifuged at 100g for 10 minutes. The medium was completely removed after centrifugation and resuspended in Lonza electroporation buffer. Meanwhile, RNP complexes of cas9 and sgRNA were prepared and incubated for 15 min at room temperature. Then, after mixing the ssDNA and RNP complexes, add the cell suspension, transfer it to a Lonza 16-well electroporation cup, put it into the 4D-Nucleofector TM X unit, and select the corresponding program for electroporation according to the cell type. After electroporation, carefully pipette the cell suspension to a 48-well plate, and continue to culture in 1640 complete medium or special medium for hematopoietic stem.
  • CD33 there is a SNP site in the second exon region of CD33.
  • SNP site When the SNP is C, CD33 is expressed normally (denoted as M); when the SNP site is T, CD33 produces alternative splicing, and the second exon is deleted and not expressed (denoted as m).
  • the hCD33 genome sequence was downloaded from the UCSC genome browser, and the sgRNA was designed through the CHOPCHOP website. Excluding easily off-target and low-efficiency sgRNAs, the final selected sgRNAs are shown in Table 4 below.
  • the cryopreserved healthy PBMCs were recovered, 20 ⁇ L/10 7 of CD3 microbeads were added, and CD3-positive T cells were sorted by LS sorting column. T cells were activated with CD3/CD28 antibody magnetic beads on the day of sorting, and viral transduction was performed 24-48 hours after activation.
  • the MOI of the lentivirus packaged with the CAR molecule was 3, and 800 ng/ ⁇ L PolyBrene and 1 ⁇ g/ ⁇ L DEAE were added for auxiliary infection. 24 hours after infection, the virus was removed by centrifugation at 300g for 10 minutes and replaced with fresh T medium.
  • the CAR positive rate was detected by flow cytometry.
  • the CD33-CAR molecule was labeled with EGFR, so the EGFR antibody detection could reflect the CAR positive rate.
  • the two cells were mixed according to the effector-target ratio of 1:30 (take 3e6 target cells as an example, the number of effector CAR-positive cells should be 1e5, If CAR-positive in CAR-T cells is 10%, the number of CD33 CAR-T cells should be 1e6, that is, the co-incubation system contains 3e6 target cells, and the number of CD33 CAR-T cells is 1e6).
  • the T cell culture medium was used for 96 hours, and the proportion of CD3 positive cells was detected during the period, which could reflect the changes of CAR-T cells.
  • the proportion of CD3-negative cells can reflect changes in the proportion of target cells after mutation.
  • Example 1 The SNP genotype of the second exon of CD33 is related to the alternative splicing of this exon
  • the proportion of m band is larger than that of M band, indicating that K562 and HL60 cells have higher CD33 exon2 deletion; in NB4 and THP1 cells, the intensity of m band and M band is similar Larger, indicating that the alternative splicing of CD33 exon2 in NB4 and THP1 cells is lower than that in K562 and HL60 cells; in Molm13 and KG1 cells, the proportion of M band is higher, indicating the degree of alternative splicing of CD33 exon2 in these two cells lower.
  • RT-qPCR primers CD33-E2F and CD33-E3R can be specific Recognize the deletion of exon2, (marked as m); the probe (CD33-VIC-E2) is on exon2, RT-qPCR primers CD33-E1F and CD33-E3R can specifically indicate the normal expression of exon2 (marked as M), as shown in the figure 3A.
  • the ratio of CD33 exon2m/M in each cell line of AML/CML relative to Raji cells was calculated.
  • the m/M ratio was more than 5 times that of the control group.
  • THP-1 and NB4 cells with SNP genotype C were used as a control.
  • the m/M ratio of KG1 and Molm13 cells with SNP genotype C was no different from the control group, or even lower ( ⁇ 1-fold).
  • the P67.6 clone-derived antibody can specifically bind to the CD33 V domain, so it can be used to indicate the expression of exon2 (M). Cells with exon2 alternative splicing deletion cannot bind to P67.6.
  • the HIM3-4 clone-derived antibody can specifically recognize the CD33 C domain, and it can be normally bound (Total) regardless of whether exon2 is deleted or not.
  • Gray is the isotype control of each flow antibody, the ability to bind to P67.6 can reflect the expression of CD33 M, and it can be seen that the expression of CD33 exon2 is not alternatively spliced:
  • CD33 exon2 was the highest in K562 cells, and the deletion of Molm13 exon2 was the lowest. It was basically consistent with the expression of CD33 exon2 detected at the mRNA level.
  • a method system that can effectively identify the genotype and alternative splicing phenotype of SNP loci is preliminarily established.
  • Sanger sequencing can clearly indicate the genotype of SNP locus, RT-PCR/RT-qPCR and P67.6 clone source
  • the method of flow antibody detection can indicate the proportion of CD33 exon2 alternative splicing deletion, which lays the foundation for the next step of mutating CD33 exon2 SNP to change the alternative splicing of CD33 for proof-of-concept.
  • the preliminary conclusion obtained in this example is that the degree of variable splicing of cells with SNP T genotype is higher, which provides a basis for the next step to design SNP C to T mutation to increase the jump expression of CD33 exon2.
  • HL60, K562, and NB4 are more suitable cells for electro-editing in AML/CML cells.
  • the cell viability was counted with a Countstar cytometer, and the GFP positive rate of the cells was detected by FACS flow.
  • the 24-hour viability of K562 and HL60 cells after electroporation was high, above 95%, and the positive rate of K562, HL60GFP was also above 95%, which was the highest;
  • the positive rate of GFP is about 65%;
  • the cell viability rate of KG1 cells after electroporation is about 60%, but the positive rate of GFP is less than 40%, and it is slightly more difficult to edit and mutate the cells later; THP1 and Molm13 cells
  • the viability of cells after electrotransformation is only about 10%, and the GFP positive rate is less than 10%, which is not suitable for later electrotransformation editing mutations.
  • a single-base cytosine editor A3A-CBE3 based on CRISPR-Cas9 technology was designed, and sgRNAs (sg8: SEQ ID NO: 13, sg13: SEQ ID NO) that can mutate the G base at the 3'ss AG site were designed : 16), by electroporating CBE plasmid and sgRNA plasmid into cells. Collect the genomic DNA of the cells on the 7th day after electroporation, use primers CD33-F/CD33-R to PCR out the CD33 exon2 region, sanger the PCR product, open the sequencing file with SnapGene, and compare the CD33 exon2 3'ss AG site G Base mutation status.
  • the shaded part is the exon sequence of CD33 exon2, and the first two bases of exon2, AG, are the 3'ss site.
  • the CBE-only cells were used as a blank control group, and the 3'ss site was AG; under the mediation of sgRNA8 or sg13, the base C on the complementary strand of sg8 and sg13 was The CBE is mutated to T, so the G base at the 3'ss AG site appears as a mutation to the A base.
  • the inventors further used the method of RT-qPCR in Example 2 to identify exon2 deletion to detect the effect of 3'ss mutation on exon2 alternative splicing.
  • the ratio of E13/E2 in the sg8 group was higher than that in the sg13 group, and the sg8 and 13 in the edited group were higher than those in the blank control group, indicating that the exon2 variable splicing in the edited cells was increased.
  • the 3'ss mutation efficiency of sg8 group is higher than that of sg13, so the exon2 deletion caused by it is also higher.
  • Example 3 Sg4-mediated SNP site and 3'ss mutation can effectively induce CD33 exon2 deletion in NB4
  • Cas9 protein and sgRNA are used to form RNP, and ssDNA repair template is provided, the SNP site on ssDNA is T, and the 3'ss site is CT, and RNP and ssDNA are delivered to cells by electroporation Inside.
  • HDR homologous recombination
  • Sg4 can effectively mutate SNPs and 3'ss sites
  • NB4 cells are superior to THP1, KG1 and other SNP C-type cells in terms of electroporation activation rate and efficiency.
  • sgRNA and corresponding ssDNA are designed for SNP sites on NB4 cells, including sg4: SEQ ID NO: 10 and its ssDNA: SEQ ID NO: 21, sg9: SEQ ID NO: 14 and its ssDNA : SEQ ID NO: 23, sg12: SEQ ID NO: 15 and its ssDNA: SEQ ID NO: 24.
  • ssDNA SEQ ID NO: 22
  • ssDNA SEQ ID NO. 28
  • CD33 exon2 had a frameshift mutation in the transcription and translation of CD33 due to the insertion mutation of T to TT, and then CD33 was knocked out.
  • sg4 can effectively mutate SNP from C (blue peak) to T (red peak), sg12 wild-type C genotype (blue peak) and SNP mutation
  • T genotypes (red peaks) accounted for about 50%, respectively, while the lowest in sg9 was from C (blue peaks) to T (red peaks).
  • sg4 could efficiently mutate the SNP from C (blue peak) to T (red peak), while the 3'ss site was mutated by AG is mutated to CT. Since the PAM end of Sg12 is farther from the 3'ss than sg9, the mediated mutation of the 3'ss is not as good as that of sg9. Therefore, in this example, sg9 was selected to mediate the simultaneous mutation of the SNP and the 3'ss site.
  • the results show that the editing efficiency of sg9 combined with SNP and 3'ss mutated ssDNA is not high.
  • the sequencing peak map there is basically no mutation at the SNP site, and the 3'ss site shows AG wild type and CT mutant. Set of peaks, the mutation efficiency is less than 20%.
  • RT-qPCR and FACS were used to detect the effect of SNP and 3'ss mutation on the expression of NB4 CD33 exon2.
  • RT-qPCR results showed that the CD33 E13/E2 ratio of the mutant SNP site (RNP-C) was higher than that of the unmutated group, and the CD33 E13/E2 ratio of the mutant SNP and 3'ss site (RNP-C+ag) was the highest, indicating that Both SNP mutation and 3'ss mutation promoted alternative splicing of CD33 exon2 (Fig. 8A).
  • the strategy of mutating SNP and 3'ss is not intended to affect the expression of the total CD33 protein, and only hopes to mutate a few bases to affect the expression of the CD33 V domain, in order to escape the CAR-T targeting the CD33 V domain of killing.
  • the results showed that there was no difference between cells after mutation and complete knockout (KO group), which did not bind to CD33 HIM-3-4 antibody. After mutation, only about 10% of cells did not bind to P67.6 but still bound HIM3-4. type to be further identified.
  • Example 4 AATCC mutation results in deletion of CD33 exon2 without affecting the C domain
  • the strategy of mutating the SNP site or 3'ss site in the early stage can cause the CD33 V domain to not be expressed, the CD33 C domain (HIM-3-4 antibody binding ability) is also affected, so the inventors used Tyto flow cytometry. Sorting, the NB4 cells that do not bind to P67.6 but still bind to HIM3-4 after mutation are sorted (PE+), and the cells that do not bind to HIM3-4 are used as a control (PE-), as shown in Figure I in Figure 9 , which are genotyped in relatively more homogeneous cell populations after sorting.
  • PE+ cell populations 4.85% of the PE+ cell populations were selected as the wild-type C genotype at the SNP site, but the mutation of TTTCT to AATCC at the TTTCT site;
  • PE-cells have C-to-T mutations at SNP sites, and there are also a large number of indels, mainly frameshift mutations caused by Cas9 protein cleavage.
  • Example 5 The sg4 targeting SNP and 3'ss mutation is off-target, and the modified sg4-14 ⁇ sg4-15 can greatly reduce the risk of off-target
  • the inventors compared CD33 with the sequence (homologous sequence) at the possible off-target position of sgRNA on the genome to detect whether there may be the existence of endogenous repair. , that is, after the CD33 exon2 position is cleaved by Cas9 protein and then broken, part of it is repaired with the CD33 homologous sequence as the template, and then the original sequence will be replaced by the homologous sequence.
  • a segment of human chromosome 19 has a sequence that is highly similar to CD33 (Fig. 10A).
  • the introduction of AATCC mutation is actually after being cut and broken by Cas9 protein, and part of it is repaired using the homologous sequence as a template.
  • the original TTTCT was Mutated to AATCC.
  • SNP and 3'ss mutation genotypes dominated, and the mutation of AATCC only the inventors sorted a small group of cells. It was detected by sanger sequencing.
  • CD33 homology region when only the cas9 protein was transferred, no mutation was generated; when the Cas9 protein and sg4 were transferred, there was also a small frameshift in the CD33 homologous region, which proved that sg4 was indeed partially off-target, resulting in Cas9 protein. Nonspecific cleavage occurs at CD33-like sequence positions.
  • sgRNA was used to form RNP complex with cas9, and ssDNA with SNP C to T mutation was provided, which was electroporated into NB4 cells, and the CD33 and CD33 homologous sequences were PCR and sanger sequencing to identify the efficiency of the modified sg4 to mediate SNP mutation. and off-target potential.
  • Sg4-14 and sg4-15 mediate the SNP site on CD33 exon2 appearing as a set of peaks of C and T, indicating that even if Sg4 is substituted at 14 or 15 bases, it can still normally bind to the CD33 region and mediate Mutation of the SNP site (Fig. 11A).
  • Example 6 Sg3 and sg10-mediated mutation of AATCC can efficiently induce CD33 exon2 deletion in NB4
  • the AATCC mutation is a promising mutation site that the inventors unexpectedly harvested.
  • the inventors designed sgRNAs for the TTTCT site (sg3: SEQ ID NO: 18, sg7: SEQ ID NO: 19, sg10: SEQ ID NO: 20), and provided ssDNA (SEQ ID NO: 26) containing the AATCC mutation as sg3 And sg7 repair template, provide ssDNA containing AATCC mutation (SEQ ID NO: 27) as sg10 repair template, 72 hours after electroporation into NB4 cells, PCR out the CD33 exon2 region, and sequencing to detect the mutation efficiency.
  • the results of genome sanger sequencing showed that in the sg3- or sg10-mediated mutation group, TTTCT on the CD33 region was mutated to AATCC, and in the CD33 homologous region, the sequencing peak was single and had no difference from the electro-cas9-only group, indicating that sg3, sg10 does not off-target to CD33 homologous sequences to produce non-specific cleavage.
  • sg7 has almost no mutation at the TTTCT site, but produces a partial set of peaks in the CD33 homology region, indicating that sg7 will partially mismatch to the CD33 homology region, resulting in non-specific cleavage.
  • the FACS results showed (Fig. 12B) that the cells after sg3 mutation did not bind P67.6 and normally bound HIM3-4 up to 73.2%, and the cells after sg10 mutation did not bind P67.6 and normally bound HIM3-4 up to 62.2%.
  • the sg4-14 or sg4-15 mutant SNP in the strategy does not bind to P67.6 and normally binds to HIM3-4 only 20%-30%.
  • Example 7 sg10-mediated AATCC mutation is more resistant to killing by Anti-CD33-CAR-T
  • engineered sg4 (sg4-14, sg4-15) can efficiently mutate SNP or 3'ss sites and lead to alternative splicing of CD33 exon2 with minimal off-target to CD33 homology regions, where V region binding is lost Only 20%-30% of the ability does not affect the binding of the C region.
  • sg10 can effectively mutate AATCC, the V region of the mutated cells almost loses the ability to bind antibodies, and the C region is still 60%-70% unaffected. Therefore, the inventors selected the AATCC mutation for later killing tolerance experiments and editing and differentiation of HSCs.
  • the inventors In order to further explore whether the mutated cells can tolerate the killing of CD33-CAR-T, the inventors first prepared CAR-T cells targeting CD33, and infected the activated T cells with lentiviruses containing CAR molecules to obtain stable CAR-expressing CAR-T cells. CD33-CAR-T. The conversion of CAR was detected by flow cytometry 72 hours after infection.
  • the inventors further designed the co-incubation experiment of NB4 cells after AATCC (sg3, sg10) or SNP mutation (sg4-14, sg4-5) and Anti-CD33-CAR-T, CAR-positive cells in CD33-CAR-T cells
  • the effector-target ratio with NB4 target cells is 1:30.
  • CD33-CAR-T cells From the time of co-incubation of CD33-CAR-T cells and edited NB4 cells, CD3-positive cells were detected by flow cytometry to represent CAR-T cells; CD3-negative cells were used to represent target cells. Simultaneous flow staining of CD33 P67.6 and HIM-3-4 antibodies verified whether NB4 resistant to CAR-T killing was a mutant cell population.
  • the ratio of CAR-T and edited NB4 cells was detected at 0 hours after co-incubation, CD3-negative cells were NB4 cells, and CD3-positive cells were CD33-CAR-T cells. 66.3% of CAR-positive CAR-T cells and edited NB4 cells were calculated according to the effect-target ratio of 1:30, and theoretically, NB4 cells accounted for 95.2%. Under the actual operation error, the FACS results showed that the initial proportion of NB4 target cells in each group was between 93.1% and 95.2%, which was an acceptable fluctuation (Fig. 14A).
  • the antibody binding ability of the CD33 V region and C region of the mutated target cells was detected after co-incubation for 0 hours. The results showed that at 0 hours, 67.5% of the cells in the CD33 V region of the unmutated cells could still be recognized by the P67.6 antibody.
  • 10.07% of sg4-15 edited NB4 cells were recognized by P67.6 antibody
  • 13.3% of sg4-14 edited NB4 cells were recognized by P67.6 antibody
  • 4.11% of sg3 edited NB4 cells were recognized by P67.6 antibody
  • 0.84% of sg10 edited NB4 cells were recognized by P67.6 antibody
  • 4.49% of CD33 KO NB4 cells were recognized by P67.6 antibody (Fig. 14C).
  • Example 8 Sg10-mediated AATCC mutation has no effect on HSC directed differentiation in vitro
  • the present invention has verified that the combination of RNP complex and ssDNA electroporation can produce site-directed mutation in NB4 AML cells, in which sg10-mediated AATCC mutation can affect the binding ability of CD33 P67.6 antibody, and the edited NB4 cells Resistant to Anti-CD33 CAR-T killing.
  • the mutation strategy was further applied to the editing of HSCs of hematopoietic stem cells to detect whether RNP could effectively induce the AATCC mutation of HSCs, and to detect whether the antibody binding ability of the CD33 V region of HSCs after mutation was destroyed by flow cytometry .
  • the TTTCT site showed a set of peaks of TTTCT wild type and AATCC mutant, but the peak of AATCC after mutation was lower than the original TTTCT, so in HSC cells The mutagenesis efficiency on , remains to be optimized.
  • HSCs To further explore whether edited HSCs have normal differentiation ability, the inventors assessed the ability of HSCs to differentiate into monocytes in vitro at the cellular level. The proportion of CD14 positive cells was detected on the 1st, 5th, 9th and 14th day of differentiation by adding the medium for inducing the myeloid mononuclear differentiation of HSC in vitro.
  • HSCs in the Sg10 editing group had normal in vitro myeloid monocyte differentiation ability:
  • the monocytes of the unmutated group (orange) and AATCC-mutated HSC cells (purple) on day 14 were detected by CD14-PE, and the results showed that the cells of the mutant group and the unmutated group were both in vitro under the action of the mononuclear differentiation medium. Normal differentiation to monocytes was possible (FIG. 15C).

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Abstract

提供了改变细胞抗原表位的方法以及利用该方法制备的细胞。还提供了所述细胞与CAR-T细胞或抗体类药物联合应用治疗肿瘤的方法和药物组合物。具有抗原表位改变的细胞不被CAR-T或抗体类药物杀伤，可输入患者体内，以缓解CAR-T产品或抗体类药物在肿瘤治疗中的副作用。

Description

一种基因编辑的造血干细胞及其与CAR-T细胞的联合应用 技术领域
本发明属于肿瘤治疗技术领域，具体涉及基因编辑的造血干细胞移植及嵌合抗原受体T细胞或抗体类药物的联合应用，尤其涉及一种免受嵌合抗原受体T细胞作用的造血干细胞或祖细胞及其制备方法以及联合CAR-T细胞或抗体类药物在治疗肿瘤中的应用。
背景技术
造血干细胞(HSC)是一类具有自我更新和多向分化潜能的造血组织干细胞，通过定向分化产生各系造血祖细胞(如淋巴系祖细胞和髓系祖细胞等)，再大量增殖分化成为各种成熟的功能性血细胞，从而维持整个机体的造血稳态。在疾病治疗领域，通过应用健康的自体或异体HSC代替患者的HSC，重建患者的造血功能和免疫功能以治疗疾病的方法被称为造血干细胞移植。经过六十多年的发展和应用，至今已有超过100万患者接受和得益于造血干细胞移植。作为第一个进入临床实践的再生方法，造血干细胞移植对细胞治疗相关的临床医学发展产生了深远的影响。
然而，过去的研究和临床实验证明，造血干细胞移植在临床应用的普及和拓展方面仍然受到极大的限制，面临着肿瘤的复发和移植并发症这两大亟需解决的问题。对于自体移植的复发患者，主要是由于恶性肿瘤患者的骨髓或外周血中残存的癌细胞污染了获取的自体干细胞，从而导致恶性疾病的复发。对于异体造血干细胞移植的患者而言，其发生移植物抗宿主病(GVHD)的概率很高。其中，急性GVHD发生概率为20％-60％，慢性GVHD发生率为50％左右，致死性GVHD发生率为5％-20％。重度的GVHD很可能危及生命，一旦发生需要尽快治疗。此外，造血干细胞移植后，早期植入对移植后造成和免疫长期重建至关重要。移植前的预处理损伤受体骨髓微环境会对移植入的供体造血干细胞产生“旁观者效应”，导致供体的造血干细胞植入不良和自我更新能力下降，导致移植疗效不佳。
随着科学和技术的不断突破，细胞治疗领域也发生了诸多重大变革，现代癌症的免疫治疗方法在一定程度上得益于造血干细胞移植的发展，同时在提高造血干细胞移植的适用性、安全性和疗效方面也有很大的前景，也有很多新的适用领域。
嵌合抗原受体T细胞(CAR-T)疗法是一种通过T细胞基因改造实现肿瘤靶向杀伤的免疫治疗技术。通过在T细胞表面表达能够识别特定抗原的受体，使得编辑后的CAR-T细胞能够定向清除肿瘤细胞。与传统移植技术相比，现在常用的CAR-T产品均是自体来源，这部分的CAR-T细胞不会导致GVHD的发生。多项临床试验证实，其对复发/难治性血液肿瘤患者具有很好的疗效。
然而，缺乏特异性的肿瘤表面抗原一直是利用CAR-T进行抗原特异性免疫治疗的难点之一。例如，靶向CD33抗原的CAR-T细胞在治疗急性髓系白血病中会对机体正常的髓系祖先 细胞和髓系细胞进行杀伤，靶向CD19抗原的CAR-T细胞不能区分恶性肿瘤B细胞和正常B细胞。因此，这些治疗中的患者必须忍受髓系发育障碍(CD33)或长期的B细胞缺失(CD19)导致的免疫缺陷，生存质量受到极大影响。此外，CAR-T细胞在体内的存续时间不如移植中完全嵌合的供者细胞持久，一旦失去CAR-T细胞对肿瘤细胞的杀伤作用，原发病就有可能卷土重来。
综合造血干细胞移植与CAR-T治疗各自的优缺点，将二者进行联合或可扬长避短。实验数据显示，将CAR-T治疗作为异体造血干细胞移植前调节治疗的一部分，可以减少高剂量化疗的毒性，并对生命质量产生积极影响。对于复发/难治性患者来说，通过前期的CAR-T治疗达到完全缓解，则可以最佳状态顺利桥接至移植，取得最好疗效。造血干细胞移植联合CAR-T治疗则可提高抗肿瘤靶向性，降低复发率和不良反应及GVHD发生率。移植后复发患者选择CAR-T治疗具有良好的有效性及安全性。因此，在移植各阶段中联合应用CAR-T治疗，可实现互补或协同，优化治疗效果，扩大适用者范围，有望成为一种新的值得推广的治疗策略。
CD33是唾液酸结合免疫球蛋白样凝集素家族成员，其在髓系祖先细胞(CMP)向后分化的所有髓系细胞中均有表达，因此CD33也是AML(急性髓细胞白血病，acute myelocytic leukemia)诊断和治疗中的经典靶点。但在使用靶向CD33的CAR-T和ADC药物治疗AML患者时，会伴随严重的毒副作用，正常的髓系细胞和髓系祖先细胞均会被靶向药物杀伤。目前，研究人员利用CRISPR-Cas9系统敲除人来源的造血干细胞中的CD33基因，CD33敲除后的造血干细胞能够向髓系细胞分化并行使功能，同时不会被anti-CD33 CAR-T细胞识别和杀伤。
因此，通过基因编辑技术对造血干细胞的靶点抗原进行改造，联合相应的CAR-T产品为细胞治疗提供了全新的思路和模式。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种免受嵌合抗原受体T细胞作用的造血干细胞或祖细胞及其应用。本发明中通过改造患者自体的造血干细胞，通过基因编辑技术对靶点抗原进行表位改造，使得改造后的造血干细胞不被CAR-T细胞或抗体类药物杀伤，再通过造血干细胞移植回输到患者体内，以缓解CAR-T产品或抗体类药物在肿瘤治疗中的副作用。
为此目的，本发明提供了以下技术方案。
在一个方面，本文提供了一种细胞，其具有细胞表面蛋白，其中所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述细胞能够抗CAR-T或抗体类药物杀伤。
在一些实施方案中，所述细胞为造血干细胞或其分化细胞。在一些具体实施方案中，所述细胞为淋巴系祖细胞、髓系祖细胞、淋巴系细胞或髓系细胞。
在一些实施方案中，所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特 异性细胞表面蛋白。
在一些实施方案中，所述细胞的抗原表位改变是由于所述细胞表面蛋白的编码基因中存在单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失引起。
在一些实施方案中，所述细胞表面蛋白在表达时存在外显子缺失。
在一些实施方案中，所述细胞表面蛋白为CD33、CD19或BCMA。
在一些实施方案中，所述细胞表面蛋白为CD33，其缺失CD33基因中第二外显子编码的氨基酸片段。
在一些实施方案中，所述细胞表面蛋白为CD33，所述细胞的CD33基因中存在如下特征之一或它们的任意组合：1)rs12459419位点为核苷酸T；2)第二外显子序列之前的3’ss位点为AA或CT；以及3)第二外显子内的TTTCT替换为AATCC。
在另一方面，本文提供了上文所述细胞与CAR-T或抗体类药物联合在制备抗肿瘤药物中的用途。
在一些实施方案中，所述CAR-T或抗体类药物能够靶向肿瘤细胞的细胞表面蛋白而杀伤所述肿瘤细胞，所述细胞表达的所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述细胞能够抗所述CAR-T或抗体类药物杀伤。
在一些实施方案中，所述肿瘤为血液肿瘤，优选AML。
另一方面，本文提供了制备细胞的方法，其中所述细胞的细胞表面蛋白具有能够抗CAR-T或抗体类药物杀伤的抗原表位改变，所述方法包括对所述细胞表面蛋白的编码基因进行基因编辑。
在一些实施方案中，所述基因编辑包括向所述编码基因中引入单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失。
在一些实施方案中，所述基因编辑通过向所述细胞引入CRISPR基因编辑系统进行。
在一些实施方案中，所述细胞为造血干细胞或其分化细胞。在一些具体实施方案中，所述细胞为淋巴系祖细胞、髓系祖细胞、淋巴系细胞或髓系细胞。
在一些实施方案中，所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
在一些实施方案中，所述细胞表面蛋白在表达时存在外显子缺失。
在一些实施方案中，所述细胞表面蛋白为CD33、CD19或BCMA。
在一些实施方案中，所述细胞表面蛋白为CD33，所述基因编辑包括如下任一种或其任意组合：1)将rs12459419位点核苷酸C突变为T；2)将第二外显子序列之前的3’ss位点核苷酸AG突变为AA或CT；以及3)将第二外显子内的核苷酸TTTCT突变为AATCC。
在一些实施方案中，将rs12459419位点核苷酸C突变为T的crispr基因编辑系统包括靶序列为SEQ ID NO：10、11、12、14或15的sgRNA。
在一些实施方案中，将rs12459419位点核苷酸C突变为核苷酸T的crispr基因编辑系统包括靶序列为SEQ ID NO：10、11或12的sgRNA和包含SEQ ID NO：21的ssDNA；靶序 列为SEQ ID NO：14的sgRNA和包含SEQ ID NO：23的ssDNA；或靶序列为SEQ ID NO：15的sgRNA和包含SEQ ID NO：24的ssDNA。
在一些实施方案中，将第二外显子序列之前的3’ss位点核苷酸AG突变为AA的crispr基因编辑系统包括靶序列为SEQ ID NO：13或16的sgRNA。
在一些实施方案中，将第二外显子序列之前的3’ss位点核苷酸AG突变为AA的crispr基因编辑系统包括靶序列为SEQ ID NO：13或16的sgRNA以及单碱基胞嘧啶编辑器，所述单碱基胞嘧啶编辑器优选A3A-CBE3。
在一些实施方案中，同时将rs12459419位点核苷酸C突变为T和将第二外显子序列之前的3’ss位点核苷酸AG突变为CT的crispr基因编辑系统包括靶序列为SEQ ID NO：10或14的sgRNA。
在一些实施方案中，同时将rs12459419位点核苷酸C突变为T和将第二外显子序列之前的3’ss位点核苷酸AG突变为CT的crispr基因编辑系统包括靶序列为SEQ ID NO：10的sgRNA和包含SEQ ID NO：22的ssDNA或靶序列为SEQ ID NO：14的sgRNA和包含SEQ ID NO：27的ssDNA。
在一些实施方案中，将第二外显子内的核苷酸TTTCT突变为AATCC的crispr基因编辑系统包括靶序列包含SEQ ID NO：18或20的sgRNA。
在一些实施方案中，将第二外显子内的核苷酸TTTCT突变为AATCC的crispr基因编辑系统包括靶序列包含SEQ ID NO：18或20的sgRNA和包含SEQ ID NO：26的ssDNA。
在另一方面，本文提供了在受试者中清除或减少肿瘤细胞的方法，其包括以能够杀伤所述肿瘤细胞的CAR-T或抗体类药物向所述受试者给药以及向所述受试者补充正常细胞，其中所述CAR-T或抗体类药物靶向所述肿瘤细胞的细胞表面蛋白，所述正常细胞表达的所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述正常细胞能够抗所述CAR-T或抗体类药物杀伤。
在一些实施方案中，所述肿瘤细胞为血液肿瘤细胞，所述正常细胞为血液细胞或能够分化为血液细胞的造血干细胞或祖细胞。
在一些实施方案中，所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
在一些实施方案中，所述正常细胞的抗原表位改变是由于所述细胞表面蛋白的编码基因中存在单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失引起。
在一些实施方案中，所述正常细胞的所述细胞表面蛋白在表达时存在外显子缺失。
在一些实施方案中，所述细胞表面蛋白为CD33、CD19或BCMA。
在一些实施方案中，所述细胞表面蛋白为CD33，其缺失CD33基因中第二外显子编码的氨基酸片段。
在一些实施方案中，所述细胞表面蛋白为CD33，其编码基因中存在如下特征之一或它们的任意组合：1)rs12459419位点为核苷酸T；2)第二外显子序列之前的3’ss位点为AA或 CT；以及3)第二外显子内的TTTCT替换为AATCC。
在一些实施方案中，所述肿瘤细胞为AML细胞。
另一方面，本文提供了药物组合物或药物组合，其包括CAR-T或抗体类药物和正常细胞，其中所述CAR-T或抗体类药物能够靶向肿瘤细胞的细胞表面蛋白而杀伤所述肿瘤细胞，所述正常细胞表达的所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述正常细胞能够抗所述CAR-T或抗体类药物杀伤。
在一些实施方案中，所述肿瘤细胞为血液肿瘤细胞，所述正常细胞为血液细胞或能够分化为血液细胞的造血干细胞或祖细胞。
在一些实施方案中，所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
在一些实施方案中，所述正常细胞的抗原表位改变是由于所述细胞表面蛋白的编码核苷酸序列中存在单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失引起。
在一些实施方案中，所述正常细胞的所述细胞表面蛋白在表达时存在外显子缺失。
在一些实施方案中，所述细胞表面蛋白为CD33、CD19或BCMA。
在一些实施方案中，所述细胞表面蛋白为CD33，其缺失CD33基因中第二外显子编码的氨基酸片段。
在一些实施方案中，所述细胞表面蛋白为CD33，其编码基因中存在如下特征之一或它们的任意组合：1)rs12459419位点为核苷酸T；2)第二外显子序列之前的3’ss位点为AA或CT；以及3)第二外显子内的TTTCT替换为AATCC。
在一些实施方案中，所述肿瘤细胞为AML细胞。
本文提供的经改造而具有抗原表位改变的细胞与CAR-T细胞或抗体类药物联合可用于肿瘤治疗。具有抗原表位改变的细胞不被CAR-T或抗体类药物杀伤，可输入患者体内，以缓解CAR-T产品或抗体类药物在在肿瘤治疗中的副作用。
附图说明
图1为实施例1中不同细胞系之间的CD33 exon2 SNP基因型的分析结果图。
图2为实施例1中不同细胞系的CD33 exon2可变剪切表达凝胶电泳图。
图3A为实施例1中RT-qPCR实验中跨CD33 exon2的探针引物设计原理图。
图3B为实施例1中检测不同细胞系的CD33 exon2的缺失情况的RT-qPCR结果图。
图4A为实施例1中不同细胞系的CD33 exon2及总蛋白的表达情况的流式结果对比图。
图4B为实施例1中不同细胞系的CD33 exon2及总蛋白的平均荧光强度统计图。
图5为实施例2中通过FACS流式检测不同细胞系的GFP阳性率和细胞活率柱状图，其中I图为GFP阳性率，II图为细胞活率。
图6A为实施例2中利用单碱基胞嘧啶编辑器A3A-CBE3突变CD33 exon2 3'ss AG位点后G碱基的突变状况示意图。
图6B为实施例2中利用EditR分析3'ss单碱基突变效率后所得柱状图。
图6C为实施例2中RT-qPCR检测K562细胞系中E13/E2比值的对比图。
图6D为实施例2中RT-qPCR检测HL60细胞系中E13/E2比值的对比图。
图7为实施例3中通过RNP联合ssDNA电转入细胞的方式突变SNP位点和3'ss位点后所得突变状况分析图。
图8A为实施例3中突变SNP位点和3'ss位点后E13/E2的RT-qPCR结果图。
图8B为实施例3中突变SNP位点和3'ss位点后流式抗体染色结果图。
图9为实施例4中通过Tyto流式分选PE+和PE-细胞和分选后所得细胞的突变状况分析图，其中I图为Tyto流式分选图，II图为突变状况分析图。
图10A为实施例5中CD33和CD33高度相似的DNA的序列比较图。
图10B为实施例5中sg4在CD33和脱靶区域的介导的编辑情况示意图。
图11A为实施例5中sg4-14和sg4-15介导CD33 exon2 SNP位点的突变后所得序列的分析图。
图11B为实施例5中流式检测突变后细胞由CD33 exon2编码的V区(P67.6)的表达量结果图。
图12A为实施例6中Sg3和sg10介导的AATCC突变的序列分析图。
图12B为实施例6中流式检测突变后细胞由CD33 exon2编码的V区(P67.6)的表达量结果图。
图13为实施例7中流式检测CD33 CART细胞中CAR阳性细胞比例图。
图14A为实施例7中CD33 CAR-T细胞和编辑后NB4细胞共孵育0小时后所得流式检测结果图。
图14B为实施例7中同时流式染色CD33 P67.6和HIM-3-4抗体0小时后所得检测结果图。
图14C为实施例7中CD33 CAR-T细胞和编辑后NB4细胞共孵育96小时后所得流式检测结果图。
图14D为实施例7中同时流式染色CD33 P67.6和HIM-3-4抗体96小时后所得检测结果图。
图14E为实施例7中不同细胞系与CD33 CAR-T细胞共孵育后0小时、22小时、44小时、68小时和96小时突变后靶细胞的比例曲线图。
图15A为实施例8中在HSC细胞中电转sg10和cas9的RNP复合物后所得细胞的序列分析图。
图15B为实施例8中同时流式染色CD33 P67.6和HIM-3-4抗体后所得检测结果图。
图15C为实施例8中加入诱导HSC体外髓系单核分化的培养基在分化第1、5、9、14天分别检测CD14阳性细胞的比例图。
图15D为实施例8中分化第1、5、9、14天时CD14阳性细胞的含量的曲线图。
图16为单核苷酸变化导致的CD33第二外显子缺失的示意图。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。出于方便理解本文提供的技术方案的目的，以下对部分技术术语进行简要说明。
“细胞表面蛋白”在本文中指表达后至少部分地位于细胞表面的蛋白，其通常为部分氨基酸序列嵌入细胞膜中的膜蛋白，也可以为附着在细胞表面的细胞外成分。对于来源于不同组织或器官的细胞，它们通常会表达各自特异的细胞表面蛋白，即组织特异性或细胞特异性蛋白。这些细胞表面蛋白还可以随着细胞的分化或成熟而增加或减少表达。在一些情况下，本文关注血液细胞特异性的细胞表面蛋白，例如主要在淋巴系细胞(例如T细胞或B细胞)或髓系细胞(例如各种粒细胞、单核细胞等)表面表达的蛋白，例如，CD33、CD19、CD21、BCMA、CD22、CD24等细胞表面标志物。在肿瘤细胞中，有些细胞表面蛋白的表达量上调，或者表达某些肿瘤特异性表面蛋白，它们可以作为药物结合或作用的靶点。
“嵌合抗体受体(chimeric antigen recessive，CAR)”，也称为嵌合T细胞受体、嵌合免疫受体，为一种工程化的膜蛋白受体分子，其可将期望的特异性赋予免疫效应细胞，例如与细胞表面蛋白(如肿瘤抗原)结合的能力。嵌合抗原受体通常由胞外抗原结合结构域、跨膜结构域和胞内信号结构域构成。在一些情形下，抗原结合结构域为一段scFv序列，负责识别和结合特定的抗原。胞内信号结构域通常包括免疫受体酪氨酸活化基序(ITAM)，例如来源于CD3ε分子的信号传导结构域，负责激活免疫效应细胞，产生杀伤作用。另外，嵌合抗原受体还可在氨基端包括负责新生蛋白在细胞内定位的信号肽，以及在抗原结合结构域和跨膜结构域之间包括铰链区。除了信号传导结构域，胞内信号结构域还可包括来源于例如4-1BB或CD28分子的共刺激结构域。相应地，将表达CAR的T细胞简称为CAR-T。CAR-T利用其细胞表面表达的CAR识别靶细胞，被靶细胞激活后产生对靶细胞的杀伤相应。利用CAR-T细胞对受试者(如癌症患者)进行治疗的大体过程为：从受试者采集外周血单个核细胞(PBMC)，分离并培养T细胞，通过慢病毒转导方式导入CAR编码核酸序列，继续培养并收集CAR+细胞，以及将CAR+细胞回输给该受试者。本领域技术人员已知，在一些情况下，可以利用NK细胞替代T细胞来进行该过程。因此，在提及CAR-T时，视情况也可涵盖表达CAR的NK细胞。
“抗体类药物”在本文中指能够与细胞表面蛋白结合从而导致表达该细胞表面蛋白的细胞的活性发生改变的抗体或抗体偶联药物(ADC)。例如，抗体分子与细胞表面蛋白(如受体)结合时可能导致细胞活性被抑制，生长停滞或死亡，或者由于抗体与细胞表面蛋白结合，导致细胞表面蛋白的配体不能正常结合，从而引起细胞活性被抑制，生长停滞或死亡。再例如，当抗体分子通过可降解接头与细胞毒剂结合时，可通过将抗体特异性识别其对应的抗原分子而将细胞毒剂靶向表达该抗原分子的细胞，导致细胞被杀伤。
“抗原表位(epitope)”在本文中指对应抗体的识别和结合位点。抗原表位可以为一段连续氨 基酸序列，或者由不连续的氨基酸形成的三维结构，它们通常位于蛋白分子的表面。抗体分子通过其重链可变区和轻链可变区形成的抗原结合部位与抗原表位结合。CAR分子中的抗原结合结构域通常包括scFv形式的抗原结合部位，其可识别靶细胞上细胞表面蛋白上的抗原表位。抗原表位变化至少是构成该抗原表位的一个或更多氨基酸的插入、删除和/或替换。在一些情况下，抗原表位变化为细胞表面蛋白中的连续氨基酸缺失，例如由于前体mRNA分子的可变剪接导致的外显子缺失引起。当靶细胞的细胞表面蛋白的抗原表位发生变化时，识别该表位的抗体分子会丧失或减弱结合能力，这种情况下，对应的包括来自该抗体分子的轻链可变区和重链可变区(对于单域抗体，仅重链可变区)的CAR-T也会丧失或减弱其靶细胞杀伤能力，即该靶细胞获得了“抗CAR-T杀伤”的性质。在优选的实施方案中，这种抗原表位变化仅仅是为了获得针对特定的CAR-T的抗CAR-T杀伤能力，而不影响该细胞表面蛋白的正常功能。
“单核苷酸突变”在本文中指细胞表面蛋白的编辑基因中的单个核苷酸变化，包括插入、删除或替换。该单个核苷酸变化会导致表达的细胞表面蛋白的氨基酸序列发生变化，或者如果该单个氨基酸变化出现在与控制RNA剪接有关的序列(如外显子剪接增强子、内含子剪接增强子、3’ss或5’ss)中时，产生剪接错误而导致氨基酸序列变化。这些氨基酸序列变化均可以使抗原表位发生变化，从而使表达该细胞表面蛋白的细胞获得抗CAR-T杀伤能力。
“剪接位点突变”在本文中指与前体RNA(pre-RNA)的剪接相关的突变。突变位点例如可以位于外显子剪接增强子、内含子剪接增强子、3’ss或5’ss、branch point以及其它序列中，只要它们可导致最终产生的蛋白的氨基酸序列发生变化。在一些情况下，剪接位点突变可导致至少一个外显子的编码序列不出现在合成的蛋白中。在一个具体实施方案中，CD33基因第二外显子的一个SNP位点(rs12459419)为T时，可导致第二外显子缺失(见图16)。
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)基因编辑技术是新出现的一种由RNA指导的通过Cas核酸酶对靶基因进行DNA编辑的技术。该技术所使用的CRISPR基因编辑系统包括Cas核酸酶和引导RNA(single-guide RNA,sgRNA)，视情况可还包括作为修复模板的ssDNA。sgRNA可以与Cas核酸酶结合，另外部分序列可以与靶基因的部分序列互补，借助sgRNA的识别作用使得Cas核酸酶可以在靶基因特定位点形成单链或双链切口。细胞通常会通过两种方式对断裂链进行DNA修复，这两种方式分别是同源重组修复机制(homology-directed repair,HDR)和非同源末端连接修复机制(non-homologous end joining,NHEJ)。在向细胞提供修复模板时，可以让细胞根据该修补模板进行切口修复。如果在修复模板(ssDNA)中加入突变核苷酸，则在同源重组修复后可以产生技术人员期望的突变，实现基因编辑目的。不提供修复模板情况下的NHEJ修复机制则可能会产生多种突变产物，也可以对这些突变产物进行筛选以获得技术人员期望的突变产物。最近，在上述CRISPR基因编辑系统上又发展了各种定点单碱基编辑器，例如CBE、ABE以及它们的各种改进变体等，这些碱基编辑器利用了改造的Cas酶与各种脱氨酶形成的融合蛋白，在sgRNA指导下在特定位点形成核苷酸定向改变。这种碱基编辑器可以认为是改进的CRISPR基因编辑技术，当本 文提及CRISPR基因编辑时也将它们涵盖在内。另外，本领域技术人员还可预期可采用DNA同源重组、借助核酸内切酶(如ZFN和TALEN)进行特定位点剪切以引入基因变动。只要能够产生期望的抗原表位变化，利用这些以及其它的基因改造技术也应涵盖在本发明的保护范围内。
“正常细胞”在本文中指非肿瘤细胞，尤其是指在CAR-T(或抗体类药物)治疗过程中被杀伤的非肿瘤细胞。由于CAR-T依赖其抗原结合结构域识别肿瘤细胞上的抗原表位而杀伤肿瘤细胞，但非肿瘤细胞上往往也表达或存在这些抗原表位，导致它们也被CAR-T杀伤，产生“on-target，off-tumor”问题。这些正常细胞被杀伤，导致受试者免疫系统或其它功能受损。在医疗实践中，可通过向受试者补充这些正常细胞来缓解上述问题。由于这些正常细胞也可能带有CAR-T所识别的抗原表位，为防止仍被CAR-T杀伤，可采用本文提供的方法对这些抗原表位进行改动，以避免CAR-T杀伤。因此，就防止CAR-T杀伤而言，这些正常细胞主要是进行了抗原表位改动的细胞。这些正常细胞可以来自与受试者自身，也可以来自于其他供者。这些正常细胞可以为已分化或成熟细胞，也可以是干细胞、祖细胞或前体细胞，例如造血干细胞(HSC)。
“药物组合物”在本文中指包含至少一种药物活性成分的药物制剂。药物制剂通常还可包括药学上可接受的赋形剂、缓冲剂等其它成分。当药物组合物包括至少两种活性成分时，它们通常共存于同一药物制剂中。“药物组合”在本文中指至少两种药物活性成分处于分开状态，即存在于不同的药物制剂中，但它们适合于先后或同时向受试者给药，以在受试者中产生优于药物活性成分单独给药的治疗效果。这种治疗效果可体现在药物活性成分的协同作用，也可以为一种药物活性成分减弱另一种药物活性成的副作用。以“药物组合”形式存在的药物活性成分可存在于同一药物包装盒中或分别存在于不同的药物包装盒中。
通过将CAR-T(或抗体类药物)疗法与正常细胞的输入(如HSC移植)相结合，本文提供了一种新的肿瘤、尤其是血液肿瘤(如AML)的治疗策略。基于该策略，通过向受试者补充经基因改造的正常细胞(如HSC)，可克服CAR-T在肿瘤治疗过程中同时杀伤非肿瘤细胞的副作用，使得在CAR-T疗法的设计中能够不必选择肿瘤特异性抗原(往往难以找到)作为靶点，而是也能够选择肿瘤相关抗原作为靶点。
本文提供的免受嵌合抗原受体T细胞杀伤作用的造血干细胞或祖细胞可包括用于突变编码细胞表面蛋白的核苷酸序列的基因编辑系统。所述细胞表面蛋白包括CAR-T细胞靶向的细胞表面蛋白。优选地，所述抗原表面蛋白包括CD33抗体识别表位、CD19抗体识别表位或BCMA抗体识别表位中的任意一种。作为本发明优选的技术方案，所述造血干细胞的或祖细胞的CD33抗体识别表位位点对应的第二外显子区域(exon2)的SNP(单核苷酸多态性位点，rs12459419)基因型表现为突变的T基因型；和/或，所述造血干细胞的CD33抗体识别表位位点对应的第二外显子区域的TTTCT位点表现为AATCC的突变。本发明以CD33为靶点抗原，以商品化的AML细胞进行SNP和CD33可变剪切实验，确认SNP位点突变的有效性后，筛选可有效导致CD33 V结构不结合抗体的突变；经过筛选后发现，在CD33 exon2存在SNP 位点，将SNP由C突变为T后，二号外显子缺失进而导致CD33 V结构域不表达，但是在突变后，也存在CD33 C结构域受到影响的风险；因此，本发明在进一步研究中意外发现，引入新的AATCC可突变位点，不仅可阻断CD33 V结构域的表达，且不影响CD33 C结构域的表达，所得细胞不仅耐受Anti-CD33 CAR-T的杀伤，且不影响细胞的正常体外分化。
因此，突变造血干细胞的CD33的第二外显子区域的SNP位点和/或TTTCT位点，优选为突变TTTCT位点，能够有效耐受anti-CD33 CAR-T的杀伤，且细胞能够正常进行体外分化。传统的敲除会造成基因功能的缺失，与直接敲除基因相比，通过基因编辑等手段，突变部分基因，造成抗原表位构象发生变化，在不影响蛋白功能的前提下，造成CAR-T或抗体药对其靶向的失效，对造血干细胞的影响更小，且在与CAR-T细胞的共孵育过程中，本发明提供的基因突变的细胞内耐受能力更强，存活率更高。
同时，本发明中所提供的基因编辑方法和设计思路同样能够扩展到其他靶点抗原，如淋巴系和髓系细胞(B细胞、T细胞、NK细胞、单核细胞、粒细胞等)中的特异表达抗原，极大幅度拓宽了细胞免疫治疗中对肿瘤靶点的选择，通过将造血干细胞移植和CAR-T细胞治疗联用，为肿瘤免疫治疗提供了新的治疗思路。
例如，针对CD19和BCMA等抗原，本发明可以通过碱基编辑或敲除协同的同源重组对造血干细胞的相关抗原进行编辑，分别筛选出CD19、BCMA等抗原的突变位点，能够使得分化出的B细胞或浆细胞耐受抗CD19-CAR-T细胞(或抗CD19抗体)或抗BCMA-CAR-T细胞(或抗BCMA抗体)的靶向，而具备正常的细胞功能。
优选地，所述造血干细胞的CD33基因的3’ss位点表现为AG到AA或CT的突变。
同样地，本发明中可采用与突变编码CD33的核苷酸序列的基因编辑方法来改变CD19的抗体识别表位或BCMA的抗体识别表位，所基于的实验方法或设计思路与CD33相似，不同之处在于基因编辑系统中所使用的sgRNA不同，相应地，所针对的位点也随之发生变化。
作为本发明优选的技术方案，所述基因编辑系统包括CRISPR基因编辑系统。所述CRISPR基因编辑系统可包括Cas酶和sgRNA(或它们的表达载体)。其中，所述sgRNA包括用于突变CD33抗体识别表位位点的sgRNA、用于突变CD19抗体识别表位位点的sgRNA或用于突变BCMA抗体识别表位位点的sgRNA。
本发明中，用于突变编码CD33的第二外显子区域的核苷酸序列的sgRNA包括：SEQ ID NO：10(sg4)、SEQ ID NO：11(sg4-14)、SEQ ID NO：12(sg4-15)、SEQ ID NO：18(sg3)或SEQ ID NO：20(sg10)所示的核苷酸序列中的任意一种。
在采用sg4进行基因突变SNP位点时，sg4存在错配至CD33同源序列导致脱靶的情况，因此，本发明中进一步替换sg4上的第14位(sg4-14)或15位(sg4-15)碱基，以降低sg4脱靶至CD33同源区域的可能性。sg4-14和sg4-15在CD33 exon2上均可有效介导SNP位点C向T的突变，突变后的细胞exon2缺失；sg3和sg10介导的突变组中，CD33区域上的TTTCT被突变为AATCC，且在CD33同源区域，测序峰单一且与仅电转cas9组没有差异，表明sg3，sg10不会脱靶至CD33同源序列上产生非特异性切割。
优选地，所述sgRNA为SEQ ID NO：11(Sg4-14)、SEQ ID NO：12(Sg4-15)或SEQ ID NO：20(Sg10)中的任意一种。
优选地，所述sgRNA为SEQ ID NO：20，制备得到的造血干细胞的CD33表位位点对应的第二外显子区域的TTTCT位点表现为AATCC的突变。
本发明中，针对CD19的抗体识别表位或BCMA的抗体识别表位，同样设计了相应的sgRNA，用于实现基因突变。
作为本发明优选的技术方案，所述基因编辑系统还包括修复模板ssDNA。
其中，所述修复模板ssDNA包括SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24或SEQ ID NO：26中任意一种所示的核苷酸序列。
本发明还提供了制备如上所述的造血干细胞或祖细胞的方法，所述方法包括将基因编辑系统转入待编辑的造血干细胞或祖细胞。其中，所述转入的方法包括转导、转染或电穿孔中任意一种。例如，本发明中所述的方法可以是：制备cas9和sgRNA的RNP复合物，孵育，再将所述RNP复合物与任选的修复模板ssDNA混合，加入待编辑的造血干细胞悬液，电转，所述电转结束后使用培养基培养，得到基因编辑改造的造血干细胞。
本发明还提供了所述造血干细胞或祖细胞在制备治疗肿瘤的药物中的应用。
本发明还提供了所述造血干细胞或祖细胞与CAR-T细胞联合在制备治疗肿瘤的药物、抗体或类抗体联合小分子的药物开发、表达抗体类分子或CAR分子的免疫细胞治疗药物的开发中的应用。
本发明中，经CD33表位改造的造血干细胞联合Anti-CD33 CAR-T、经CD19表位改造的造血干细胞联合Anti-CD19 CAR-T、经BCMA表位改造的造血干细胞联合Anti-BCMA CAR-T，将其应用与肿瘤细胞免疫的治疗研究中，对于缓解CAR-T产品在肿瘤治疗中的副作用、提高患者生存质量具有重要的意义。
本发明还提供了药物组合物，其包括如所述造血干细胞或祖细胞。优选地，所述药物组合物还包括药学上可接受的载体。
本发明还提供一种治疗方法，其包括对受试者施用上述药物组合物和CAR-T。
其中，所述受试者为患有肿瘤的动物或人。
与现有技术相比，本发明的有益效果为：
本发明依据基因编辑改造抗原表位的正常构象、阻断原有表面蛋白的免疫性相关结构，避免其被抗体的靶向而其仍具有正常的生物学功能的设计概念，提供设计不同的sgRNA，突变造血干细胞的CD33 exon2位点，使其exon2位点缺失进而导致CD33 V结构域不表达，逃逸anti-CD33 CAR-T细胞的靶向和杀伤，解决CAR-T细胞在杀伤肿瘤细胞的同时造成的副作用；同时，为了解决造血干细胞在基因编辑制备过程中面临的脱靶和效率低下等问题，本发明中进一步排除易脱靶和低效率的sgRNA，制备得到的细胞不仅CD33 V结构域的表达，且不影响CD33 C结构域的表达，与CAR-T细胞共孵育时，存活率较高，且高于CD33基因敲除后的细胞，同时利用所述sgRNA获得的HSC的体外分化能力不受影响；本发明是基于广 泛的基础研究得出的可行手段，通过基因编辑突变造血干细胞创造出“肿瘤特异性”抗原以便于细胞免疫治疗，解决CAR-T细胞在杀伤肿瘤细胞的同时，也会靶向患者体内正常表达靶点抗原的健康细胞而产生的“on-target，off-tumor”问题，为肿瘤免疫治疗提供了新的治疗思路。
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案，但下述的实例仅仅是本发明的简易例子，并不代表或限制本发明的权利保护范围，本发明的保护范围以权利要求书为准。
以下实施例中使用的材料如下表1所示，其余未提及材料均可购自本领域常规生产厂家。
表1实施例中使用的部分实验材料
Figure PCTCN2022089600-appb-000001
以下实施例中，使用的实验方法如下，其余未提及的实验方法均为本领域技术人员知晓的常规实验技术和手段。
1、细胞基因型鉴定
收集约1×10 5个细胞，采用PureLink TMGenomic DNA Mini Kit提取基因组DNA(对于突变细胞，一般电转后72小时收集基因组DNA)。以基因组DNA为模板，用AmpliTaq Gold TM360 Master Mix PCR，扩增出覆盖目标区域的DNA片段。将PCR产物测序，利用Sanger法测序获得碱基序列图谱，对比序列基因型。
2、mRNA水平RT-qPCR鉴定CD33 exon2的可变剪切
收集约1×10 6个细胞，采用PrimeScript TM RT reagent Kit提取细胞RNA，并用TB
Figure PCTCN2022089600-appb-000002
Premix DimerEraser TM试剂盒反转录RNA，得到cDNA。以cDNA为模板，设计跨CD33 exon2的探针引物(E13)，可特异性扩增exon2缺失的序列(m)；在exon2上的探针引物(E2)，可特异性扩增exon2正常表达的序列(M)，计算并导出RT-qPCR的ct值。
最终以m/M的ct比值反映CD33 exon2的可变剪切程度，比值越高，则exon2的缺失表达越高，可变剪切发生越高。
本文实施例中使用的普通引物、探针引物详见表2。
表2实施例中使用的引物
引物名称 序列(5'to3') SEQ ID NO.
CD33-F GAAGCTGCTTCCTCAGACATGC 1
CD33-R ATGGTTCTCTCCGTAGTCACAC 2
CD33-similar-F GAAGCCTCTGCCTCAGACATGC 3
CD33-similar-R ATGGTTCTCTCCGTGGTCACAC 4
CD33-VIC-E2 ACAGTTACAAATCTCCCCAG 5
CD33-FAM-E13 TGCTGCCCCTGCTGTGGGCAGACTTGAC 6
CD33-E2F TCTTTCGGATGGAGAGAGGA 7
CD33-E1F ACACAGGAAGCCCTGGAAG 8
CD33-E3R GAGCAGGTCAGGTTTTTGGA 9
3、流式细胞染色
收集约1×10 5个细胞，用PBS+2％胎牛血清缓冲液洗1遍，完全弃去上清。加入human FcR-blocker，4℃避光封闭孵育10分钟，清洗一遍后加入流式抗体，混匀后4℃避光孵育30分钟，洗一遍后，加入缓冲液(含有DAPI或7-AAD)上机检测。
4、电转
采用LONZA电转仪，使用LONZA官网推荐的各细胞电转程序和电转kit，具体各细胞采用的电转条件见表3。
取所需细胞100g离心10分钟。离心后完全去除培养基，并用Lonza电穿孔缓冲液重悬。与此同时，制备cas9和sgRNA的RNP复合物，室温孵育15分钟。随后将ssDNA与RNP复合物混匀后，加入细胞悬液，转移至Lonza 16孔电转杯中，放入4D-Nucleofector TMX单元，根据细胞类型选择对应程序进行电穿孔。电转后小心吸取细胞悬液至48孔板，以1640完全培养基或造血干专用培养基继续培养。
表3各种细胞电转条件
细胞类型 电转Kit 电转程序 电转体系 细胞数
K562 SF FF-120 20μL 2e5
HL60 SF EN-138 20μL 4e5
NB4 SF CZ-100 20μL 4e5
THP1 SG FF-100 20μL 2e5
KG1 SF FF-100 20μL 2e5
Molm13 SF CA-137 20μL 2e5
5、CD33可编辑表位位点sgRNA设计
通过文献资料，找到可编辑的CD33表位位点。CD33第二个外显子区域存在SNP位点，SNP是C时，CD33正常表达(记为M)；当SNP位点是T时，CD33产生可变剪切，二号外显子缺失进而不表达(记为m)。
通过UCSC genome browser下载hCD33基因组序列，通过CHOPCHOP网站设计sgRNA。排除易脱靶和低效率的sgRNA，最终挑选出的sgRNA的如下表4所示。
表4 sgRNA和ssDNA序列
Figure PCTCN2022089600-appb-000003
Figure PCTCN2022089600-appb-000004
Figure PCTCN2022089600-appb-000005
6、CD33 CAR-T细胞制备
复苏冻存的健康PBMC，加入20μL/10 7的CD3微珠，用LS分选柱分选出CD3阳性T细胞。分选当天用CD3/CD28抗体磁珠激活T细胞，激活24-48小时后，进行病毒转导。包装有CAR分子的慢病毒MOI为3，加入800ng/μL PolyBrene和1μg/μL DEAE辅助感染。感染24小时后，300g离心10分钟离心去除病毒，更换为新鲜T培养基。
T细胞感染慢病毒后5天，用流式细胞术检测CAR阳性率，CD33-CAR分子带有EGFR标签，故EGFR抗体检测可反映CAR阳性率。
7、CD33-CAR-T细胞与编辑后靶细胞共孵育检测突变耐受情况
待靶细胞电转编辑完成，并制备得到CD33 CAR-T细胞后，根据1:30效靶比混匀两种细胞(以靶细胞3e6个为例，则效应细胞CAR阳性细胞数应为1e5个，若CAR-T细胞中CAR阳性为10％，则CD33 CAR-T细胞数应为1e6个，即为共孵育体系中含有靶细胞3e6个，CD33 CAR-T细胞数为1e6个)。
使用T细胞培养基培养96小时，期间检测CD3阳性细胞比例，可反应CAR-T细胞的变化。相应的，CD3阴性细胞比例可反映突变后靶细胞比例的变化。
期间同步检测突变后靶细胞中可结合CD33 P67.6或HIM3-4抗体细胞的比例变化，若丧失结合P67.6，仍结合HIM3-4这部分突变后细胞可存活，证明突变后细胞可耐受CD33 CAR-T的杀伤。
实施例1 CD33第二个外显子的SNP位点基因型与该外显子的可变剪切相关
为探究SNP位点突变以改造CD33可变剪切的可行性，本实施例中首先选择表达CD33的AML五种细胞系(HL60，NB4，THP1，KG1，Molm13)及CML细胞K562作为研究对象。
(1)收集各细胞基因组DNA，在CD33 exon2上下游设计扩增引物对，以gDNA为模板，对CD33 exon2区域进行PCR，将PCR产物sanger测序，用SnapGene打开测序文件，比对各细胞系CD33 exon2上的SNP位点(rs12459419)基因型。
结果显示，不同AML/CML细胞系之间的CD33 exon2 SNP基因型并不完全相同(图1)：阴影部分为CD33 exon2外显子序列，其第四位碱基为SNP位点。K562，HL60的SNP位点基因型表现为T，而NB4，KG1，THP-1，Molm13则表现为C基因型。
(2)收集细胞RNA，反转录为cDNA，采用引物CD33-F/CD33-R，在mRNA水平通过RT-PCR检测不同细胞系的CD33 exon2的缺失情况，exon2缺失的PCR条带为317bp(m)，exon2存在的情况下PCR条带为698bp(M)，保证各组模板量一致的情况下，m的条带越弱，表明exon2的可变剪切缺失程度越高。
结果显示，SNP位点基因型是T的细胞(K562，HL60)CD33 exon2更容易发生可变剪切进而缺失(图2)：
在K562和HL60细胞中，m条带所占的比重大于M条带比重，表明K562和HL60细胞的CD33 exon2缺失更高；在NB4和THP1细胞中，m条带和M条带强弱相差不大，表明NB4和THP1细胞CD33 exon2的可变剪切低于K562和HL60细胞；在Molm13和KG1细胞中，M条带所占比例更高，表明这两种细胞的CD33 exon2可变剪切程度较低。
(3)为了更加精确定量exon2可变剪切缺失的比例，本实施例中进一步设计跨CD33 exon2的探针(CD33-FAM-E13)，RT-qPCR引物CD33-E2F和CD33-E3R可特异性识别exon2的缺失，(记为m)；探针(CD33-VIC-E2)在exon2上，RT-qPCR引物CD33-E1F和CD33-E3R可特异性指示exon2正常表达(记为M)，如图3A所示。
收集细胞RNA，反转录为cDNA，在mRNA水平通过RT-qPCR检测不同细胞系的CD33 exon2的缺失情况，m/M的比值越高，表明exon2的可变剪切缺失程度越高。
结果显示，SNP位点基因型是T的细胞(K562，HL60)CD33 exon2更容易发生可变剪切进而缺失(图3B)：
以Raji细胞作为对照，统计AML/CML各细胞系的CD33 exon2m/M相对于Raji细胞的比值，在SNP基因型为T的K562和HL60的细胞中，m/M比值是对照组的5倍以上，SNP基因型为C的THP-1和NB4细胞次之，SNP基因型为C的KG1和Molm13细胞m/M比值与对照组没有差别，甚至更低(<1倍)。
(4)进一步通过流式细胞技术验证其CD33 exon2蛋白水平的表达。
研究表明P67.6克隆来源抗体可特异性结合CD33 V结构域，因此其可用来指证exon2的表达(M)，发生exon2可变剪切缺失的细胞无法结合P67.6。而HIM3-4克隆来源抗体可特异性识别CD33 C结构域，无论exon2缺失与否，其均可正常被结合(Total)。
通过收集AML/CML活细胞，共染CD33-P67.6和CD33-HIM3-4直标抗体，流式检测CD33 exon2及总蛋白的表达情况，并统计其平均荧光强度(MFI)，以Total/M MFI的比值反映CD33 exon2的可变剪切，比值越大，侧面表明CD33 exon2的缺失越高。
流式结果显示，K562细胞的P67.6抗体结合能力明显低于AML/CML其他细胞系，而Molm13的表达居于最高；且K562细胞的Total/M MFI比值最高，CD33 exon2的缺失最高，Molm13 exon2的缺失最低(图4A，图4B)：
灰色为各流式抗体的同型对照，结合P67.6的能力可反映CD33 M的表达，可见CD33 exon2未被可变剪切的表达：
K562<HL60<THP1<NB4/KG1<Molm13。
结合HIM3-4的能力可反映CD33总蛋白的表达：
K562<HL60<THP1<NB4<KG1<Molm13。
统计各细胞系中CD33 Total/M MFI的比值：
K562>HL60>THP1>KG1>NB4>Molm13；
表明K562细胞的CD33 exon2的缺失程度最高，Molm13 exon2的缺失最低。与mRNA水平检测的CD33 exon2的表达基本一致。
本实施例中初步建立可有效鉴定SNP位点基因型、可变剪切表型的方法体系，利用sanger测序可清晰指示SNP位点基因型，RT-PCR/RT-qPCR和P67.6克隆来源流式抗体检测的手段可指示CD33 exon2可变剪切缺失的比例，为下一步突变CD33 exon2 SNP以改变CD33的可变剪切进行概念验证奠定方法基础。本实施例中得到的初步结论为：SNP为T基因型的细胞可变剪切程度更高，为下一步设计SNP C向T突变以增加CD33 exon2的跳跃表达提供依据。
实施例2突变CD33 exon2的3’ss和SNP均可增强其可变剪切
(1)HL60，K562，NB4是AML/CML细胞中更适合电转编辑的细胞。
在初步摸索好AML/CML几种细胞的CD33 exon2 SNP基因型及exon2的表达高低后，为了进一步在各细胞系基础上做突变编辑，我们初步用带有pmax-GFP的质粒做适合各细胞系的电转条件摸索。
采用LONZA电转仪，电转后24小时，用Countstar细胞计数仪计数细胞活率，并通过FACS流式检测细胞的GFP阳性率。
结果显示，K562，HL60，NB4电转后细胞活率较好，GFP阳性率较高，更适合后期电转编辑(图5，其中I图为GFP阳性率，II图为细胞活率)：
K562，HL60细胞电转后24小时活率都很高，在95％以上，且K562，HL60GFP的阳性率也在95％以上，居于最高；NB4细胞电转后24小时活率也很高，在95％左右，但其GFP阳性率在65％左右；KG1细胞的电转后细胞活率在60％左右，但其GFP的阳性率不及40％，后期对该细胞进行编辑突变难度略大；THP1和Molm13细胞电转后细胞的活率只有10％上下，且GFP阳性率也不足10％，不适合后期的电转编辑突变。
(2)3’ss位点突变可诱导HL60，K562的CD33 exon2跳跃表达
由于在K562和HL60细胞中，其SNP位点已经是T基因型，只能对其3’ss位点做突变方可进一步增加可变剪切。
本实施例中基于CRISPR-Cas9技术的单碱基胞嘧啶编辑器A3A-CBE3，并设计可突变3’ss AG位点G碱基的sgRNA(sg8：SEQ ID NO：13，sg13：SEQ ID NO：16)，通过将CBE质粒与sgRNA质粒电转入细胞。通过收集电转后第7天细胞的基因组DNA，采用引物CD33-F/CD33-R PCR出CD33 exon2区域，将PCR产物sanger测序，用SnapGene打开测序文件，比对CD33 exon2 3’ss AG位点G碱基的突变状况。
结果显示，在K562和HL60细胞中，sgRNA8和sgRNA13均可有效突变3’ss位点的G碱基(图6A，图6B)：
阴影部分为CD33 exon2外显子序列，exon2前两位碱基AG为3’ss位点。
在K562和HL60细胞中，以仅电转CBE的细胞作为空白对照组，其3’ss位点为AG；在sgRNA8或sg13的介导下，sg8和sg13所处的互补链上的碱基C被CBE定向突变为T， 故3’ss AG位点上的G碱基表现为向A碱基的突变。
发明人进一步采用实例2中RT-qPCR鉴定exon2缺失的方法，检测3’ss突变对exon2可变剪切的影响。
结果显示，3’ss突变可增强K562，HL60exon2的可变剪切(图6C，图6D)：
在K562细胞中，sg8组E13/E2的比例高于sg13组，编辑组的sg8，13均比空白对照组高，说明编辑后细胞的exon2可变剪切升高。且sg8组3’ss突变效率高于sg13，故其导致的exon2缺失也就更高。
在HL60细胞中，sg8和sg13组的E13/E2比例均高于空白对照组，即HL60细胞中3’ss突变也会导致其CD33 exon2跳跃表达。
实施例3 Sg4介导的SNP位点及3’ss突变可有效诱导NB4的CD33 exon2缺失
本实施例中，基于CRISPR-Cas9技术，利用Cas9蛋白和sgRNA形成RNP，并提供ssDNA修复模板，ssDNA上SNP位点为T，3’ss位点为CT，将RNP和ssDNA通过电转递送至细胞内。
利用细胞修复过程中的同源重组(HDR)，细胞基因组CD33 exon2上的sgRNA结合位置附近，经Cas9切割后，会部分以ssDNA为修复模板进行修复，则SNP和3’ss位点就会从原有的C，AG被替换为T，CT。
(1)Sg4可有效突变SNP和3’ss位点
基于前期研究，SNP基因型为C的细胞可变剪切发生较少，是探索突变SNP以改变可变剪切较好的细胞模型。且NB4细胞在电转活率和效率上均优于THP1，KG1等SNP C型细胞。
因此，本实施例在NB4细胞上，针对SNP位点设计sgRNA及相应的ssDNA，其中包括sg4：SEQ ID NO：10及其ssDNA:SEQ ID NO：21，sg9：SEQ ID NO：14及其ssDNA:SEQ ID NO：23，sg12：SEQ ID NO：15及其ssDNA:SEQ ID NO：24。为了同时突变SNP和3’ss位点，另设计新的ssDNA与原sg4的组合(ssDNA:SEQ ID NO：22)，以及ssDNA与sg9的组合(ssDNA:SEQ ID NO.28)，并以文献报道的CD33-KO的sgRNA(SEQ ID NO：17)和ssDNA(SEQ ID NO：25)作为对照。通过RNP联合ssDNA电转入细胞的方式，突变其SNP位点和3’ss位点。
结果显示，sg4可同时高效诱导NB4细胞的CD33SNP突变和3’ss突变(图7)：
以仅转入cas9蛋白的组作为对照，其基因组上CD33 exon2上的SNP位点和3’ss位点均没有突变(每个测序位点均是单一峰图)。以文献报道的KO组作为阳性对照，CD33 exon2由于发生了T向TT的插入突变，CD33的转录翻译均发生移码突变，进而CD33被敲除。
在RNP复合物联合突变SNP位点的ssDNA电转组中，sg4可有效将SNP由C(蓝色峰)突变为T(红色峰)，sg12野生型C基因型(蓝色峰)和SNP突变后T基因型(红色峰)分别约占50％，而sg9中由C(蓝色峰)向T(红色峰)的最低。
在RNP复合物联合同时突变SNP位点和3’ss位点的ssDNA电转组中，sg4可高效将SNP 由C(蓝色峰)突变为T(红色峰)，同时将3’ss位点由AG突变为CT。Sg12由于PAM端距离3’ss比sg9更远，介导对3’ss的突变不如sg9，故本实施例中选择用sg9介导SNP和3’ss位点的同时突变。
结果显示，sg9联合SNP和3’ss位点同时突变的ssDNA，其编辑效率并不高，测序峰图中，SNP位点基本没有突变，3’ss位点显示AG野生型和CT突变型的套峰，突变效率不及20％。
(2)Sg4介导的SNP和3’ss突变可有效介导CD33 exon2的可变剪切
采用RT-qPCR和FACS的鉴定方法，检测SNP和3’ss突变对NB4 CD33 exon2表达的影响。
结果显示，SNP位点及3’ss突变可有效诱导NB4的CD33 exon2可变剪切：
RT-qPCR结果显示突变SNP位点(RNP-C)的CD33 E13/E2比值比未突变组高，同时突变SNP和3’ss位点(RNP-C+ag)的CD33 E13/E2最高，表明SNP突变和3’ss突变对CD33 exon2的可变剪切均有促进作用(图8A)。
流式抗体染色结果显示，SNP突变后的细胞有60％左右丧失结合P67.6抗体的能力，SNP和3’ss同时突变的NB4有80％左右不结合P67.6抗体。表明SNP和3’ss突变均能促进CD33 exon2的缺失，进而CD33 V结构域不表达，不具备结合P67.6抗体能力(图8B)。
然而，本实施例中，突变SNP和3’ss策略旨在不影响CD33总蛋白的表达，只希望突变若干碱基影响CD33 V结构域的表达，以逃避靶向CD33 V结构域的CAR-T的杀伤。结果显示突变后细胞与完全敲除(KO组)基本无差别的不结合CD33HIM-3-4抗体，突变后细胞不结合P67.6仍结合HIM3-4的只有10％上下，这部分细胞的基因型有待进一步鉴定。
实施例4 AATCC突变导致CD33 exon2缺失同时不影响C结构域
前期突变SNP位点或3’ss位点的策略虽然可引起CD33 V结构域不表达，但CD33 C结构域(HIM-3-4抗体结合能力)也受到了影响，因此发明者通过Tyto流式分选，将突变后不结合P67.6仍结合HIM3-4的NB4细胞分选出来(PE+)，并以不结合HIM3-4的细胞为对照(PE-)，如图9中I图所示，在分选后相对更单一的细胞群中鉴定其基因型。
结果显示，导致突变后细胞缺失CD33 exon2并不影响C结构域结合(4％PE+的细胞)的实际是AATCC突变(如图9中II图所示)：
在sg4介导的SNP突变NB4细胞中：
分选出4.85％的PE+细胞群在SNP位点主要表现为野生型的C基因型，反而在TTTCT位点表现为TTTCT向AATCC的突变；
PE-细胞在SNP位点存在C向T的突变，同时还存在大量indel，主要为Cas9蛋白切割导致的移码突变。
在sg4介导的SNP和3’ss位点同时突变的NB4细胞中：
分选出4％的PE+细胞群在SNP和3’ss为位点也主要表现为野生型基因型，同样在TTTCT 位点，存在TTTCT向AATCC的突变的套峰；PE-细胞在SNP和3’ss位点存在C向T和AG向CT的突变，同时也有移码突变产生的杂峰。
即无论是突变SNP还是同时突变SNP和3’ss位点，CD33 V结构域被破坏，C结构域不受影响的一小群细胞(10％左右)，实则为AATCC的突变。
实施例5靶向SNP和3’ss突变的sg4存在脱靶，改造后的sg4-14\sg4-15可极大降低脱靶的风险
基于前期靶向SNP和3’ss突变的策略中发现有AATCC突变的引入，发明者通过比对CD33与基因组上sgRNA可能脱靶位置的序列(同源序列)，检测是否可能存在内源修复的存在，即CD33 exon2位置经Cas9蛋白切割后断裂后，部分以CD33同源序列为模板进行修复，进而原有序列会被替换为同源序列。
(1)序列比对发现，AATCC突变的引入的确为内源修复。
人第19号染色体一段基因存在与CD33高度相似的序列(图10A)，AATCC突变的引入实则是经Cas9蛋白切割后断裂后，部分以该同源序列为模板进行修复，结果原有的TTTCT被突变为AATCC。且由于内源模板修复几率低于人为提供的ssDNA模板修复，故在前期的突变策略中，SNP和3’ss突变基因型占主体，而AATCC的突变只有发明者将该一小群细胞分选出来才得以用sanger测序检测到。
(2)比对靶向SNP和3’ss sgRNA的序列，以鉴定其脱靶的可能性。
结果显示，前期用于突变SNP和3’ss的sgRNA中，除了sg4在第18位碱基处与CD33相似序列有差异，其他sgRNA均与CD33的同源序列完全匹配，存在严重的脱靶风险。
本实施例中为了进一步验证sg4是否会错配至CD33同源序列导致脱靶，在NB4细胞中仅转入Cas9蛋白和sg4。并针对CD33区域和同源区域分别设计引物，将PCR产物测序，检测Cas9蛋白在CD33和同源区域的切割情况。
结果显示，CD33区域和同源区域均存在移码突变(图10B)：
在CD33区域：仅转入cas9蛋白的情况下，没有任何突变产生；转入Cas9蛋白和sg4的情况下，CD33 exon2区域出现大量移码。
在CD33同源区域：仅转入cas9蛋白的情况下，没有任何突变产生；转入Cas9蛋白和sg4的情况下，CD33同源区域也出现少量移码，证明sg4的确会部分脱靶，导致Cas9蛋白在CD33相似序列位置产生非特异切割。
(3)进一步地，替换sg4上的第14位(sg4-14：SEQ ID NO：11)或15位(sg4-15：SEQ ID NO：2)碱基，以降低sg4脱靶至CD33同源区域的可能性。
同样采用sgRNA与cas9形成RNP复合物，并提供SNP C向T突变的ssDNA，电转入NB4细胞，并对CD33和CD33同源序列PCR，sanger测序，鉴定改造后的sg4介导SNP突变的效率和脱靶可能性。
结果显示，sg4-14和sg4-15在CD33 exon2上均可有效介导SNP位点C向T的突变，突 变后的细胞exon2缺失，无法结合CD33 P67.6抗体：
Sg4-14和sg4-15介导CD33 exon2上的SNP位点表现为C和T的套峰，表明即使Sg4被替换了14或15位碱基，其仍可正常结合到CD33区域，并介导SNP位点的突变(图11A)。
在CD33同源区域，测序indel很低，表明改造后的Sg4极少脱靶至CD33的同源区域。
FACS结果显示(图11B)，sg4-14突变组有60％细胞不结合P67.6，不结合P67.6正常结合HIM3-4的细胞约20％；sg4-15突变组有50％细胞不结合P67.6，不结合P67.6正常结合HIM3-4的细胞约26％。
故虽然前期针对SNP位点和3'ss突变的策略，其附近的sgRNA无法避免脱靶问题，但对Sg4改造后的sg4-14，sg4-15仍可担任突变SNP位点，促进CD33 exon2可变剪切的角色。
实施例6 Sg3和sg10介导的AATCC突变可高效诱导NB4的CD33 exon2缺失
除了从上调可变剪切角度考虑，突变SNP位点或3’ss位点促进exon2跳跃表达外，AATCC突变则是发明者意外收获的有希望的突变位点。
此突变为自然存在，且前期结果显示，突变后的AATCC不仅可阻断CD33 V domain的抗体结合，且不影响CD33 C domain的结合，意味着AATCC的突变可逃避靶向CD33 V区的CD33-CAR-T或GO药物的杀伤，且该突变对CD33整个蛋白的表达影响较小，可最大程度不影响CD33蛋白的正常功能。
发明者针对TTTCT位点设计sgRNA(sg3:SEQ ID NO：18,sg7:SEQ ID NO：19,sg10:SEQ ID NO：20)，并提供含有AATCC突变的ssDNA(SEQ ID NO：26)作为sg3和sg7的修复模板，提供含有AATCC突变的ssDNA(SEQ ID NO：27)作为sg10的修复模板，电转NB4细胞后72小时，PCR出CD33 exon2区域，测序检测突变效率。
结果显示sg3和sg10可有效介导AATCC突变，突变后细胞不结合CD33-P67.6克隆抗体，CD33 C结构域的结合能力基本不受影响，仅表现为部分indel导致的敲除(图12A)：
对基因组sanger测序结果显示，在sg3或sg10介导的突变组中，CD33区域上的TTTCT被突变为AATCC，且在CD33同源区域，测序峰单一且与仅电转cas9组没有差异，表明sg3，sg10不会脱靶至CD33同源序列上产生非特异性切割。sg7在TTTCT位点几乎没有突变，但在CD33同源区域上产生了部分套峰，说明sg7会部分错配至CD33同源区域，产生非特异性切割。
FACS结果显示(图12B)，sg3突变后的细胞不结合P67.6正常结合HIM3-4的高达73.2％，sg10突变后的细胞不结合P67.6正常结合HIM3-4的达62.2％，而前期策略中sg4-14或sg4-15突变SNP，不结合P67.6正常结合HIM3-4只有20％-30％。
实施例7 sg10介导的AATCC突变更加耐受Anti-CD33-CAR-T的杀伤
尽管用改造后的sg4(sg4-14，sg4-15)可有效突变SNP或3'ss位点并导致CD33 exon2可变剪切，且极少脱靶至CD33同源区域，但其中丧失V区结合能力，不影响C区结合的只有 20％-30％。
而同样不脱靶的情况下，sg10可有效突变AATCC，突变后的细胞V区几乎丧失抗体结合能力，且C区不受影响的仍有60％-70％。故发明者选择AATCC突变，用于后期杀伤耐受实验和HSC的编辑分化。
为了进一步探究突变后细胞是否可耐受CD33-CAR-T的杀伤，发明者首先制备靶向CD33的CAR-T细胞，通过用含有CAR分子的慢病毒感染激活的T细胞，得到稳定表达CAR的CD33-CAR-T。感染后72小时流式检测CAR的转效。
FACS结果显示，CAR-T细胞中CAR阳性细胞占据66.3％(图13)。
发明者进一步设计AATCC(sg3，sg10)或SNP突变(sg4-14，sg4-5)后的NB4细胞与Anti-CD33-CAR-T的共孵育实验，CD33-CAR-T细胞中的CAR阳性细胞与NB4靶细胞的效靶比为1:30。
从CD33-CAR-T细胞和编辑后NB4细胞共孵育之时起，用流式检测CD3阳性细胞，代表CAR-T细胞；用CD3阴性细胞代表靶细胞。同时流式染色CD33 P67.6和HIM-3-4抗体，验证耐受CAR-T杀伤的NB4是否是突变的细胞群。
共孵育结果显示，sg10编辑后细胞可耐受CD33-CAR-T杀伤：
在共孵育起0小时检测CAR-T和编辑后NB4细胞的比例，CD3阴性细胞即为NB4细胞，CD3阳性细胞为CD33-CAR-T细胞。66.3％CAR阳性的CAR-T细胞与编辑后NB4细胞按照1：30效靶比计算，理论上NB4细胞占比95.2％。实际操作误差下，FACS结果显示每组NB4靶细胞初始比例在93.1％-95.2％之间，属于可接受波动(图14A)。
经共孵育96小时以后，仅电转cas9未编辑组细胞占总细胞数的比例从起始的95.2％逐渐被CD33-CAR-T杀死至2.87％，sg4-15编辑的NB4细胞被杀死至23.4％，sg4-14编辑的NB4细胞被杀死至57.4％，sg3编辑的NB4细胞被杀死至71.2％，而sg10编辑的NB4细胞仍有94.6％，高于CD33 KO的NB4细胞的90.7％(图14B)。
共孵育0小时起检测突变后靶细胞的CD33 V区和C区的抗体结合能力，结果显示在0小时时候，未突变组细胞的CD33 V区仍有67.5％细胞可被P67.6抗体识别，sg4-15编辑的NB4细胞有10.07％可被P67.6抗体识别，sg4-14编辑的NB4细胞有13.3％可结合P67.6抗体，sg3编辑的NB4细胞有4.11％可被P67.6抗体识别，sg10编辑的NB4细胞有0.84％可被P67.6抗体识别，CD33 KO的NB4细胞有4.49％可被P67.6抗体识别(图14C)。
96小时孵育后检测耐受CD33-CAR-T杀伤的突变靶细胞群，结果显示，能结合CD33 P67.6抗体的NB4细胞均被CD33-CAR-T靶向杀死，突变后仍具备正常C结构域抗体结合能力的细胞可耐受杀伤(图14D)。
通过检测共孵育后0小时，22小时，44小时，68小时，96小时突变后细胞的比例，发现随着CAR-T细胞的杀伤，sg10突变组细胞逃避CD33-CAR-T的杀伤，而sg3，sg4-14，sg4-15突变组细胞仍会激活CD33-CAR-T细胞，引起CAR-T细胞的杀伤和快速增殖，进而靶细胞被杀伤至死或靶细胞占整体比例不断下降(图14E)。
实施例8 Sg10介导的AATCC突变对HSC的体外定向分化没有影响
基于前期实验，本发明已在NB4 AML细胞中验证了RNP复合物联合ssDNA电转的方式可制造定点突变，其中sg10介导的AATCC突变可影响CD33 P67.6抗体的结合能力，且编辑后NB4细胞耐受Anti-CD33 CAR-T的杀伤。
因此，本实施例中进一步将该突变策略应用于造血干细胞HSC的编辑，检测RNP的方式能否有效诱导HSC的AATCC突变，并用流式检测突变后HSC的CD33 V区的抗体结合能力是否被破坏。
结果显示Cas9和sg10形成RNP复合物，并提供含AATCC突变的ssDNA模板，电转入HSC后，同样可在HSC中引起部分AATCC的突变(图15A)，且突变后细胞的CD33 V区抗体结合能力丧失(图15B)：
在TTTCT位点上，在仅电转入cas9蛋白的对照组中，没有sgRNA介导，故没有cas9蛋白的切割，TTTCT位点测序表现单一峰(图15A)；
在转入cas9蛋白和sg10，并提供AATCC修复模板的情况下，TTTCT位点上表现为TTTCT野生型和AATCC突变型的套峰，但突变后AATCC的峰值低于原有TTTCT，故在HSC细胞上的突变效率有待优化。
流式检测显示，未突变组细胞的CD33 V区具备正常的抗体结合能力(P67.6阳性)，C区仍可正常结合抗体(HIM3-4阳性)；突变后细胞C区不受影响(HIM3-4阳性)，V区的抗体结合能力被破化(P67.6阴性)，表明突变后细胞V区结构改变，可逃避靶向V区的CD33 CAR-T的杀伤，且其C区不受影响(图15B)。
为了进一步探究编辑后的HSC是否具备正常分化能力，发明者在细胞水平评估了HSC体外分化为单核细胞的能力。通过加入诱导HSC体外髓系单核分化的培养基，在分化第1，5，9，14天分别检测CD14阳性细胞的比例。
结果显示Sg10编辑组HSC具备正常的体外髓系单核细胞分化能力：
用CD14-PE检测未突变组(橙色)和AATCC突变的HSC细胞(紫色)后第14天的单核细胞，结果显示突变组细胞和未突变组细胞在体外单核分化培养基的作用下均可正常分化至单核细胞(图15C)。
记录分化第1、5、9、14天CD14阳性细胞的比例，结果显示突变后细胞(红色)的分化趋势与未编辑组(黑色)基本一致，且在分化终点第14天，突变后HSC向CD14单核细胞分化的比例甚至高于未编辑组的CD14单核分化(图15D)。
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，均落在本发明的保护范围和公开范围之内。

Claims (45)

  1. 细胞，其具有细胞表面蛋白，其中所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述细胞能够抗CAR-T或抗体类药物杀伤。
  2. 如权利要求1所述的细胞，其中所述细胞为造血干细胞或其分化细胞，如淋巴系祖细胞、髓系祖细胞、淋巴系细胞或髓系细胞。
  3. 如权利要求1或2所述的细胞，其中所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
  4. 如权利要求1-3任一项所述的细胞，其中所述细胞的抗原表位改变是由于所述细胞表面蛋白的编码基因中存在单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失引起。
  5. 如权利要求1-4任一项所述的细胞，其中所述细胞表面蛋白在表达时存在外显子缺失。
  6. 如权利要求1-5任一项所述的细胞，其中所述细胞表面蛋白为CD33、CD19或BCMA。
  7. 如权利要求1-6任一项所述的细胞，其中所述细胞表面蛋白为CD33，其缺失CD33基因中第二外显子编码的氨基酸片段。
  8. 如权利要求1-7任一项所述的细胞，其中所述细胞表面蛋白为CD33，所述细胞的CD33基因中存在如下特征之一或它们的任意组合：
    1)rs12459419位点为核苷酸T；
    2)第二外显子序列之前的3’ss位点为AA或CT；以及
    3)第二外显子内的TTTCT替换为AATCC。
  9. 权利要求1-8的细胞与CAR-T或抗体类药物联合在制备抗肿瘤药物中的用途。
  10. 如权利要求9所述的用途，其中所述CAR-T或抗体类药物能够靶向肿瘤细胞的细胞表面蛋白而杀伤所述肿瘤细胞，所述细胞表达的所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述细胞能够抗所述CAR-T或抗体类药物杀伤。
  11. 如权利要求9或10所述的用途，所述肿瘤为血液肿瘤，优选AML。
  12. 制备细胞的方法，其中所述细胞的细胞表面蛋白具有能够抗CAR-T或抗体类药物杀伤的抗原表位改变，所述方法包括对所述细胞表面蛋白的编码基因进行基因编辑。
  13. 如权利要求12所述的方法，其中所述基因编辑包括向所述编码基因中引入单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失。
  14. 如权利要求12或13所述的方法，其中所述基因编辑通过向所述细胞引入CRISPR基因编辑系统进行。
  15. 如权利要求12-14任一项所述的方法，其中所述细胞为造血干细胞或其分化细胞，如淋巴系祖细胞、髓系祖细胞、淋巴系细胞或髓系细胞。
  16. 如权利要求12-15任一项所述的方法，其中所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
  17. 如权利要求12-16任一项所述的方法，其中所述细胞表面蛋白在表达时存在外显子 缺失。
  18. 如权利要求12-17任一项所述的方法，其中所述细胞表面蛋白为CD33、CD19或BCMA。
  19. 如权利要求12-18任一项所述的方法，其中所述细胞表面蛋白为CD33，所述基因编辑包括如下任一种或其任意组合：
    1)将rs12459419位点核苷酸C突变为T；
    2)将第二外显子序列之前的3’ss位点核苷酸AG突变为AA或CT；以及
    3)将第二外显子内的核苷酸TTTCT突变为AATCC。
  20. 如权利要求12-19任一项所述的方法，其中将rs12459419位点核苷酸C突变为T的crispr基因编辑系统包括靶序列为SEQ ID NO：10、11、12、14或15的sgRNA。
  21. 如权利要求12-20任一项所述的方法，其中将rs12459419位点核苷酸C突变为核苷酸T的crispr基因编辑系统包括靶序列为SEQ ID NO：10、11或12的sgRNA和包含SEQ ID NO：21的ssDNA；靶序列为SEQ ID NO：14的sgRNA和包含SEQ ID NO：23的ssDNA；或靶序列为SEQ ID NO：15的sgRNA和包含SEQ ID NO：24的ssDNA。
  22. 如权利要求12-21任一项所述的方法，其中将第二外显子序列之前的3’ss位点核苷酸AG突变为AA的crispr基因编辑系统包括靶序列为SEQ ID NO：13或16的sgRNA。
  23. 如权利要求12-22任一项所述的方法，其中将第二外显子序列之前的3’ss位点核苷酸AG突变为AA的crispr基因编辑系统包括靶序列为SEQ ID NO：13或16的sgRNA以及单碱基胞嘧啶编辑器，所述单碱基胞嘧啶编辑器优选A3A-CBE3。
  24. 如权利要求12-23任一项所述的方法，其中同时将rs12459419位点核苷酸C突变为T和将第二外显子序列之前的3’ss位点核苷酸AG突变为CT的crispr基因编辑系统包括靶序列为SEQ ID NO：10或14的sgRNA。
  25. 如权利要求12-24任一项所述的方法，其中同时将rs12459419位点核苷酸C突变为T和将第二外显子序列之前的3’ss位点核苷酸AG突变为CT的crispr基因编辑系统包括靶序列为SEQ ID NO：10的sgRNA和包含SEQ ID NO：22的ssDNA、或靶序列为SEQ ID NO：14的sgRNA和包含SEQ ID NO：27的ssDNA。
  26. 如权利要求12-25任一项所述的方法，其中将第二外显子内的核苷酸TTTCT突变为AATCC的crispr基因编辑系统包括靶序列包含SEQ ID NO：18或20的sgRNA。
  27. 如权利要求12-26任一项所述的方法，其中将第二外显子内的核苷酸TTTCT突变为AATCC的crispr基因编辑系统包括靶序列包含SEQ ID NO：18或20的sgRNA和包含SEQ ID NO：26的ssDNA。
  28. 在受试者中清除或减少肿瘤细胞的方法，包括以能够杀伤所述肿瘤细胞的CAR-T或抗体类药物向所述受试者给药以及向所述受试者补充正常细胞，其中所述CAR-T或抗体类药物靶向所述肿瘤细胞的细胞表面蛋白，所述正常细胞表达的所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述正常细胞能够抗所述CAR-T或抗体类药物杀 伤。
  29. 如权利要求28所述的方法，其中所述肿瘤细胞为血液肿瘤细胞，所述正常细胞为血液细胞或能够分化为血液细胞的造血干细胞或祖细胞。
  30. 如权利要求28或29所述的方法，其中所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
  31. 如权利要求28-30任一项所述的方法，其中所述正常细胞的抗原表位改变是由于所述细胞表面蛋白的编码基因中存在单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失引起。
  32. 如权利要求28-31任一项所述的方法，其中所述正常细胞的所述细胞表面蛋白在表达时存在外显子缺失。
  33. 如权利要求28-32任一项所述的方法，其中所述细胞表面蛋白为CD33、CD19或BCMA。
  34. 如权利要求28-33任一项所述的方法，其中所述细胞表面蛋白为CD33，其缺失CD33基因中第二外显子编码的氨基酸片段。
  35. 如权利要求28-34任一项所述的方法，其中所述细胞表面蛋白为CD33，其编码基因中存在如下特征之一或它们的任意组合：
    1)rs12459419位点为核苷酸T；
    2)第二外显子序列之前的3’ss位点为AA或CT；以及
    3)第二外显子内的TTTCT替换为AATCC。
  36. 如权利要求28-35任一项所述的方法，其中所述肿瘤细胞为AML细胞。
  37. 药物组合物或药物组合，包括CAR-T或抗体类药物和正常细胞，其中所述CAR-T或抗体类药物能够靶向肿瘤细胞的细胞表面蛋白而杀伤所述肿瘤细胞，所述正常细胞表达的所述细胞表面蛋白经改造而具有抗原表位改变，所述抗原表位改变使得所述正常细胞能够抗所述CAR-T或抗体类药物杀伤。
  38. 如权利要求37所述的药物组合物或药物组合，其中所述肿瘤细胞为血液肿瘤细胞，所述正常细胞为血液细胞或能够分化为血液细胞的造血干细胞或祖细胞。
  39. 如权利要求37或38所述的药物组合物或药物组合，其中所述细胞表面蛋白为淋巴系细胞特异性细胞表面蛋白或髓系细胞特异性细胞表面蛋白。
  40. 如权利要求37-39任一项所述的药物组合物或药物组合，其中所述正常细胞的抗原表位改变是由于所述细胞表面蛋白的编码核苷酸序列中存在单核苷酸突变、剪接位点突变和/或核苷酸片段替换、插入和/或缺失引起。
  41. 如权利要求37-40任一项所述的药物组合物或药物组合，其中所述正常细胞的所述细胞表面蛋白在表达时存在外显子缺失。
  42. 如权利要求37-41任一项所述的药物组合物或药物组合，其中所述细胞表面蛋白为CD33、CD19或BCMA。
  43. 如权利要求37-42任一项所述的药物组合物或药物组合，其中所述细胞表面蛋白为CD33，其缺失CD33基因中第二外显子编码的氨基酸片段。
  44. 如权利要求37-42任一项所述的药物组合物或药物组合，其中所述细胞表面蛋白为CD33，其编码基因中存在如下特征之一或它们的任意组合：
    1)rs12459419位点为核苷酸T；
    2)第二外显子序列之前的3’ss位点为AA或CT；以及
    3)第二外显子内的TTTCT替换为AATCC。
  45. 如权利要求37-43任一项所述的药物组合物或药物组合，其中所述肿瘤细胞为AML细胞。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116516007A (zh) * 2023-04-18 2023-08-01 华中科技大学同济医学院附属协和医院 Cd19表达的检测方法
CN116850210A (zh) * 2023-01-17 2023-10-10 武汉大学 用于治疗急性髓系白血病的成分和方法
CN116850210B (zh) * 2023-01-17 2024-05-31 武汉大学 用于治疗急性髓系白血病的成分和方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108290939A (zh) * 2015-10-16 2018-07-17 纽约市哥伦比亚大学理事会 用于抑制谱系特异性抗原的组合物和方法
CN108463227A (zh) * 2015-11-04 2018-08-28 宾夕法尼亚大学董事会 在造血干细胞中基因编辑的方法和组合物
CN110662554A (zh) * 2017-02-28 2020-01-07 Vor生物制药股份有限公司 用于抑制谱系特异性蛋白质的组合物和方法
WO2020150478A1 (en) * 2019-01-16 2020-07-23 The Trustees Of Columbia University In The City Of New York Compositions and methods for inhibition of lineage specific antigens
WO2021062227A2 (en) * 2019-09-27 2021-04-01 Beam Therapeutics Inc. Compositions and methods for treatment of liquid cancers

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JOP20190116A1 (ar) * 2018-05-24 2019-11-24 Janssen Biotech Inc الأجسام المضادة لتكتل التمايز 33 (cd33)، والأجسام المضادة ثنائية النوعية لتكتل التمايز 33 (cd33)/تكتل التمايز 3 (cd3) واستخداماتها

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108290939A (zh) * 2015-10-16 2018-07-17 纽约市哥伦比亚大学理事会 用于抑制谱系特异性抗原的组合物和方法
CN108463227A (zh) * 2015-11-04 2018-08-28 宾夕法尼亚大学董事会 在造血干细胞中基因编辑的方法和组合物
CN110662554A (zh) * 2017-02-28 2020-01-07 Vor生物制药股份有限公司 用于抑制谱系特异性蛋白质的组合物和方法
WO2020150478A1 (en) * 2019-01-16 2020-07-23 The Trustees Of Columbia University In The City Of New York Compositions and methods for inhibition of lineage specific antigens
WO2021062227A2 (en) * 2019-09-27 2021-04-01 Beam Therapeutics Inc. Compositions and methods for treatment of liquid cancers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MIRIAM Y. KIM, KYUNG-ROK YU, SAAD S. KENDERIAN, MARCO RUELLA, SHIRLEY CHEN, TAE-HOON SHIN, AISHA A. ALJANAHI, DANIEL SCHREEDER, MI: "Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia", CELL, vol. 173, no. 6, 1 May 2018 (2018-05-01), Amsterdam NL , pages 1439 - 1453.e19, XP055568383, ISSN: 0092-8674, DOI: 10.1016/j.cell.2018.05.013 *
TAO ZHONGFEI; WANG MIN; WANG JIANXIANG: "Advances in Immunotherapy of Acute Myeloid Leukemia by Using Chimeric Antigen Receptor Modified T Cells", CHINESE JOURNAL OF HEMATOLOGY, vol. 37, no. 2, 14 February 2016 (2016-02-14), CN , pages 160 - 163, XP009540774, ISSN: 0253-2727, DOI: 10.3760/cma.j.issn.0253-2727.2016.02.017 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116850210A (zh) * 2023-01-17 2023-10-10 武汉大学 用于治疗急性髓系白血病的成分和方法
CN116850210B (zh) * 2023-01-17 2024-05-31 武汉大学 用于治疗急性髓系白血病的成分和方法
CN116516007A (zh) * 2023-04-18 2023-08-01 华中科技大学同济医学院附属协和医院 Cd19表达的检测方法
CN116516007B (zh) * 2023-04-18 2024-01-19 华中科技大学同济医学院附属协和医院 Cd19表达的检测方法

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