Composition and method for inhibiting lineage-specific antigen

Abstract

To provide a cell population to be used in a method for treating hematopoietic organ malignant tumor.SOLUTION: Provided is a cell population to be used in a method for treating a hematopoietic organ malignant tumor, where the method includes administering the cell population to a subject who needs the method, the cell population includes a hematopoietic cell genetically engineered so as to lack CD33 or have a CD33 expression level lower than a CD33 expression level in a naturally generating hematopoietic cell, and the hematopoietic cell is selected from the group consisting of a hematopoietic stem cell, a precursor cell, a bone marrow precursor cell, a lymphoid precursor cell, a bone marrow cell, a lymphoid cell and a combination thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Cross-references to related cases This application is a continuation of U.S. Provisional Patent Application No. 62 / 242,685, filed October 16, 2015. Priority is claimed under USC § 119, which is incorporated herein by reference in its entirety. can be. [Background technology]

[0002] Despite decades of attempts, curative immunotherapy for cancer remains extremely elusive. It is difficult and fundamentally based on either antibodies or T cells (via the T cell receptor). This is the antigen recognition ability of the immune system (Cousin-Frankel, Science (2013) 342:1432). Antibody-based immunotherapy is a method to target cancer by targeting antigens on tumor cells. When upregulated compared to normal cells (e.g., Her-2 in proliferating breast cancers), r-2), or the tumor cells express an antigen that is recognized by an antibody or an antibody-toxin conjugate. In cases where therapies are used (e.g., rituximab against CD20), they are widely used (Base Iga et al., Annals Oncology (2001) 12:S35). Antibody-based Clinical trials using targeted immunotherapy have shown improved patient outcomes in a limited number of cancer types. Although there have been some indications of efficacy (usually when combined with standard chemotherapy), these effects are often limited. have serious safety and efficacy issues (Cousin-Frankel Cancer , Science (2013) 342:1432).

[0003] Effective T cell therapy for cancer has been even more difficult to achieve clinically (Sch Mitt et al., Hum. Gene Ther. (2009) 20(11):1240. Cancer Effective T cell therapy for cancer requires T cells with high affinity binding to antigens on cancer cells. Chimeric antigen receptor T cells (CAR T cells) are widely used and are HLA antigens, which recognize and "present" peptides with high affinity and specificity. No auxiliary recognition molecule is required. The T cell receptor of CAR T cells binds antigen-binding heavy chains and The light chain is "swapped" and thus eliminates the need for HLA accessory molecules. The T receptor mediates T cell activation by binding of the CAR T receptor to the target antigen. It is fused to a signal region.

[0004] Clinical use of CAR T cells is limited to targeting a narrow range of cell surface antigens This further supports the need for new and improved methods in the treatment of cancer. The method has been shown to improve outcomes in elderly patients who cannot undergo intensive chemotherapy, the current standard of care. , acute myeloid leukemia (AML), which remains very poor with a median survival of only 5-10 months. (Dohner et al., NEJM (2015) 373:113 6).

[0005] A novel approach to cancer immunotherapy that targets specific classes of lineage-specific cell surface antigens on tumor cells Disclosed herein are methods for the treatment of CAR T cell therapies that target lineage-specific cell surface antigens. Combined with replacement of non-tumor cells by injecting or reinjecting the missing engineered cell population Tumor recurrence is prevented by maintaining patient surveillance in vivo with CAR T cells. The signal is stopped or reduced. Summary of the Invention

[0006] The present disclosure provides agents comprising antigen-binding fragments that bind lineage-specific cell surface antigens ( For example, immune cells expressing chimeric receptors targeting CD33 ( Selectively induces cell death of cells expressing the antigen, while inhibiting cell death of cells lacking the antigen (e.g. The discovery that genetically engineered hematopoietic cells can escape the resulting cell death has led to Based on these findings, we aimed to target lineage-specific cell surface antigens. Targeting agents, e.g., CD33-targeting CAR-T cells and lineage-specific cell antigens Immunotherapy involving the combination of hematopoietic cells lacking a marker (e.g., CD33) has been shown to be effective in treating hematopoietic malignancies. It is hoped that this will lead to an effective treatment for tumors.

[0007] One aspect of the present disclosure provides a method for treating hematopoietic malignancies, the method comprising: The present invention relates to a method for treating a subject in need of such treatment, the method comprising administering to said subject (i) an effective dose of an agent that targets a lineage-specific cell surface antigen. an amount of the agent that binds the lineage-specific cell surface antigen, and (ii) a population of hematopoietic cells lacking a lineage-specific cell surface antigen. In some embodiments, the agent is an antigen-binding fragment that binds a lineage-specific cell surface antigen. The target cell may be an immune cell (e.g., a T cell) that expresses a chimeric receptor that includes the target segment. In some embodiments, the immune cells, hematopoietic cells, or both, are allogeneic or autologous. In some embodiments, the hematopoietic cells are hematopoietic stem cells (e.g., CD34 + / CD33 - In some embodiments, the hematopoietic stem cells are bone marrow cells or peripheral blood mononuclear cells. It can be obtained from PBMCs.

[0008] In some embodiments, the antigen-binding fragment is a type 2 lineage-specific cell surface antigen (e.g., In some embodiments, the antibody binds a lineage-specific cell surface antigen, such as CD33. The original binding fragment is a lineage-specific type 1 cell surface antigen (e.g., CD19). It binds target cell surface antigens.

[0009] In some embodiments, the antigen-binding fragment in the chimeric receptor is human CD33. or specifically binds lineage-specific cell surface antigens, which may be human proteins such as CD19 In some embodiments, the scFv is a single chain antibody fragment (scFv) that binds to the human and binds to human CD33 and has the same complementarity determining regions (CDRs) as those in SEQ ID NO:12. ), and a heavy chain variable region having the same CDRs as in SEQ ID NO:13. In one example, the scFv comprises a light chain variable region having the amino acid sequence of SEQ ID NO:12. A heavy chain variable region having the sequence of SEQ ID NO:13 and a light chain having the amino acid sequence of SEQ ID NO:14. Contains a variable region.

[0010] The chimeric receptor further comprises: (a) a hinge region; (b) a transmembrane region; and (c) at least one (d) a cytoplasmic signaling domain, or (e) a combination thereof. In some embodiments, the chimeric receptor comprises at least one co-stimulatory signaling domain. These include CD27, CD28, 4-1BB, OX40, CD30, CD40, P D-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIG Co-stimulation of HT, NKG2C, B7-H3, GITR, HVEM, or a combination of these In some embodiments, at least one co-stimulatory signal may be derived from a co-stimulatory receptor. The signaling domain is a high-expressing domain containing the signaling domain of CD28 and the signaling domain of ICOS. In one example, at least one costimulatory signaling domain is a hybrid costimulatory domain. The chimeric receptor is derived from CD28 and is a second co-stimulatory signaling molecule derived from 4-1BB or ICOS. It further includes a loop transmission region.

[0011] In some embodiments, the chimeric receptor comprises a cytoplasmic signaling domain that is derived from CD3ζ. In some embodiments, the chimeric receptor is derived from CD8α or CD28α. In some embodiments, the chimeric receptor antibody comprises a CD8, CD2 8, or contains a transmembrane domain derived from ICOS.

[0012] In some embodiments, the chimeric receptor comprises, from the N-terminus to the C-terminus, (i) a lineage-specific cell scFv that binds to surface antigens (e.g., CD33 or CD19), hinge derived from CD8α domain, CD8-derived transmembrane domain, 4-1BB-derived costimulatory domain, and CD3ζ-derived cytoplasmic synthase domain. (ii) a lineage-specific cell surface antigen (e.g., CD33 or CD19). scFv that binds to CD8α, CD8α-derived hinge region, CD28-derived transmembrane region, CD28-derived costimulatory domain, and CD3ζ-derived cytoplasmic signaling domain, or (iii) lineage-specific scFv that binds to cell surface antigens (e.g., CD33 or CD19), CD8α-derived human Fv CD28-derived transmembrane region, CD28-derived first costimulatory region, 4-1BB-derived second It contains a costimulatory domain, and a CD3ζ-derived cytoplasmic signaling domain.

[0013] In any of the methods described herein, the subject is diagnosed with Hodgkin's lymphoma, non-Hodgkin's lymphoma, In some examples, the subject may have acute myeloma, leukemia, or multiple myeloma. myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia have leukemia.

[0014] In another aspect, the disclosure provides a nucleic acid encoding any of the chimeric receptors described herein. The chimeric receptor is an antigen-binding domain that binds CD33. The CD3ζ-derived cytoplasmic signaling domain, which is a fragment of the CD3 gene, An antigen-binding fragment (e.g., an scFv fragment) may comprise a null transfer region. A heavy chain variable region having the same CDRs as those in SEQ ID NO:12, and SEQ The light chain variable region has the same CDRs as those in ID NO: 13. In one embodiment, the scFv comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:12, and a light chain variable region having the amino acid sequence of SEQ ID NO:13.

[0015] Another aspect of the disclosure provides a vector comprising any of the nucleic acids presented herein. Chimeric receptors encoded by the nucleic acids described herein and such chimeric receptors Immune cells (e.g., T cells) expressing the receptor are also within the scope of the present disclosure. In embodiments, the immune cells may be obtained from a patient with a hematopoietic malignancy.

[0016] Another aspect of the present disclosure is the detection of lineage-specific cell surface antigens that are present on hematopoietic cells prior to genetic engineering. Genetically engineered hematopoietic cells (e.g., H In some embodiments, endogenous SCs encoding lineage-specific cell surface antigens are provided. All or part of a sex gene can be modified, for example, by genome editing (e.g., zinc finger nucleic acid insertion). ZFN, Transcription Activator-Like Effector-Based Nuclease (TALEN), or a CRISPR-Cas system. In some embodiments, the system The lineage-specific cell surface antigen is CD33, and the immunoglobulin constant (IgC) domain of CD33 In some embodiments, the hematopoietic cells are hematopoietic stem cells (e.g., CD3 4 + / CD33 - cells).

[0017] In some embodiments, the hematopoietic stem cells are derived from bone marrow cells or peripheral blood mononuclear cells (PBMCs). can be obtained from.

[0018] Methods for producing cells lacking the lineage-specific cell surface antigens described herein are also described herein. The method includes providing a cell, and in the cell, (i) a lineage-specific cell expression vector. Hybridization occurs to a portion of the nucleotide sequence encoding a surface antigen (e.g., CD33, CD19). Contains the nucleotide sequence of the soybean CRISPR-Cas guide RNA (gRNA) a nucleic acid, and (ii) a Cas endonuclease (e.g., Cas9 or Cpf1). A nucleic acid comprising a nucleotide sequence of a CRISPR-Cas system gRNA and The Cas nuclease and the Cas nuclease can be, for example, encoded by the same nucleic acid or different nucleic acids, or can be introduced into cells as preformed ribonucleoprotein complexes. In an embodiment, the portion of the nucleotide sequence to which the gRNA hybridizes is 18 to 22 amino acids. In some instances, the gRNA is comprised of SEQ ID NO:11 or Contains the nucleotide sequence of NO:28 to 31.

[0019] In some embodiments, the cells are hematopoietic stem cells (e.g., CD34 + ) and other hematopoietic cells. be.

[0020] (i) Antigens that target and bind lineage-specific cell surface antigens (ii) an immune cell comprising an agent comprising a binding fragment, and Also within the scope of the disclosure are kits that include a population of hematopoietic cells (e.g., hematopoietic stem cells) that lack a source. In some embodiments, agents that target lineage-specific cell surface antigens are These immune cells express antigen-binding flags that bind lineage-specific cell surface antigens. Includes ment.

[0021] Additionally, the present disclosure provides methods for targeting lineage-specific surface antigens for use in treating hematopoietic malignancies. and / or hematopoietic cells lacking lineage-specific cell surface antigens. The present invention relates to a pharmaceutical composition comprising either one of the above-mentioned compounds, and a pharmaceutical agent for use in treating hematopoietic malignancies. The present invention also provides uses of said immune and hematopoietic cells for the treatment of cancer.

[0022] The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the disclosure This will be apparent from the detailed description of several embodiments and the appended claims.

[0023] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure: It will be understood that the present invention is not limited to the disclosed embodiments, and may be modified in any manner without departing from the spirit and scope of the present invention. Please refer to the above for better understanding. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows an exemplary illustration of type 0, type 1, type 2, and type 3 lineage specific antigens. [Diagram 2] FIG. 13 Schematic representation of immune cells expressing chimeric receptors targeting the type 0 lineage-specific cell surface antigen, CD307. Multiple myeloma (MM) cells expressing CD307, as well as other cells expressing CD307, such as plasma cells, are targeted by immune cells expressing anti-CD307 chimeric receptors. [Diagram 3] Schematic representation of immune cells expressing chimeric receptors targeting the type 2 lineage-specific cell surface antigen, CD33. Acute myeloid leukemia (AML) cells express CD33. Human hematopoietic stem cells (HSCs) are genetically engineered to lack CD33 and therefore are not recognized by immune cells expressing anti-CD33 chimeric receptors. HSCs can expand myeloid cells. [Figure 4] Schematic of genome editing using the CRISPR / Cas system. The sgRNA hybridizes to a portion of an exon of a lineage-specific cell surface antigen, and the Cas9 endonuclease cleaves upstream of the protospacer adjacent motif (PAM) sequence (5'-NGG-3'). The sequences, from top to bottom, correspond to SEQ ID NOs: 45 and 46. [Diagram 5] Schematic diagram of genome editing method using CRISPR / Cas9 system to disrupt CD33. PX458 vector encoding Cas9 protein and guide RNA targeting CD33 were transfected into K-562 cells, a human leukemia cell line. Flow cytometry was performed on cell populations using anti-CD33 antibody before (top) and after (bottom) delivery of Cas9 and guide RNA to cells. Genome editing resulted in deletion of the coding region of the gene and significant reduction of CD33 from the cell surface. [Figure 6]Figure 1 shows a schematic of a genome editing method using the CRISPR / Cas9 system to disrupt CD45RA. The PX458 vector encoding the Cas9 protein and a guide RNA targeting CD45RA were nuclear-transfected into TIB-67 reticulum cell sarcoma mouse macrophage-like cells. Flow cytometry was performed on cell populations using an anti-CD45RA antibody before (top) and after (bottom) delivery of Cas9 and guide RNA into the cells. Genome editing resulted in the deletion of the coding region of the gene and a significant reduction of CD45RA from the cell surface. [Figure 7] 1 shows an overview of an exemplary chimeric receptor comprising an antigen-binding fragment targeting CD33. A: A comprehensive chimeric receptor targeting CD33, comprising an anti-CD33scFv, a hinge region, a transmembrane region, a costimulatory region, and a signaling region. B: A chimeric receptor targeting CD33, comprising an anti-CD33scFv, a hinge region derived from CD8, a transmembrane region derived from CD8, and an intracellular region derived from CD28 and CD3ζ. C: A chimeric receptor targeting CD33, comprising an anti-CD33scFv, a hinge region derived from CD8, a transmembrane region derived from CD8, and an intracellular region derived from ICOS (or CD27, 4-1BB, or OX-40) and CD3ζ. D: A chimeric receptor targeting CD33, comprising an anti-CD33scFv, a hinge region derived from CD8, a transmembrane region derived from CD8, and an intracellular region derived from OX-40, CD28, and CD3ζ. [Figure 8] FIG. 1 is a schematic representation of immunotoxins. [Figure 9] Figure 1: Expression of anti-CD33 chimeric receptors expressed in K562 cells transduced with empty vector or vector encoding anti-CD33 chimeric receptor. A: Western blot with a primary antibody recognizing CD3ζ. The table shows the estimated molecular weight of each of the chimeric receptors tested. B: Flow cytometry analysis showing an increased population of cells staining positively with anti-CD33 chimeric receptor. [Figure 10]Anti-CD33 chimeric receptor binding to CD33. A: Ponceau stained protein gel. Lanes 1, 3, 5: CD33 molecules. Lanes 2, 4, 6: CD33 molecules + APC conjugate. B: Western blot with a primary antibody that recognizes CD3ζ. Lanes 1, 3, and 5 contain chimeric receptor co-incubated with CD33 molecules, and lanes 2, 4, and 6 contain chimeric receptor co-incubated with CD33-APC conjugate. C: Flow cytometry analysis showing an increase in the population of cells expressing anti-CD33 chimeric receptor and binding CD33. [Figure 11] Cytotoxicity of K562 cells by NK92 cells expressing the indicated chimeric receptors. A: CART1 and CART2 compared to empty HIVzsG vector. B: CART3 compared to empty HIVzsG vector. [Figure 12] Cytotoxicity of K562 cells lacking CD33 by NK92 cells expressing the indicated chimeric receptors (shown as percent cytotoxicity on the y-axis). A: Unsorted population of K562 cells pretreated with CD33-targeting CRISPR / Cas reagents. B: Single clones of K562 cells lacking CD33. Bars represent, from left to right, empty HIVzsG vector, CART1, CART2, and CART3. [Figure 13] Flow cytometric analysis of primary T cell populations. A: Cell sorting based on expression of T cell markers C4+, CD8+, or both CD4+CD8+. B: Relative expression of CD33 in the indicated populations of primary T cells. [Figure 14] Cytotoxicity of K562 cells by primary T cells expressing the indicated chimeric receptors. A: CD4+ T cells, B: CD4+ / CD8+ (CD4 / 8) and CD8+ (CD8). [Figure 15] FIG. 1 shows flow cytometry analysis of CD33 editing in K652 cells using the CRISPR / Cas9 system and two different gRNAs (Crispr3, top right panel, and Crispr5, bottom right panel). [Figure 16]Figure 1 shows that K562 cells lacking CD33 exhibit normal cell proliferation and erythropoietic differentiation. A: Flow cytometric analysis of the indicated cell populations + 50 μM hemin on day 1. B: Flow cytometric analysis of the indicated cell populations on day 9. C: MTT cell proliferation assay. [Figure 17] Flow cytometric analysis of CD33 editing in human CD34+ cells using the CRISPR / Cas9 system and two different gRNAs (crispr3, bottom left, and crispr5, bottom right). A: Flow cytometric analysis of CD33 editing in human CD34+ cells using the CRISPR / Cas9 system. B: crispr3. C: crispr5. [Figure 18] Figure 1 shows colony formation of human CD34+ / CD33- cells compared to human CD34+ / CD33+. Bars (left to right) represent uninfected lentivirus, empty vector control, crispr1, crispr3, and crispr5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Cancer immunotherapy, which targets antigens present on the cell surface of cancer cells, has been shown to improve the development and / or survival of patients. Target antigens are also present on the surface of normal non-cancer cells that are essential or highly involved in the survival of the tumor. Targeting these antigens is particularly challenging when the targeting site is a single target of such cells in addition to the cancer cells. The cytotoxic effects of immunotherapy on the cells may result in adverse effects in the subject. .

[0026] The methods, nucleic acids, and cells described herein can be used to inhibit not only cancer cells, but also the development and progression of a subject. and / or target antigens that are also present on cells critical for survival (e.g., type 1 or type 2 antigens) The method enables: (1) counting cells bearing a target lineage-specific cell surface antigen; (2) reducing the amount of antigen presenting the antigen using an agent that targets the antigen; Therefore, lineage-specific expression of normal cells (e.g., non-cancerous cells) that can be killed by administration of the agent is The methods described herein involve replacing hematopoietic cells lacking a target cell surface antigen with hematopoietic cells lacking a target cell surface antigen. and maintaining surveillance of target cells, including cancer cells, that express lineage-specific cell surface antigens. A non-cancerous cell population expressing a lineage-specific antigen that may be important for the development and / or survival of a subject. You can maintain the group.

[0027] Therefore, lineage-specific markers such as CD33 or CD19 may be useful for treating hematopoietic malignancies. Immune cells expressing chimeric receptors containing antigen-binding fragments that target cell surface antigens - Patents.com , and the co-use of hematopoietic cells, such as hematopoietic stem cells (HSCs), that lack lineage-specific cell surface antigens. Also described herein are chimeric receptors, nucleic acids encoding same, and vectors containing same. and immune cells (e.g., T cells) expressing such chimeric receptors are described herein. The present disclosure also provides genetically engineered strains lacking lineage-specific antigens as described herein. The present invention provides genetically engineered hematopoietic cells, as well as methods for producing the same (e.g., genome editing methods).

[0028] definition The terms "subject," "individual," and "patient" are used interchangeably and refer to a vertebrate, preferably a Mammals include, but are not limited to, human primates, non-human primates, and the like. In the context of this disclosure, the term " A "subject" also refers to a tissue that can be cultured in vitro or ex vivo, or that can be manipulated in vivo. The term "subject" may be used interchangeably with the term "organism."

[0029] The terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and The terms "oligonucleotide" and "oligonucleotide" are used interchangeably. Ribonucleotides or ribonucleotides, or their analogues, Examples of polynucleotides include, but are not limited to, genes or gene fragments. The coding or non-coding regions of the fragment, exons, introns, messenger RNA ( mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), Short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA A. Recombinant polynucleotides, branched polynucleotides, plasmids, vectors, any sequence Sequences of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers One or more nucleotides within the polynucleotide may be further modified. The sequence of a polynucleotide may be interrupted by non-nucleotide components. The molecules may be modified after polymerization, such as by conjugation with a labeling agent.

[0030] The term "hybridization" refers to the reaction of one or more polynucleotides to form a nucleic acid. This refers to a reaction that forms a complex stabilized by hydrogen bonds between the bases of the oxidase residues. Binding may occur via Watson-Crick base pairing, Hoogsteen binding, or any other conjugation. The complex can be generated in a sequence-specific manner. The complex consists of two strands that form a double-stranded structure, Three or more strands forming a multistranded complex, single self-hybridization The hybridization step may comprise a hybridization step, ... The reaction may then proceed to more extensive reactions, such as initiating PCR or cleaving a polynucleotide with an enzyme. A sequence capable of hybridizing to a given sequence may constitute a step in a process. It is called the "complement" of the sequence.

[0031] The term "recombinant expression vector" refers to a construct that expresses an mRNA, protein, polypeptide, or comprises a nucleotide sequence encoding a peptide, and the vector is expressed in a cell and cells under conditions sufficient to produce RNA, proteins, polypeptides, or peptides. When contacted, the mRNA, protein, polypeptide, or peptide is produced by the host cell. Genetically modified oligonucleotide or polynucleotide constructs that enable expression of the peptide The vectors of the present disclosure are generally not naturally occurring. The non-naturally occurring recombinant expression vectors of the present disclosure can be any of the nucleotide sequences. The types of nucleic acids may include, but are not limited to, DNA and RNA, which may be single stranded or may be double-stranded, may be synthetic or may be derived in part from natural sources, and may be naturally occurring, non-naturally occurring, or may contain mutated nucleotides.

[0032] As used herein, "transfection," "transformation," or "transduction" The incorporation of one or more exogenous polynucleotides into a host cell by physical or chemical means This refers to the introduction of.

[0033] "Antibody", "Fragment of an antibody", "Antibody fragment", "Functional fragment of an antibody" The terms "antigen-binding moiety" and "antigen-binding portion" are used interchangeably and refer to a molecule capable of specifically binding to a particular antigen. (Holliger et al. , Nat. Biotech. (2005) 23(9):1126). This antibody is an antibody and Antibody fragments may be Fab, F(ab'), s cFv, disulfide-linked Fv, Fc, or variants and / or mixtures. The antibodies can be chimeric, humanized, single chain, or bispecific. All antibody isotypes are encompassed by the present disclosure, including IgA, IgD, IgE, IgG, and IgM. The relevant IgG subtypes include IgG1, IgG2, IgG3, and IgG4. The chain or heavy chain variable region is determined by three hypervariable regions called complementarity determining regions (CDRs). The CDRs of the antibody or antigen-binding portion thereof are comprised of a framework interrupted by a non-human or of human origin. The antibodies may be human, humanized, or non-human (e.g., antibodies modified to reduce antigenicity in humans). The framework may be a Zumi framework, or a synthetic framework (e.g., a consensus sequence).

[0034] The antibody or antigen-binding portion is about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 M not yet Full, about 10 -10 Less than M, about 10 -11 Less than M or about 10 -12 Dissociation constant less than M ( K D The affinity of the antibodies of the present disclosure can be readily measured using conventional techniques. (Scatchard et al., Ann. NY Acad. Sci. (1949) 5 1:660, and U.S. Patent Nos. 5,283,173, 5,468,614, or (equivalent literature).

[0035] The term "chimeric receptor," "chimeric antigen receptor," or alternatively, "CAR" refers to a chimeric antigen receptor that is a Used interchangeably throughout the body, they refer to at least the extracellular antigen binding domain, the transmembrane domain, and the subdomain. A cytoplasmic signaling domain containing a functional signaling domain derived from a stimulatory molecule as defined above ( A recombinant polypeptide construct comprising an intracellular signaling domain (also referred to herein as an "intracellular signaling domain"). Lee et al., Clin. Cancer Res. (2012) 18(10):278 0, Jensen et al., Immunol Rev. (2014) 257(1):127, w www.cancer.gov / about-cancer / treatment / res In one embodiment, the stimulatory molecule is a T cell receptor complex. In one embodiment, the cytoplasmic signaling region is a zeta chain associated with fusion. and further comprising one or more functional signaling domains from at least one costimulatory molecule that is Costimulatory molecules also include 4-1BB (e.g., CD137), CD27, and / or The CAR can be CD28, or a fragment of these molecules. A cellular antigen-binding domain that contains an extracellular antigen recognition domain, a transmembrane domain, and a functional signal transduction domain derived from a stimulatory molecule. The CAR comprises a chimeric fusion protein that includes an intracellular signaling region. domains, transmembrane domains, and functional signal transduction domains derived from costimulatory molecules and stimulatory molecule-derived domains A chimeric fusion protein comprising an intracellular signaling domain that includes a functional signaling domain of the present invention. Alternatively, the CAR may comprise an extracellular antigen recognition region, a transmembrane region, and one or more Two functional signaling domains derived from costimulatory molecules and one functional signaling domain derived from stimulatory molecules The CAR also includes a chimeric fusion protein comprising an intracellular signaling region comprising a An extracellular antigen recognition domain, a transmembrane domain, and at least two domains derived from one or more costimulatory molecules. and a functional signaling domain from a stimulatory molecule. The antigen recognition portion of the CAR, which is encoded by a nucleic acid sequence, can be any The antibody fragment may comprise a lineage-specific antigen-binding antibody fragment. CDR, variable region (or a portion thereof), constant region (or a portion thereof), or any of the above It may include any combination.

[0036] The term "signal transduction region" refers to a molecule that transmits information within a cell by acting as a second messenger. by producing effects or by responding to such messengers. They act as regulators to regulate cellular activity through defined signaling pathways. It refers to a functional portion of a protein that is used for

[0037] The term "zeta" or alternatively "zeta chain", "CD3-zeta", or "TC "R-zeta" is available under GenBank accession numbers NP_932170, NP_000725, and or XP_011508447, or a protein from a non-human species, e.g., mouse The zeta stimulatory domain is defined as the equivalent residues from rodents, monkeys, and other animals. "CD3-zeta stimulatory region" or alternatively "TCR-zeta stimulatory region" The "region" is a region that is suitable for functionally transmitting the early signals required for T cell activation. The amino acid residues are defined as those derived from the cytoplasmic domain of the data chain.

[0038] The terms "genetically engineered" or "genetically modified" refer to organisms that have been produced by genetic engineering techniques. This refers to cells that have been engineered, for example by genome editing, to Heterologous sequences that do not occur naturally in the vector system or ribosomes. The heterologous nucleic acid molecule may be introduced into the cell via other means for introducing the nucleic acid molecule into the cell. The term may be integrated into the genome or may exist extrachromosomally, such as in the form of a plasmid. Also included are embodiments in which an engineered and isolated CAR polypeptide is introduced into a cell. nothing.

[0039] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the same individual.

[0040] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into whom the material is introduced. Two or more individuals are said to be homozygous when the genes at one or more loci are not identical. It is said to be a system.

[0041] The term "cell lineage" refers to specific identifiable cells that have a common ancestor and are derived from the same type of identifiable cell. As used herein, cell lineage refers to cells that have developed into competent / functional cells. However, it is not known whether it is a cancer, but it is also known as a cancer of the respiratory tract, prostate, breast, kidney, intestine, nervous system, skeleton, blood vessels, liver, hematopoietic system, muscle, or contains cardiac cell lineages.

[0042] The term "inhibition," when used in reference to gene expression or function of a lineage-specific antigen, Inhibition refers to a decrease in the amount or function of gene expression of a lineage-specific antigen, where inhibition is the Inhibition can be complete, resulting in no detectable expression or function. The inhibition may be partial, absent or absent. Partial inhibition is almost complete inhibition. By eliminating specific target cells, AR T cells can effectively inhibit the overall expression of a particular cell lineage.

[0043] Cells such as hematopoietic cells that are "lacking lineage-specific antigens" lack their naturally occurring counterparts, e.g. For example, they have significantly reduced expression of lineage-specific antigens compared to endogenous hematopoietic cells of the same type. or cells that do not express lineage-specific antigens, i.e., by routine measurements such as FACS. In some instances, the lineage-specific antigen of an "antigen-depleted" cell is not detectable by the The expression level of the same lineage-specific antigen is less than about 40% of that of its naturally occurring counterpart (e.g. , 30%, 20%, 15%, 10%, 5%, or lower). As used herein, the term "about" refers to ±5% of a particular value. For example, an expression level of about 40% is , the expression level may be anywhere between 35% and 45%.

[0044] Agents targeting lineage-specific cell surface antigens Aspects of the present disclosure include agents that target lineage-specific cell surface antigens, e.g., on target cancer cells. Such agents provide antigen binding and targeting of lineage-specific cell surface antigens. In some instances, the antigen-binding fragment may comprise a lineage-specific antigen. The antibody may be a single chain antibody (scFv) that specifically binds to the antigen.

[0045] A. Lineage-specific cell surface antigens As used herein, the terms "lineage-specific cell surface antigen" and "cell surface lineage-specific "Heterologous antigen" may be used interchangeably and refers to a specific antigen that is present on the surface of a cell and is specific to one or more types of cell lineage. The term refers to any antigen that is associated with a population. For example, an antigen may be present in one or more populations of cell lineages. and may be absent (or in reduced amounts) on the cell surface of other cell populations.

[0046] In general, lineage-specific cell surface antigens are characterized by the antigen and / or the cell population that presents the antigen. Based on many factors, such as whether it is required for the survival and / or development of the host organism, A summary of exemplary types of lineage-specific antigens is provided in Table 1 below. See also FIG. do. [Table 1]

[0047] As shown in Table 1 and Figure 1, type 0 lineage-specific cell surface antigens play important roles in tissue homeostasis and survival. and cell types bearing type 0 lineage-specific cell surface antigens are also required for the survival of the subject. Therefore, the role of type 0 lineage-specific cell surface antigens in homeostasis and survival, or Given the importance of cells bearing type 0 or 1 lineage-specific cell surface antigens, Since inhibition or elimination of cells bearing such antigens may be detrimental to the survival of the subject, Targeting this category of antigens has been difficult using conventional CAR T cell immunotherapy. As a result, lineage-specific cell surface antigens (such as type 0 lineage-specific antigens) and / or A cell bearing such an antigen may be, for example, a cell that performs a non-redundant function of life in a subject. For this purpose, lineage-specific antigens may be required for survival in patients with CAR T cells. They may be poor targets for cell-based immunotherapy.

[0048] In contrast to type 0 antigens, type 1 cell surface lineage-specific antigens and type 1 cell surface lineage-specific antigens are present. The cells that express the type 1 cell surface lineage are not required for tissue homeostasis or survival of the subject. Targeting a specific antigen is unlikely to have adverse consequences for the subject. For example, It is a type 1 antigen that is specifically expressed on both normal plasma cells and multiple myeloma (MM) cells. CAR T cells engineered to target CD307, which mediates the elimination of both cell types, (Figure 2) (Elkins et al., Mol Cancer Ther. 10:2222 ( 2012). However, because the plasma cell lineage may be sacrificed for the survival of the organism, CD 307 and other type 1 lineage-specific antigens are suitable for CAR T cell-based immunotherapy The type 1 class of lineage-specific antigens are expressed in the ovary, testis, prostate, breast, and uterus. It can be expressed in a wide variety of different tissues, including the membrane, and pancreas. The agents target cell surface lineage-specific antigens that are type 1 antigens.

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

[0050] A wide variety of antigens can be targeted by the methods and compositions of the present disclosure. Monoclonal antibodies against the target antigen can be purchased commercially or, as described above, can be prepared by incubating the antibody with a suitable antibody. Standard monoclonal antibody methodologies following immunization of animals with Standard somatic cell hybrids from Milstein, Nature (1975) 256:495 The antibody or antibodies may be produced using standard methods, including immunoprecipitation techniques. The nucleic acid to be read can be sequenced using any standard DNA or protein sequencing technique. The sequences can be sequenced.

[0051] In some embodiments, the cells are targeted using the methods and cells described herein. Cell surface lineage-specific antigens are cell surface lineage-specific antigens on leukocytes or subpopulations of leukocytes. In some embodiments, the cell surface lineage specific antigen is an antigen associated with a myeloid cell. In some embodiments, the cell surface lineage-specific antigen is a cluster of differentiation (CD). Examples of D antigens include, but are not limited to, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, C D8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD1 1d, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32a, CD32 b, CD32c, CD33, CD34, CD35, CD36, CD37, CD38, CD 39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD4 3, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、C D49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55 、CD56、CD57、CD58、CD59、CD60a、CD61、CD62E、CD 62L、CD62P、CD63、CD64a、CD65、CD65s、CD66a、CD 66b、CD66c、CD66F、CD68、CD69、CD70、CD71、CD72 、CD73、CD74、CD75、CD75S、CD77、CD79a、CD79b、C D80、CD81、CD82、CD83、CD84、CD85A、CD85C、CD85 D、CD85E、CD85F、CD85G、CD85H、CD85I、CD85J、CD 85K、CD86、CD87、CD88、CD89、CD90、CD91、CD92、C D93、CD94、CD95、CD96、CD97、CD98、CD99、CD99R、 CD100、CD101、CD102、CD103、CD104、CD105、CD10 6、CD107a、CD107b、CD108、CD109、CD110、CD111、 CD112、CD113、CD114、CD115、CD116、CD117、CD11 8、CD119、CD120a、CD120b、CD121a、CD121b、CD12 1a、CD121b、CD122、CD123、CD124、CD125、CD126、 CD127、CD129、CD130、CD131、CD132、CD133、CD13 4、CD135、CD136、CD137、CD138、CD139、CD140a、C D140b、CD141、CD142、CD143、CD144、CDw145、CD1 46、CD147、CD148、CD150、CD152、CD152、CD153、C D154、CD155、CD156a、CD156b、CD156c、CD157、CD 158b1、CD158b2、CD158d、CD158e1 / e2、CD158f、C D158g、CD158h、CD158i、CD158j、CD158k、CD159a 、CD159c、CD160、CD161、CD163、CD164、CD165、CD 166、CD167a、CD168、CD169、CD170、CD171、CD172 a、CD172b、CD172g、CD173、CD174、CD175、CD175s 、CD176、CD177、CD178、CD179a、CD179b、CD180、C D181、CD182、CD183、CD184、CD185、CD186、CD191 、CD192、CD193、CD194、CD195、CD196、CD197、CDw 198、CDw199、CD200、CD201、CD202b、CD203c、CD2 04、CD205、CD206、CD207、CD208、CD209、CD210a、 CDw210b、CD212、CD213a1、CD213a2、CD215、CD21 7、CD218a、CD218b、CD220、CD221、CD222、CD223、 CD224、CD225、CD226、CD227、CD228、CD229、CD23 0、CD231、CD232、CD233、CD234、CD235a、CD235b、 CD236、CD236R、CD238、CD239、CD240、CD241、CD2 42、CD243、CD244、CD245、CD246、CD247、CD248、C D249、CD252、CD253、CD254、CD256、CD257、CD258 , CD261, CD262, CD263, CD264, CD265, CD266, CD2 67, CD268, CD269, CD270, CD271, CD272, CD273, C D274, CD275, CD276, CD277, CD278, CD279, CD280 , CD281, CD282, CD283, CD284, CD286, CD288, CD2 89, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, C D301, CD302, CD303, CD304, CD305, 306, CD307a, CD307b, CD307c, D307d, CD307e, CD309, CD312, C D314, CD315, CD316, CD317, CD318, CD319, CD320 , CD321, CD322, CD324, CD325, CD326, CD327, CD3 28, CD329, CD331, CD332, CD333, CD334, CD335, C D336, CD337, CD338, CD339, CD340, CD344, CD349 , CD350, CD351, CD352, CD353, CD354, CD355, CD3 57, CD358, CD359, CD360, CD361, CD362, and CD36 3. www.bdbiosciences.com / documents / BD_ See Reagents_CDMarkerHuman_Poster.pdf.

[0052] In some embodiments, the cell surface lineage specific antigen is CD19, CD20, CD11, CD123, CD56, CD34, CD14, CD33, CD66b, CD41, CD6 1, CD62, CD235a, CD146, CD326, LMP2, CD22, CD52 , CD10, CD3 / TCR, CD79 / BCR, and CD26. In embodiments, the cell surface lineage specific antigen is CD33 or CD19.

[0053] Alternatively, or in addition, the cell surface lineage-specific antigen may be a cancer antigen, e.g., a cell surface antigen that is differentially expressed on cancer cells. In some embodiments, the cancer antigen may be a cell surface lineage-specific antigen that is present next to the cancer antigen. These are tissue or cell lineage specific antigens. Cell surface lineage specific antigens are associated with certain types of cancer. Examples of target antigens include, but are not limited to, CD20, CD22 (non-Hodgkin's lymphoma, B-cell lymphoma) and CD52 (B-cell CLL), CD33 (acute osteomyelitis, chronic lymphocytic leukemia (CLL)), Myeloid leukemia (AML), CD10 (gp100) (common (precursor B cell) acute lymphoblastic leukemia hematologic malignancies and malignant myeloma), CD3 / T cell receptor (TCR) (T cell lymphoma and leukemia disease), CD79 / B cell receptor (BCR) (B cell lymphoma and leukemia), CD26 ( epithelial and lymphoid malignancies), human lymphocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (lymphoid malignancies), RCAS1 (gynecologic cancers, bile duct adenocarcinoma, and pancreatic In some embodiments, the cell surface antigen (CTA) is a cytoplasmic antigen (CTA) that is a cytoplasmic antigen. The surface antigen CD33 is associated with AML cells.

[0054] B. Antigen-binding fragments Any of the antibodies or antigen-binding fragments thereof can be administered to the lineage-specific cells described herein. Such antibodies or antigens can be used to construct agents that target surface antigens. Binding fragments can be prepared by conventional methods, for example, hybridoma or recombinant techniques. It can be manufactured.

[0055] For example, antibodies specific for lineage-specific antigens of interest can be generated by conventional hybridoma technology. can be made using lineage-specific antigens that can be linked to a carrier protein such as KLH. A host animal can then be immunized to produce antibodies that bind to the complex. The vaccination route or schedule may be selected based on the efficacy and safety of the antibody stimulation and production, as further described herein. The methods generally follow established conventional techniques for the production of murine, humanized, and human antibodies. General techniques for production are known in the art and are described herein. Any mammalian subject or antibody-producing cells derived therefrom may be used as a hiPSC for the treatment of mammalian hiPSCs, including humans. It is contemplated that these may be engineered to serve as a basis for the production of brinoma cell lines. The host animal is then injected intraperitoneally, intramuscularly, with an amount of an immunogen, including those described herein. Inoculation may be by intramuscular, oral, subcutaneous, intraplantar, and / or intradermal route.

[0056] Hybridomas are Kohler, B. and Milstein, C. (1975) Nature 256:495-497, or Buck, DW et al., In Vitr A general somatic cell hybridization assay improved by They can be prepared from lymphocytes and immortalized myeloma cells using immunohistochemistry. are available from, but not limited to, X63-Ag8.653 and San Diego, California, USA. Salk Institute, Cell Distribution Center, Hybridization Generally, the technique involves the use of a fusogen such as polyethylene glycol, or The myeloma cells and lymphocytes are fused by electrical methods well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and incubated with hypoxanthine-amine. The cells are grown in a selective growth medium such as heptamer-thymidine (HAT) medium and hybridized. The parent cells that have not been cultured are removed. Either of these media is used to culture hybridomas secreting monoclonal antibodies. As an alternative to cell fusion techniques, EBV-immortalized B cells can be used to The hybridomas can be expanded and used to produce the TCR-like monoclonal antibodies described herein. Subcloning is performed as necessary, and the supernatant is subjected to conventional immunoassay methods (e.g., radioimmunoassay). Anti-immunogen activity was measured by immunoassay, enzyme immunoassay, or fluorescent immunoassay. and measure.

[0057] Hybridomas that can be used as a source of antibodies are monoclonal antibodies capable of binding to lineage-specific antigens. The term "hybridoma" includes all derivatives that are descendants of the parent hybridoma that produces the cloned antibody. Hybridomas producing such antibodies can be cultured in vitro or in vivo using known methods. Monoclonal antibodies can be grown, if desired, by ammonium sulfate precipitation, gel electrophoresis, or the like. Traditional immunoglobulin purification methods such as electrophoresis, dialysis, chromatography, and ultrafiltration It can be separated from the culture medium or body fluid by methods. If unwanted activity is present, e.g. The preparation is then passed over an adsorbent consisting of the immunogen attached to a solid phase to release the desired antibodies from the immunogen. It can be removed by elution or release. Proteins that are prophylactic, such as keyhole limpet hemocyanin, serum albumin, Bovine thyroglobulin or soybean trypsin inhibitor, bifunctional or derivative Maleimidobenzoyl sulfosuccinimide ester (which converts cysteine ​​residues to conjugation via lysine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde aldehyde, succinic anhydride, SOCl, or R1N=C=NR where R and R1 are independent The target antigen or fragment containing the target amino acid sequence conjugated with the alkyl group Immunization of host cells with the antigen produces a population of antibodies (e.g., monoclonal antibodies). can be done.

[0058] Optionally, the antibody of interest (e.g., produced by a hybridoma) may be sequenced. The polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and The mouse cells can then be expanded and frozen for future use. The sequences may be used to "humanize" an antibody or to improve the affinity (affinity maturation) or other properties of the antibody. For example, antibodies may be used in genetic engineering to improve clinical trials or in humans. When used in therapeutic intervention, the constant regions may be engineered to more closely resemble human constant regions to enhance immune response. The antibody sequence can be engineered to achieve high affinity for lineage-specific antigens. It may be desirable to make one or more polynucleotide alterations to the antibody and to detect the target. It will be apparent to one skilled in the art that the antibody can still maintain its binding specificity for the target antigen. Wax.

[0059] In other embodiments, fully human antibodies can be produced using recombinant human antibodies that express specific human immunoglobulin proteins. This can be achieved by using commercially available mice that have been engineered to Human antibodies) or transgenic mice engineered to generate more robust immune responses. The techniques may also be used to produce humanized or human antibodies. Examples of such techniques Xenomouse ( Registered trademark of Medarex, Inc. (Princeton, New Jersey) uMAb-Mouse® and TC Mouse®. Alternatively, antibodies can be produced by phage display or recombinantly by yeast technology. See, for example, U.S. Patent Nos. 5,565,332, 5,580,717, and 5,733 ,743, and 6,265,150, and Winter et al., (1994) A See, e.g., nnu. Rev. Immunol. 12:433-455. Display technology (McCafferty et al., (1990) Nature 348:55 2-553) was used to analyze immunoglobulin variable (V) region gene replicas from non-immunized donors. It is possible to produce human antibodies and antibody fragments in vitro from the truncated human antibodies.

[0060] Antigen-binding fragments of intact antibodies are prepared by standard methods. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules. Fab can be produced by reducing the disulfide bonds of F(ab')2 fragments. can occur.

[0061] Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies, and bispecific antibodies The antibodies can be produced, for example, by conventional recombinant techniques. DNA encoding suitable monoclonal antibodies can be readily isolated and sequenced using conventional techniques. sequence (e.g., specific for genes encoding the heavy and light chains of a monoclonal antibody) By using oligonucleotide probes that can specifically bind to hybridoma cells. Cells serve as a preferred source of such DNA. Once isolated, the DNA is The vector can then be placed into an expression vector of the same or similar type, which can then be transformed into E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or other forms of immunoglobulin protein Transfected into host cells, such as myeloma cells that do not produce Obtaining synthesis of monoclonal antibodies in cells. See, e.g., PCT Publication No. WO87 / 0446 2. The DNA is then modified, for example, to include human heavy and light chains in place of the homologous murine sequences. By replacing the sequences encoding the nucleotide constant regions of the ribozyme, Morrison et al. (1984) ) Proc. Nat. Acad. Sci. 81:6851, or immunoglobulin code Covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the sequence In this way, "chimeric" or "hybrid" antibodies, etc. can be produced. Engineered antibodies can be generated that have the binding specificity of a target antigen.

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

[0063] Methods for constructing humanized antibodies are well known in the art. et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033( In one example, the VH and VL variable regions of a parent non-human antibody can be synthesized using the following: The resulting product is then subjected to three-dimensional molecular modeling analysis by methods known in the art. Framework amino acid residues predicted to be important for the formation of the structure were modeled on the same molecule. In parallel, human antibodies having amino acid sequences that are homologous to the parent non-human antibody are identified. HV and HL chains were determined from any antibody genetic material using the parent VH and VL sequences as search queries. From the child database, human VH and VL acceptor genes are then selected. .

[0064] The CDR regions in the selected human acceptor gene are then cloned into the CDR regions from the parent non-human antibody. or a functional variant thereof. Residues in the framework regions of the parent chain that are predicted to be important were used to identify the human acceptor -Can be substituted for the corresponding residue in the gene.

[0065] Single chain antibodies can be produced by recombinantly synthesizing nucleotide sequences encoding the heavy chain variable region and the light chain variable region. It can be prepared by linking nucleotide sequences encoding the chain variable regions. In the case of single chain antibodies, a flexible linker is incorporated between the two variable regions. The techniques reported for the production of glycerol (U.S. Pat. Nos. 4,946,778 and 4,704 ,692) can be adapted to generate phage or yeast scFv libraries, Lineage-specific antigen-specific scFv clones were isolated from the library using routine procedures. Positive clones are subjected to further screening to determine whether they bind lineage-specific antigens. Can identify things.

[0066] In some instances, the lineage specific antigen of interest is CD33 and the antigen binding fragment is Specific binding of CD33, e.g., human CD33. Exemplary weight groups of anti-human CD33 antibodies The amino acid and nucleic acid sequences of the light and low chain variable regions are shown below. These are shown in bold and underlined in the amino acid sequence. [ka] [ka] [ka] [ka]

[0067] Anti-CD3 antibodies for use in constructing the CD33-targeting agents described herein 3 antibody binding fragments are SEQ ID NO:12 and SEQ ID NO:13 Such antibodies may contain the same heavy and / or light chain CDRs as those of one or more of the frames. In some instances, the anti-CD33 antibody flagellar region may include an amino acid residue mutation. The nucleotide sequence of the nucleotide sequence of the present invention is at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95% or more) of the heavy chain variable region and / or has at least 70% sequence identity with SEQ ID NO:13 (e.g., 75%, 80%, 85%, 90%, 95% or more) of the same light chain variable region It may include a region.

[0068] The "percent identity" of two amino acid sequences is determined by the formula: ul,Proc.Natl.Acad.Sci.USA87:2264-68,1990 Improved Karlin and Altschul Proc. Natl.A Determined using the algorithm in cad.Sci.USA90:5873-77,1993. Such an algorithm is described in Altschul et al., J. Mol. Biol. 215 :403-10, 1990, NBLAST and XBLAST programs (version 2 BLAST protein searches are integrated into the XBLAST program, The experiment was performed with a word length of 3 and a word length of 50 to find amino acid sequences that are homologous to the protein molecules of the present disclosure. If there are gaps between the two sequences, a gapped BLAST search can be performed. Altschul et al., Nucleic Acids Res. 25(17):3389- 3402, 1997. BLAST and Gapped B When using the LAST program, each program (e.g., XBLAST and NBL AST) can be used as default parameters.

[0069] C. Immune cells expressing chimeric receptors In some embodiments, the methods described herein target lineage-specific cell surface antigens. The agent is an antigen-binding fragment capable of binding to a lineage-specific antigen (e.g., CD33 or CD19). Chimeric receptors that contain fragments (e.g., single chain antibodies) are immune cells that express chimeric receptors. Target cells bearing lineage-specific antigens on their cell surface ( Recognition of a target cell (e.g., a cancer cell) is mediated by a signaling domain (e.g., a costimulatory signaling domain) of the chimeric receptor. Transduction domain and / or cytoplasmic signaling domain) which transmits activation signals to the kinase domain. It can activate effector function in immune cells that express the mela receptor.

[0070] As used herein, a chimeric receptor is capable of being expressed on the surface of a host cell and is a receptor that is capable of being expressed on the surface of a host cell. It refers to a non-naturally occurring molecule that contains an antigen-binding fragment that binds to a surface lineage-specific antigen. Generally, a chimeric receptor contains at least two regions that are derived from different molecules. In addition to the antigen-binding fragments described in, the chimeric receptors contain a hinge region, a transmembrane region, and a small may further comprise at least one costimulatory domain and one or more cytoplasmic signaling domains. In some embodiments, the chimeric receptor comprises, from the N-terminus to the C-terminus, a cell surface lineage-specific antigen. The antigen-binding fragment that binds to the antigen, the hinge region, the transmembrane region, and the cytoplasmic signal transduction domain are In some embodiments, the chimeric receptor comprises at least one costimulatory domain. Further includes:

[0071] In some embodiments, the chimeric receptor described herein comprises a hinge region, It may be located between the antigen-binding fragment and the transmembrane domain. The hinge region is generally It is found between two regions of a protein and depends on the flexibility of the protein and the phase of one or both of the regions. Such flexibility and antigen binding to separate regions of the chimeric receptor may allow for relative movement. Any amino acid sequence that results in the movement of the ligated fragment can be used.

[0072] The hinge region is about 10 to 200 amino acids, e.g., 15 to 150 amino acids, 20 It may contain from 100 amino acids, or from 30 to 60 amino acids. So, the hinge region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 4 0, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, or 20 There may be 0 amino acids.

[0073] In some embodiments, the hinge region is a hinge region of a naturally occurring protein. The hinge region of any protein known in the art to contain a hinge region is not limited to the present invention. The chimeric receptors are suitable for use in the chimeric receptors described herein. The hinge region is at least a portion of the hinge region of the naturally occurring protein and provides flexibility to the chimeric receptor. In some embodiments, the hinge region is that of CD8α or CD28α. In some embodiments, the hinge region is a portion of the hinge region of CD8α, e.g. For example, at least 15 (e.g., 20, 25) amino acids of the hinge region of CD8α or CD28α. , 30, 35, or 40 consecutive amino acids.

[0074] The hinge region of an antibody, such as an IgG, IgA, IgM, IgE, or IgD antibody, may also be Suitable for use in the chimeric receptors described herein. In some embodiments, The hinge region is the region that connects the antibody constant regions CH1 and CH2. In one embodiment, the hinge region is that of an antibody, and the hinge region of the antibody and one or more of the antibody's In some embodiments, the hinge region comprises a hinge region of an antibody and a C region of an antibody. In some embodiments, the hinge region comprises the hinge region of an antibody and an anti-H3 constant region. In some embodiments, the antibody comprises an IgG, an Ig, or a combination of the CH2 and CH3 constant regions. In some embodiments, the antibody is an IgG, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgA antibody. In some embodiments, the hinge region is an IgG1 antibody. In some embodiments, the hinge region comprises an Ig It contains the hinge region and CH3 constant region of the G1 antibody.

[0075] Chimeric receptors that contain hinge regions that are non-naturally occurring peptides are also within the scope of this disclosure. In some embodiments, the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain of the Fc receptor The hinge region between the where x and n are independently 3 to 1, including 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. It may be an integer of 2 or greater.

[0076] Additional peptide phospholipids that may be used in the hinge region of the chimeric receptors described herein. Carr is known in the art. Wriggers et al., Current Trends in Peptide Science(2005)80(6):736-746, and PCT Publication WO2012 / 088461.

[0077] In some embodiments, the chimeric receptors described herein may include a transmembrane domain. The transmembrane domain for use in the chimeric receptor may be any of the transmembrane domains known in the art. As used herein, a "transmembrane domain" refers to a domain that translocates through a cell membrane, preferably the cytoplasm. It refers to any protein structure that is thermodynamically stable in the nuclear cell membrane. Transmembrane regions suitable for use in melanoma receptors can be obtained from naturally occurring proteins. Alternatively, the transmembrane region can be a synthetic, non-naturally occurring protein segment, e.g. It may be a hydrophobic protein segment that is thermodynamically stable in the membrane.

[0078] The transmembrane domain determines the structure of the membrane, including the number of passes the transmembrane domain makes across the membrane and the orientation of the protein. They are classified based on the topology of their transmembrane domain. For example, single-pass membrane proteins cross the cell membrane once. Multipassage membrane proteins are proteins that pass through the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, In some embodiments, the transmembrane region has a single traverse. In some embodiments, the transmembrane domain is a transmembrane domain that is located at the N-terminus of the chimeric receptor. A single-pass transmembrane domain on the extracellular side of the vesicle, with the C-terminus of the chimeric receptor facing the intracellular side of the cell In some embodiments, the transmembrane region is derived from a single-pass transmembrane protein. In some embodiments, the transmembrane domain is that of CD8α. In some embodiments, the transmembrane domain is that of CD28. In some embodiments, the transmembrane domain is that of ICO It belongs to S.

[0079] In some embodiments, the chimeric receptors described herein are capable of binding to one or more co-stimulatory signals. The term "costimulatory signaling region" as used herein includes a co-stimulatory signaling region. , a protein that mediates intracellular signaling and induces immune responses, including effector functions The costimulatory signaling region of the chimeric receptor described herein refers to at least a portion of the The co-stimulatory protein may be a cytoplasmic signaling domain from a co-stimulatory protein, which transmits a signal. These are then transported to the brain by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. Regulates responses mediated by

[0080] In some embodiments, the chimeric receptor comprises more than one (at least two, three, four, In some embodiments, the co-stimulatory signaling region comprises one or more co-stimulatory signaling regions. Mela receptors express more than one costimulatory signaling domain derived from different costimulatory proteins In some embodiments, the chimeric receptor does not include a costimulatory signaling region.

[0081] In general, many immune cells are involved in the regulation of immune function, such as promoting cell proliferation, differentiation and survival, as well as in the regulation of immune function. In addition to stimulating with antigen-specific signals, co-stimulation is required to activate the effector functions of cells. Activation of costimulatory signaling regions in host cells (e.g., immune cells) The study showed that cells produce and secrete cytokines, have macrophage characteristics, proliferation, differentiation, survival, and The addition of any co-stimulatory protein may increase or decrease the cytotoxicity and / or cytotoxicity of the target protein. The costimulatory signaling region may be adapted for use in the chimeric receptors described herein. The type of costimulatory signaling domain will depend on the type of immune cell on which the chimeric receptor is expressed ( e.g., primary T cells, T cell lines, NK cell lines) and desired immune effector functions (e.g., The covalent linker for use in the chimeric receptor is selected based on factors such as the ability to bind to the covalent linker and the ability to bind to the covalent linker. An example of a stimulatory signaling domain can be the cytoplasmic signaling domain of a costimulatory protein. , including but not limited to CD27, CD28, 4-1BB, OX40, CD30, Cd40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LI GHT, NKG2C, B7-H3. In some embodiments, the costimulatory region is 4-1 In some embodiments, the costimulatory region is derived from CBB, CD28, or ICOS. D28, and the chimeric receptor contains a second costimulatory domain derived from 4-1BB or ICOS. nothing.

[0082] In some embodiments, the costimulatory region comprises more than one costimulatory region or more than one co-stimulatory region. In some embodiments, the costimulatory domain is a fusion domain that includes a portion of a stimulatory domain. and a fusion of the costimulatory domain from ICOS.

[0083] In some embodiments, the chimeric receptors described herein comprise a cytoplasmic signaling domain. Any cytoplasmic signaling region can be used in the chimeric receptors described herein. Generally, the cytoplasmic signaling domain is connected to the extracellular ligand-binding domain and its ligand. Transmitting signals such as interactions to induce cellular effector functions (e.g., cytotoxicity) Stimulates cellular responses such as

[0084] As will be appreciated by those skilled in the art, factors involved in T cell activation include cytoplasmic signal transduction. The phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the target region. Any known ITAM-containing region can be used to construct the chimeric receptors described herein. Generally, ITAM motifs consist of amino acid sequences separated by 6–8 amino acids. It contains two repeats of the string YxxL / I, where each x is independently any amino acid, and The motif YxxL / Ix(6-8)YxxL / I may occur. In some embodiments, The cytoplasmic signaling domain is derived from CD3ζ.

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

[0086] Exemplary building block nucleic acid sequences for construction of chimeric receptors are shown below. CD28 intracellular signaling domain-DNA-human (SEQ ID NO:3) ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGA AGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCT TTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTT TGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAG GAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAAC ATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACC AGCCCTAGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC CAGCAGGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGG CCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAAC CCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGA TGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTC AGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGC ICOS intracellular signaling region - DNA - human (SEQ ID NO:4) CTATCAATTTTTGATCCTCCTCCTTTTAAAGTAACTCTTA CAGGAGGATATTTGCATATTTATGAATCACAACTTTGTTG CCAGCTGAAGTTCTGGTTACCCATAGGATGTGCAGCCTTT GTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGC TTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAA CGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAA AAATCTAGACTCACAGATGTGACCCTAAGAGTGAAGTTCA GCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCT GTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGC CD28 / ICOS costimulatory signaling region - DNA - human (SEQ ID NO:5) ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGA AGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCT TTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTT TGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAG GAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGTTC ATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAG ATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGC CCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAG CTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAG AAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGC

[0087] In some embodiments, the nucleic acid encodes an antigen-binding fragment that binds to CD33. A heavy chain variable region having CDRs identical to those in SEQ ID NO:12 and a SEQ ID NO:13. Q ID NO: 13. In this embodiment, the antigen-binding fragment has the sequence shown by SEQ ID NO:12. The antibody comprises a light chain variable region as shown by SEQ ID NO:13 and a light chain variable region as shown by SEQ ID NO:14. In some embodiments, the chimeric receptor comprises at least a transmembrane domain and a cytoplasmic signaling domain. In some embodiments, the chimeric receptor further comprises a hinge region and / or a cognate region. It further comprises a stimulatory signaling domain.

[0088] Table 3 shows exemplary chimeric receptors described herein. Exemplary constructs include To the C-terminus, it contains an antigen-binding fragment, a transmembrane domain, and a cytoplasmic signaling domain. In some instances, the chimeric receptor has a structure located between the antigen-binding fragment and the transmembrane domain. In some instances, the chimeric receptor further comprises one or more costimulatory domains. and which may be located between the transmembrane domain and the cytoplasmic signaling domain. [Table 3]

[0089] The amino acid sequences of the exemplary chimeric receptors listed in Table 3 above are shown below. CART1 amino acid sequence (SEQ ID NO:20) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSC KSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTF GQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVV KPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIY PGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSA VYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGG AVHTRGLDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR CART2 amino acid sequence (SEQ ID NO:21) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSC KSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTF GQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVV KPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIY PGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSA VYYCAREVRLRYFDVWGQGTTVTVSALSNSSIMYFSHFVPV FLPAKPTTTPAPRPPPTPAPTIASQPLSLRPEASRPAAGGA VHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR CART3 amino acid sequence (SEQ ID NO:22) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSC KSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTF GQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVV KPGASVKMSCKASGYFTSYYIHWIKQTPGQGLEWVGVIY PGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSA VYYCAREVRLRYFDVWGQGTTVTVSSALSNSSIMYFSHFVP VFLAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGG AVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNTPRRPGPTRKHYQPYAPPRDFAAYRSKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR CART8 amino acid sequence (SEQ ID NO:23) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSC KSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTF GQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVV KPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIY PGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSA VYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGG AVHTRGLDFWLPIGCAAFVVVCILGCILICWLTKKKYSSS VHDPNGEYMFMRAVNTAKKSRLTDVTLTKRGRKKLLYIFK QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR CART4dual amino acid sequence (SEQ ID NO:24) MWLQSLLLLGTVACSISIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EIGSTSGSGKPGSGEGSTKGLQESGPGLVAPSQSLSVTCT VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGG SYAMDYWGQGTSVTVSALSNSIMYFSHFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKP FWVLVVVGGVLACYSLLVTVAFIIFWVRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR CART5dual amino acid sequence (SEQ ID NO:25) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSC KSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTF GQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVV KPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIY PGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSA VYYCAREVRLRYFDVWGQGTTVTVSSALSNSIMYFSHFVP VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGG AVHTRGLDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CART6 amino acid sequence (SEQ ID NO:26) MWLQSLLLLGTVACSISIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EIGSTSGSGKPGSGEGSTKGLQESGPGLVAPSQSLSVTCT VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGG SYAMDYWGQGTSVTVSALSNSIMYFSHFVPVFLPAKPTTT PAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDKP FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR CART7 amino acid sequence (SEQ ID NO:27) MWLQSLLLLGTVACSISEIVLTQSPGSLAVSPGERVTMSC KSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESG VPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSSRTF GQGTKLEIKRGSTSGSGKPGSGEGSTKGQVQLQQPGAEVV KPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIY PGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSA VYYCAREVRLRYFDVWGQGTTVTVSSIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR

[0090] The nucleic acid sequences of the exemplary chimeric receptors listed in Table 3 above are provided below. CART1 nucleic acid sequence (SEQ ID NO:38) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTTCTCTGCTGCTGCTGGGCACCGTGGCCTGTAGCATCA GCGAGATCGTGCTGACCCAGAGCCCTGGCTCTCTGGCTGT GTCTCCTGGCGAGCGCGTGACCATGAGCTGCAAGAGCAGC CAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGG CCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCT GATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGAT AGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGA CAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTA CTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGC ACCAAGCTGGAAATCAAGCGGGGCAGCACAAGCGGCAGCG GAAAGCCTGGATCTGGCGAGGGCTCTACCAAGGGCCAGGT GCAGCTGCAGCAGCCTGGCGCCGAAGTCGTGAAACCTGGC GCCTCCGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCT TCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGG ACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAAC GACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCA CCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCA GCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTAC TGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGG GCCAGGGAACCACCGTGACCGTGTCTAGCGCCCTGAGCAA CAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTG CCCGCCAAGCCTACCACAACCCCTGCCCCTAGACCTCCTA CCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAG GCCCGAGGCTTCTAGACCAGCTGCTGGCGGAGCCGTGCAC ACCAGAGGCCTGGATATCTACATCTGGGCCCACTGGCCG GCACCTGTGGCGTGCTGCTGCTGTCTCTCGTGATCACCAA GAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGCGGCCCGTGCAGACCACCCAGGAAGAGGACGGCT GTAGCTGCCGGTTCCCCGAGGAAGAAGAAGGGGCTGCGA GCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCC TATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACC TGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAG AGGCAGGGACCCTGAGATGGCGGCAAGCCCAGACGGAAG AACCCTCAGGAAGGCCTGTATAACGAACTGCAGAAAGACA AGATGGCCGAGGCCTACTCCGAGATCGGAATGAAGGGCGA GCGGAGAAGGAGGCAAGGGCCACGATGGACTGTACCAGGGC CTGAGCACCGCCACCAAGGACACCTATGACGCCCTGCACA TGCAGGCCCTGCCCCCAGAATGAAATTCATCGACGTTAAC TATTCTAG CART2 nucleic acid sequence (SEQ ID NO:39) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTCTCTGCTGCTGCTGGGCACCGTGGCCTGTAGCATCA GCGAGATCGTGCTGACCCAGAGCCCTGGCTCTCTGGCTGT GTCTCCTGGCGAGCGCGTGACCATGAGCTGCAAGAGCAGC CAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGG CCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCT GATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGAT AGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGA CAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTA CTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGC ACCAAGCTGGAAATCAAGCGGGGCAGCACAAGCGGCAGCG GAAAGCCTGGATCTGGCGAGGGCTCTACCAAGGGCCAGGT GCAGCTGCAGCAGCCTGGCGCCGAAGTCGTGAAACCTGGC GCCTCCGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCT TCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGG ACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAAC GACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCA CCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCA GCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTAC TGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGG GCCAGGGAACCACCGTGACCGTGTCTGCCCTGAGCAACAG CATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTGCCC GCCAAGCCTACCACAACCCCTGCCCCTAGACCTCCTACCC CAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCC CGAGGCTTCTAGACCAGCTGCTGGCGGAGCCGTGCACACC AGAGGACTGGACAAGCCCTTCTGGGTGCTGGTGGTCGTGG GCGGAGTGCTGGCCTGTTACAGCCTGCTCGTGACAGTGGC CTTCATCATCTTTTGGGTGCGCAGCAAGCGGTCTAGACTG CTGCACAGCGACTACATGAACATGACCCCCAGAAGGCCAG GCCCCACCCGGAAGCACTATCAGCCTTACGCCCCTCCCAG AGACTTCGCCGCCTACCGGTCCAGAGTGAAGTTCAGCAGA AGCGCCGACGCCCCTGCCTATCAGCAGGGCCAGAACCAGC TGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGA CGTGCTGGACAAGAGAAGAGGCCGGGACCCTGAGATGGGC GGCAAGCCCAGACGGAAGAACCCTCAGGAAGGCCTGTATA ACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACTCCGA GATCGGCATGAAGGGCGAACGGCGGAGAGGCAAGGGACAC GATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGACA CCTATGACGCCCTGCACATGCAGGCCCTGCCCCCCAGATG AAATTCATCGACGTTAACTATTCTAG CART3 nucleic acid sequence (SEQ ID NO: 40) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTCTCTGCTGCTGCTGGGCACCGTGGCCTGTAGCATCA GCGAGATCGTGCTGACCCAGAGCCCTGGCTCTCTGGCTGT GTCTCCTGGCGAGCGCGTGACCATGAGCTGCAAGAGCAGC CAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGG CCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCT GATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGAT AGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGA CAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTA CTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGC ACCAAGCTGGAAATCAAGCGGGGCAGCACAAGCGGCAGCG GAAAGCCTGGATCTGGCGAGGGCTCTACCAAGGGCCAGGT GCAGCTGCAGCAGCCTGGCGCCGAAGTCGTGAAACCTGGC GCCTCCGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCT TCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGG ACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAAC GACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCA CCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCA GCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTAC TGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGG GCCAGGGAACCACCGTGACCGTGTCTAGCGCCCTGAGCAA CAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTG CCCGCCAAGCCTACCACAACCCCTGCCCCTAGACCTCCTA CCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAG GCCCGAGGCTTCTAGACCAGCTGCTGGCGGAGCCGTGCAC ACCAGAGGACTGGACAAGCCCTTCTGGGTGCTGGTGGTCG TGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTGACAGT GGCCTTCATCATCTTTTGGGTGCGCAGCAAGCGGTCTAGA CTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGGC CAGGCCCCACCCGGAAGCACTATCAGCCTTACGCCCCTCC CAGAGACTTCGCCGCCTACAGATCCAAGAGAGGCCGGAAG AAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCG TGCAGACCACCCAGGAAGAGGACGGCTGTAGCTGCCGGTT CCCCGAGGAAGAAGAAGGGGGCTGCGAGCTGAGAGTGAAG TTCAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGCC AGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGA AGAGTACGACGTGCTGGACAAGAGAAGAGGCCGGGACCCT GAGATGGGCGGCAAGCCCAGACGGAAGAACCCTCAGGAAG GCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGC CTACTCCGAGATCGGAATGAAGGGCGAGCGGCGGAGAGGC AAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCA CCAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCC CCCCAGATGAAATTCATCGACGTTAACTATTCTAG CART4 dual nucleic acid sequence (SEQ ID NO:41) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTCTCTGCTGCTGCTGGGCACCGTGGCCTGCAGCATCA GCATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAG CCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAG GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCG ACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACT GCACAGCGGCGTGCCCTCTAGATTTTCCGGCAGCGGCTCC GGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGG AAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCGGC AGCACAAGCGGCTCTGGCAAGCCTGGATCTGGCGAGGGCT CTACCAAGGGCCTGCAGGAATCTGGCCCTGGACTGGTGGC CCCTAGCCAGAGCCTGTCTGTGACCTGTACCGTGTCCGGC GTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGC CCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGG CAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCC TGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTA CTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCC ATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTG CCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCC CGTGTTTCTGCCCGCCAAGCCTACCACAACCCCTGCCCCT AGACCTCCTACCCCAGCCCCTACAATCGCCAGCCAGCCTC TGTCTCTGAGGCCCGAGGCTTCTAGACCAGCTGCTGGCGG AGCCGTGCACACCAGAGGACTGGACAAGCCCTTCTGGGTG CTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTATAGCCTGC TCGTGACAGTGGCCTTCATCATCTTTTGGGTGCGCGTGAA GTTCAGCCGCAGCGCCGATGCCCCTGCCTATCAGCAGGGA CAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGG AAGAGTACGACGTGCTGGACAAGAGAAGAGGCCGGGACCC TGAGATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAGGAA GGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGG CCTACAGCGAGATCGGCATGAAGGGCGAACGGCGGAGAGG CAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACCGCC ACCAAGGACACCTATGACGCCCTGCACATGCAGGCTCTGC CCCCTCGCTGAAATTCATCGACGTTAACTATTCTAG CART5 dual nucleic acid sequence (SEQ ID NO:42) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTCTCTGCTGCTGCTGGGCACCGTGGCCTGTAGCATCA GCGAGATCGTGCTGACCCAGAGCCCTGGCTCTCTGGCTGT GTCTCCTGGCGAGCCGTGACCATGAGCTGCAAGAGCAGC CAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGG CCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCT GATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGAT AGATTCACCGGCAGCGGCTCTGGCACCGACTTCCACCCTGA CAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTA CTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGC ACCAAGCTGGAAATCAAGCGGGGCAGCACAAGCGGCAGCG GAAAGCCTGGATCTGGCGAGGGCTCTACCAAGGGCCAGGT GCAGCTGCAGCAGCCTGGCGCCGAAGTCGTGAAACCTGGC GCCTCCGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCT TCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGG ACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAAC GACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCA CCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCA GCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTAC TGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGG GCCAGGGAACCACCGTGACCGTGTCTAGCGCCCTGAGCAA CAGCATCATGTACTTCAGCCACTTCGTGCCCGTGTTTCTG CCCGCCAAGCCTACCACAACCCCTGCCCCTAGACCTCCTA CCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAG GCCCGAGGCTTCTAGACCAGCTGCTGGCGGAGCCGTGCAC ACCAGAGGACTGGACAAGCCCTTCTGGGTGCTGGTGGTCG TGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTGACAGT GGCCTTCATCATCTTTTGGGTGCGCAGCAAGCGGTCTAGA CTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGGC CAGGCCCCACCCGGAAGCACTATCAGCCTTACGCCCCTCC CAGAGACTTCGCCGCCTACAGAAGCTGAAATTCATCGACG TTAACTATTCTAG CART6 nucleic acid sequence (SEQ ID NO: 43) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTCTCTGCTGCTGCTGGGCACCGTGGCCTGCAGCATCA GCATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAG CCTGGGCGATAGAGTGACCATCAGCTGCAGAGCCAGCCAG GACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCG ACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACT GCACAGCGGCGTGCCCTCTAGATTTTCCGGCAGCGGCTCC GGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGG AAGATATCGCTACCTACTTCTGTCAGCAAGGCAACACCCT GCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCGGC AGCACAAGCGGCTCTGGCAAGCCTGGATCTGGCGAGGGCT CTACCAAGGGCCTGCAGGAATCTGGCCCTGGACTGGTGGC CCCTAGCCAGAGCCTGTCTGTGACCTGTACCGTGTCCGGC GTGTCCCTGCCTGACTATGGCGTGTCCTGGATCAGACAGC CCCCCAGAAAGGGCCTGGAATGGCTGGGAGTGATCTGGGG CAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGG CTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCC TGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTA CTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCC ATGGACTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTG CCCTGAGCAACAGCATCATGTACTTCAGCCACTTCGTGCC CGTGTTTCTGCCCGCCAAGCCTACCACAACCCCTGCCCCT AGACCTCCTACCCCAGCCCCTACAATCGCCAGCCAGCCTC TGTCTCTGAGGCCCGAGGCTTCTAGACCAGCTGCTGGCGG AGCCGTGCACACCAGAGGACTGGACAAGCCCTTCTGGGTG CTGGTGGTCGTGGGCGGAGTGCTGGCCTGTTATAGCCTGC TCGTGACAGTGGCCTTCATCATCTTTTGGGTGCGCAGCAA GCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACC CCCAGACGGCCAGGCCCCACCCGGAAACACTATCAGCCTT ACGCCCCTCCCAGAGACTTCGCCGCCTACCGGTCCAGAGT GAAGTTCAGCAGATCCGCCGACGCCCCTGCCTATCAGCAG GGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAC GGGAAGAGTACGACGTGCTGGACAAGAGAAGAGGCCGGGA CCCTGAGATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAG GAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCG AGGCCTACAGCGAGATCGGCATGAAGGGCGAACGGCGGAG AGGCAAGGGCCACGATGGACTGTATCAGGGCCTGAGCACC GCCACCAAGGACACCTATGACGCCCTGCACATGCAGGCTC TGCCCCCTCGCTGAAATTCATCGACGTTAACTATTCTAG CART7 nucleic acid sequence (SEQ ID NO:44) GGTGTCGTGAGCGGCCGCTGAACTGGCCACCATGTGGCTG CAGTCTCTGCTGCTGCTGGGCACCGTGGCCTGTAGCATCA GCGAGATCGTGCTGACCCAGAGCCCTGGCTCTCTGGCTGT GTCTCCTGGCGAGCGCGTGACCATGAGCTGCAAGAGCAGC CAGAGCGTGTTCTTCAGCAGCTCCCAGAAGAACTACCTGG CCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAGACTGCT GATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGAT AGATTCACCGGCAGCGGCTCTGGCACCGACTTCACCCTGA CAATCAGCAGCGTGCAGCCCGAGGACCTGGCCATCTACTA CTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCCAGGGC ACCAAGCTGGAAATCAAGCGGGGCAGCACAAGCGGCAGCG GAAAGCCTGGATCTGGCGAGGGCTCTACCAAGGGCCAGGT GCAGCTGCAGCAGCCTGGCGCCGAAGTCGTGAAACCTGGC GCCTCCGTGAAGATGTCCTGCAAGGCCAGCGGCTACACCT TCACCAGCTACTACATCCACTGGATCAAGCAGACCCCTGG ACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAAC GACGACATCAGCTACAACCAGAAGTTCCAGGGCAAGGCCA CCCTGACCGCCGACAAGTCTAGCACCACCGCCTACATGCA GCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTAC TGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGG GCCAGGGAACCACCGTGACCGTGTCCAGCATCGAAGTGAT GTACCCCCCTCCCTACCTGGACAACGAGAAGTCCAACGGC ACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCC CTCTGTTTCCTGGCCCTAGCAAGCCCTTCTGGGTGCTGGT GGTCGTGGGCGGAGTGCTGGCCTGTTACAGCCTGCTCGTG ACAGTGGCCTTCATCATCTTTTGGGTGCGCAGCAAGCGGT CTAGACTGCTGCACAGCGACTACATGAACATGACCCCCAG AAGGCCAGGCCCCACCCGGAAGCACTATCAGCCTTACGCC CCTCCCAGAGACTTCGCCGCCTACCGGTCCAGAGTGAAGT TCAGCAGAAGCGCCGACGCCCCTGCCTATCAGCAGGGCCA GAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAA GAGTACGACGTGCTGGACAAGCGGAGAGGCAGGGACCCTG AGATGGGCGGCAAGCCCAGACGGAAGAACCCTCAGGAAGG CCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCC TACTCCGAGATCGGCATGAAGGGCGAGCGGAGAAGAGGCA AGGGCCACGATGGACTGTACCAGGGCCTGAGCACCGCCAC CAAGGACACCTATGACGCCCTGCACATGCAGGCCCTGCCC CCCAGATGAAATTCATCGACGTTAACTATTCTAG

[0091] Any of the chimeric receptors described herein can be produced by routine methods, such as recombinant techniques. The method for preparing the chimeric receptor herein can be carried out by using an antigen-binding flag. ment, and optionally a hinge region, a transmembrane region, at least one costimulatory signal transduction region, A polyclonal antibody comprising each of the chimeric receptor domains, including the cytoplasmic signaling domain and the cytoplasmic signaling domain. In some embodiments, the construction of a chimeric receptor includes the creation of a nucleic acid encoding the peptide. The nucleic acids encoding each of the elements are linked together using recombinant techniques.

[0092] The sequences of each of the components of the chimeric receptor can be determined by routine techniques, e.g., by the methods known in the art. It can be obtained by PCR amplification from any one of a variety of sources. In this embodiment, one or more of the sequences of the components of the chimeric receptor are derived from a human cell. Alternatively, one or more components of the chimeric receptor can be synthesized. For example, the region) can be directly or by amplification using methods such as PCR amplification or ligation. is indirectly linked (e.g., by using a nucleic acid encoding a peptide linker) to form a chimeric receptor Alternatively, the nucleic acid encoding the chimeric receptor can be synthetic. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is R It is NA.

[0093] One or more constructs of the chimeric receptor, either before or after linking the sequences of each of the components. Mutation of one or more residues in a structural component (e.g., an antigen-binding fragment). In embodiments, one or more mutations in the chimeric receptor components enhance the binding of the components to the target. Modulate (increase or decrease) the affinity of (e.g., an antigen-binding fragment) for a target antigen is decreased) and / or may be engineered to modulate the activity of a component.

[0094] Any of the chimeric receptors described herein may be prepared in a suitable vector for expression by conventional techniques. In some embodiments, the immune cells can be primary T cells or T cells. Alternatively, the immune cells can be T cells, such as an established NK cell line (e.g., NK In some embodiments, the immune cells may be NK cells, such as CD4+-92 cells. 8(CD8 + ) or CD8 and CD4 (CD8 + / CD4 + )-expressing T cells In some embodiments, the T cells are T cells of an established T cell line, e.g., 293 T cells or Jurkat cells.

[0095] Primary T cells are derived from peripheral blood mononuclear cells (PBMCs), bone marrow, tissues such as the spleen, lymph nodes, and thymus. The immune cells can be obtained from any source, such as human, mouse, or tumor tissue. Suitable sources for providing the glycerol will be apparent to those of ordinary skill in the art. The immune cell population may be derived from bone marrow or PBMCs obtained from patients with hematopoietic malignancies. In some embodiments, the immune cell population is derived from a human patient. In some embodiments, the immune cells are subsequently administered immune cells expressing the chimeric receptor. Immune cells administered to the same subject from which they were obtained are derived from autologous These cells are called homeopathic cells, whereas immune cells obtained from a subject other than the one to whom the cells are being administered are called homeopathic cells. , called allogeneic cells.

[0096] The desired host cell type is obtained by co-culturing the cells with stimulatory molecules. In a cell population, for example, anti-CD3 and anti-CD28 antibodies can be used to expand T cells. can be used for.

[0097] To construct immune cells expressing any of the chimeric receptor constructs described herein, To this end, vectors for stable or transient expression of chimeric receptors are provided as described herein. The chimeric receptor may be constructed and introduced into immune host cells by conventional methods. The nucleic acid to be loaded can be a viral vector or the like in operable linkage to a suitable promoter. The nucleic acid and the vector can be cloned into an appropriate expression vector. by contacting each molecule with a restriction enzyme at 200 .mu.m to generate complementary ends on each molecule that can pair with each other and be joined with ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the nucleic acid encoding the chimeric receptor. The synthetic linker may contain a nucleic acid sequence that corresponds to a particular restriction site in the vector. The choice of expression vector / plasmid / viral vector is important for the expression of chimeric receptors. Depending on the host cell type, it should be suitable for integration and replication in eukaryotic cells. do.

[0098] A variety of promoters can be used for the expression of the chimeric receptors described herein, including, but not limited to, Although not yet determined, the cytomegalovirus (CMV) intermediate early promoter and viral LTR Rous sarcoma LTR, HIV-LTR, HTLV-1 LTR, Moloney murine leukemia virus MMLV LTR, myeloproliferative sarcoma virus (MPSV) LTR, spleen-limited focus-forming virus Simian Virus 40 (SV40) middle early promoter, single Pure herpes tk virus promoter, with or without the EF1-α intron Contains the long-acting elongation factor 1-α (EF1-α) promoter. Additional promoter for expression of chimeric receptors. The motor may include any constitutively active promoter in immune cells. Any regulatable promoter may be used, such that its expression can be regulated in the immune cell.

[0099] Furthermore, vectors can be used to, for example, effect stable or transient transformation in a host cell. A selectable marker gene, such as the neomycin gene, for selection of transfectants; Enhancer / promoter sequence derived from human CMV mid-early gene for mRNA stability SV40-derived transcription termination and RNA processing signals for α-globin or β-globin 5'- and 5'-receptor domains for mRNA stability and transcription from highly expressed genes such as bins 3'-untranslated region; SV40 polyoma origin of replication and C for proper episomal replication olE1; internal ribosome binding site (IRES), versatile multiple cloning site; sense T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA a "suicide switch" or "suicide vector" that, when induced, causes death in cells that carry the vector Killing genes" (e.g., HSV thymidine kinase, inducible caspases such as iCasp9) , as well as some or all of the reporter genes for assessing expression of the chimeric receptor. See Section VI, infra. Methods for constructing vectors containing the chimeric vectors are well known and available in the art. Examples of the preparation of vectors for expression of the La receptor are described, for example, in US2014 / 0106449 , which is incorporated herein by reference in its entirety.

[0100] In some embodiments, the chimeric receptor construct or the nucleic acid encoding the chimeric receptor The acid is a DNA molecule. In some embodiments, the chimeric receptor construct or chimera thereof The nucleic acid encoding the receptor is a DNA vector that is electroporated into immune cells. (e.g., Till et al., Blood (2012) 119(17):3940-39 In some embodiments, the nucleic acid encoding the chimeric receptor is an mRNA. molecule, which can be electroporated into immune cells.

[0101] Any of the vectors containing a nucleic acid sequence encoding the chimeric receptor constructs described herein. Such vectors can be used to infect hosts by any suitable method. The vector can be delivered into a host cell, such as an immune cell. Methods for delivering the vector into an immune cell have been conventionally described. are well known in the art and can be used for DNA, RNA, or transposon electroporation. liposomes or nanoparticles that deliver antibodies, DNA, RNA, or transposons Transfection reagents; DNA, RNA, or transposons by mechanical deformation or protein delivery (see, e.g., Sharei et al., Proc. Natl. Acad. S ci.USA(2013)110(6):2082-2087); or In some embodiments, the vector for expression of the chimeric receptor comprises: It is delivered to the host cell by viral transduction. Exemplary viral methods for delivery include , including but not limited to, recombinant retroviruses (e.g., those described in PCT Publication No. WO90 / 0793 6, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93 / 10218, WO91 / 02805, U.S. Pat. 40 and 4,777,127, GB Patent No. 2,200,651, and European Patent No. (see Patent No. 0345242), Arifa virus-based vectors, and Adenovirus-based vectors. Nono-associated virus (AAV) vectors (see, e.g., PCT Publication No. WO94 / 12649, W O93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 119 84 and WO 95 / 00655). In some embodiments, the chimeric recipient In some embodiments, the expression vector for the chimeric receptor is a retrovirus. The expression vector is a lentivirus. In some embodiments, expression of the chimeric receptor The vector used is an adeno-associated virus.

[0102] A vector encoding the chimeric receptor is introduced into a host cell using a viral vector. In one example, viral particles carrying vectors capable of infecting immune cells are known in the art. , e.g., PCT Application Nos. WO1991 / 002805A2 and WO1998 / 00927 1A1, and any of the methods found in U.S. Pat. No. 6,194,191. The viral particles can be harvested from the cell culture supernatant and contacted with immune cells. The components may be separated and / or purified prior to processing.

[0103] Methods for preparing host cells expressing any of the chimeric receptors described herein include This may include activating and / or increasing immune cells in vitro. Activating a cell activates the cell so that it can carry out effector functions (e.g., cytotoxicity). The method of activating host cells is to stimulate the host cells into an activated state. The expression of the receptor depends on the type of host cell used. The method of transfecting cells can include any method that results in an increase in the number of cells expressing the chimeric receptor, e.g. , causing host cells to grow or stimulating host cells to grow. The method for stimulating expression of the chimeric receptor depends on the type of host cell used for expression of the chimeric receptor. It will depend on the particulars of the invention and will be apparent to one of ordinary skill in the art. Host cells expressing any of the chimeric receptors described herein may be activated in vitro prior to administration to a subject. be sexualized and / or expanded.

[0104] In some embodiments, the agent targeting the cell surface lineage-specific antigen is an antibody-drug. As will be appreciated by those of ordinary skill in the art, the term "antibody-drug conjugate" is used herein as a generic term. " can be used interchangeably with "immunotoxin" and refers to an antibody (or antibodies) conjugated to a toxin or drug molecule. Binding of an antibody to its corresponding antigen. is the delivery of a toxin or drug molecule to a cell that presents an antigen on its cell surface (e.g., a target cell). This allows for delivery, thereby resulting in the death of the targeted cell.

[0105] In some embodiments, the agent is an antibody-drug conjugate. The antibody-drug conjugates contain an antigen-binding fragment and a toxin that induces cytotoxicity in target cells. In some embodiments, the antibody-drug conjugate comprises an antibody or drug that targets a type 2 antigen. In some embodiments, the antibody-drug conjugate targets CD33 or CD19. .

[0106] In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the sequence represented by SEQ ID NO: The heavy chain CDRs are identical to the heavy chain variable region shown in SEQ ID NO:12, and The light chain variable region has the same light chain CDRs as that shown in SEQ ID NO:13. In one embodiment, the antigen-binding fragment of the antibody-drug conjugate has the structure set forth in SEQ ID NO:12. and a light chain variable region represented by SEQ ID NO:13. Has an area.

[0107] Toxins or drugs suitable for use in antibody-drug conjugates are well known in the art. and will be apparent to those skilled in the art. Peters et al., Biosci. Rep. ( 2015) 35(4):e00225. In some embodiments, the antibody-drug The conjugate may include a linker (e.g., a peptide such as a cleavable linker) that attaches the antibody and the drug molecule. The beverage may further comprise a drinker.

[0108] The ADCs described herein may be administered to subjects receiving the combination therapy described herein. It may be used as a continuous treatment.

[0109] Hematopoietic cells lacking lineage-specific cell surface antigens The present disclosure also provides methods for producing lineage-specific cell surface antigens, such as HSCs, that have been genetically modified to lack lineage-specific cell surface antigens. In some embodiments, the hematopoietic cells are hematopoietic stem cells. HSCs can give rise to both myeloid and lymphoid progenitor cells, which They respectively differentiated into myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, red blood cells, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells) HSCs are then enriched in cell surface antigens that can be used to identify and / or isolate HSCs. The marker CD34 (e.g., CD34 + ) expression and cell lineage commitment They are characterized by the absence of cell surface markers.

[0110] In some embodiments, the HSCs are obtained from a subject, such as a mammalian subject. In some embodiments, the mammalian subject is a non-human primate, a rodent (e.g., a mouse or a rat). In some embodiments, the HSCs are In some embodiments, the antibody is obtained from a human patient, such as a human patient with a hematopoietic malignancy. In some embodiments, the HSCs are obtained from a healthy donor. The immune cells expressing the gene are then obtained from the subject to be administered. HSCs administered to a subject are called autologous cells, whereas HSCs administered to a subject are called autologous cells. The HSCs obtained from the subject are called allogeneic cells.

[0111] HSCs may be obtained from any suitable source using standard procedures known in the art. In some embodiments, the HSCs can be isolated from a bone marrow sample or a blood sample, etc. Alternatively, or in addition, the HSCs are obtained from an umbilical cord sample. In some embodiments, HSCs can be obtained from bone marrow or peripheral blood mononuclear cells (PB Typically, bone marrow cells are obtained from the iliac crest, femur, tibia, vertebral column, rib cage, or other bone marrow of a subject. Bone marrow may be obtained from the bone or other marrow cavity. Bone marrow may be harvested from a patient and processed using techniques known in the art. The cells can be isolated by a variety of separation and washing methods that have been described in detail herein. The method involves the following steps: a) extraction of bone marrow samples; b) centrifugation of the bone marrow suspension into three fractions. c) separation and collection of the intermediate fraction and the buffy coat from step (b); The bone marrow fraction is centrifuged once more in a separation solution, typically Ficoll®, to separate the bone marrow. d) collect the intermediate fraction containing marrow cells for recovery of reinjectable bone marrow cells ( c) washing of the collected fractions.

[0112] HSCs are generally present in the bone marrow, but to collect HSCs from peripheral blood, mobilization agents are used. In some embodiments, HSCs can be mobilized into the circulation by administration of The recipient is administered a mobilizing agent such as granulocyte colony stimulating factor (G-CSF). The number of HSCs collected by mobilization using a mobilization agent is compared to the number of cells obtained without the use of a mobilization agent. is generally greater than the number of

[0113] In some embodiments, a sample is obtained from a subject and a desired cell type (e.g., CD34 + / CD33 - The cells are then enriched for, for example, PBMCs and / or CD34 + Hematopoietic cells can be isolated from blood as described herein. from the cells of interest, e.g., with an antibody that binds to a cell surface epitope of the desired cell type. Another method that can be used is the isolation of cellular functions by receptor-mediated cell proliferation and / or activation. Antibodies against cell surface markers to selectively enrich for specific cell types without cell activation This involves negative selection using the body.

[0114] HSC populations have been genetically engineered to lack lineage-specific cell surface antigens. The cells are then transfected with stem cell factor (SCF), Flt-3 ligand (Flt3L), and then expanded. ), thrombopoietin (TPO), interleukin 3 (IL-3), or interleukin 2 (IL-2) Conditions including an expansion medium containing one or more cytokines, such as leukin 6 (IL-6). The cells may be cultured at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th In some embodiments, the HSCs are expanded for any length of time necessary. Isolation of desired cell populations (e.g., CD34) from samples obtained from the body + / CD33 - ) minutes In some embodiments, the HSCs are expanded after isolation and prior to genetic engineering. Cells expanded after treatment and thus genetically modified to lack lineage-specific cell surface antigens In some embodiments, the desired trait (e.g., A single cell (a "clone") or several cells with the same phenotype or genotype can be selected and expanded independently.

[0115] In some embodiments, the hematopoietic cells are genetically modified to lack cell surface lineage-specific antigens. In some embodiments, the hematopoietic cells are genetically modified to be targeted by the agent. As used herein, hematopoietic cells lack the same cell surface lineage-specific antigens that are expressed by the gene Naturally occurring hematopoietic cells of the same type as the modified hematopoietic cells (e.g., the same The presence of cell surface lineage-specific antigens compared with If the hematopoietic cells have significantly reduced expression of the cell surface lineage-specific antigens, they are considered to lack the cell surface lineage-specific antigens. In some embodiments, the hematopoietic cells exhibit detectable expression of cell surface lineage-specific antigens. The expression level of cell surface lineage-specific antigens can be determined by any means known in the art. For example, the expression level of cell surface lineage-specific antigens can be evaluated using antigen-specific antibodies. The antigen can be detected by detection (e.g., flow cytometry, Western Blotting).

[0116] In some embodiments, expression of cell surface lineage-specific antigens on genetically modified hematopoietic cells is The findings are compared to the expression of cell surface lineage-specific antigens on naturally occurring hematopoietic cells. In one embodiment, the genetic engineering involves the expression of cell surface lineage-specific antigens on naturally occurring hematopoietic cells. At least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% cell surface lineage specific This results in a decrease in the expression of the antigen.

[0117] In some embodiments, the hematopoietic cells contain all endogenous genes encoding cell surface lineage-specific antigens. In some embodiments, the entire endogenous gene encoding a cell surface lineage-specific antigen is missing. In some embodiments, the hematopoietic cells express cell surface lineage-specific antigens. In some embodiments, the hematopoietic cells include a portion of an endogenous gene encoding a cell surface system. In another embodiment, the IL-16 expression vector expresses a portion of the IL-16 specific antigen (e.g., a truncated protein). In these mice, some of the endogenous genes encoding cell surface lineage-specific antigens have been deleted. In some embodiments, at least 10%, 20%, or more of the genes encoding cell surface lineage-specific antigens are 0%, 30%, 40%, 50%, 60%, 70% or more has been removed.

[0118] As will be appreciated by those of skill in the art, nucleic acids encoding cell surface lineage-specific antigens are A portion of the nucleotide sequence may be removed or may be one or more non-coding sequences. Therefore, the hematopoietic cells lack the antigen (eg, have significantly reduced expression of the antigen).

[0119] In some embodiments, the cell surface lineage specific antigen is CD33. The structure shown contains two immunoglobulin domains, an IgV domain and an IgC2 domain. In some embodiments, a portion of the immunoglobulin C region of CD33 is deleted.

[0120] Any genetically engineered hematopoietic cell, such as HSC, that lacks cell surface lineage-specific antigens. Some can be prepared by conventional methods or by the methods described herein. In some embodiments, the genetic engineering is performed using genome editing. When used in this context, "genome editing" refers to the modification of any protein-coding or non-coding nucleotide sequence of an organism. It generally refers to a method of modifying a genome, including a transcription factor sequence, to disrupt the expression of a target gene. Genome editing techniques involve the creation of endogenous genes that can cleave the nucleic acids of a genome, for example at a targeted nucleotide sequence. The repair of double-strand breaks in the genome involves the use of endonucleases. and / or foreign nucleic acid can be inserted at the targeted site.

[0121] Genome editing methods generally involve the generation of endonucleases that generate double-stranded breaks in the target nucleic acid. These methods are classified based on the type of enzyme used. ZFNs, transcription activator-like effector-based nucleases (TALENs) ), meganucleases, and the use of CRISPR / Cas systems.

[0122] In one aspect of the present disclosure, the replacement of tumor cells with an altered population of normal cells is lineage-specific. Such modifications are carried out using normal cells in which target antigens are modified. This may include depletion or inhibition of any lineage-specific antigen using the as9 system, where clustering The Critical Interdigitated Short Palindromic Repeats (CRISPR) Cas9 system can be used to engineer non-naturally occurring C This is the RISPR-Cas9 system (Figure 4).

[0123] CRISPR-Cas systems have been used in a variety of applications including, but not limited to, bacteria, humans, Drosophila, zebrafish, and It has been successfully used to edit the genomes of a variety of organisms, including fish and plants. For example, Jiang et al., Nature Biotechnology (2013) 31 (3):233, Qi et al., Cell (2013) 5:1173, DiCarlo et al., Nu cleic Acids Res.(2013)7:4336, Hwang et al., Nat. Biotechnol (2013), 3:227), Gratz et al., Genetics ( 2013)194:1029, Cong et al., Science(2013)6121:81 9. Mali et al., Science (2013) 6121:823, Cho et al. Nat. Biology otechnol (2013) 3:230, and Jiang et al., Nucleic Acid See ids Research (2013) 41 (20): el88.

[0124] The present disclosure relates to a CRIS that hybridizes to a target sequence in a lineage-specific antigen polynucleotide. The CRISPR / Cas9 system uses the Cas9 nuclease and and engineered crRNA / tracrRNA (or single-stranded guide RNA). The CRISPR / Cas9 complex can bind to the lineage-specific antigen polynucleotide and This allows for the cleavage of nucleotides, thereby modifying the polynucleotide.

[0125] The CRISPR / Cas system of the present disclosure expresses cell surface lineage-specific Within the target antigen, within or near the gene, e.g., in the leader sequence, trailer sequence, or introns, or within non-transcribed regions, either upstream or downstream of the coding region. The guideline used in this disclosure is capable of binding to and / or cleaving a region of interest. The gRNA guides the Cas gene to a specific cleavage site (target site) in the genome. The cleavage site can be designed to direct the binding of the 9-gRNA complex to the unknown sequence region. or regions containing SNPs, nucleotide insertions, nucleotide deletions, rearrangements, etc. The ion exchange material may be selected to release fragments.

[0126] The gene region is cut by Cas enzymes, which cut either the single or double strand at the target sequence. In one embodiment, such cleavage can include reducing transcription of the target gene. In another embodiment, cleavage may result in homologous recombination with an exogenous template polynucleotide. and repairing the cleaved target polynucleotide by recombination, wherein the repair The amplification results in the insertion, deletion, or substitution of one or more nucleotides in the target polynucleotide. do.

[0127] The terms "gRNA," "guide RNA," and "CRISPR guide sequence" are used throughout. and the Cas DNA binding protein of the CRISPR / Cas system. gRNA refers to a nucleic acid that contains a sequence that determines specificity. Hybridize (complementary, partially, or completely) to a sequence; hybridize to a target nucleic acid The gRNA or a portion thereof may be 15–25 nucleotides in length, 18–22 nucleotides in length, or In some embodiments, the amino acid sequence may be 1 to 2 nucleotides, or 19 to 21 nucleotides. The gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In this embodiment, the gRNA sequence that hybridizes to the target nucleic acid sequence is 10 to 30 amino acids in length, or is 15 to 25 nucleotides.

[0128] In addition to a sequence that binds to a target nucleic acid, in some embodiments, the gRNA comprises a scaffold sequence. The gRNA also includes a sequence that encodes both a sequence complementary to the target nucleic acid and a scaffold sequence. Expression of the nucleotide sequence is essential for the function of binding (hybridizing) to a target nucleic acid and endonuclease binding to the target nucleic acid. It has the dual function of recruiting clease, which results in site-specific CRISPR activity. In some embodiments, such chimeric gRNAs comprise a single-stranded guide RNA (sg RNA).

[0129] As used herein, the "scaffold sequence" is also referred to as tracrRNA. The Cas endonuclease binds to the target nucleic acid that is hybridized to the complementary gRNA sequence. It refers to a nucleic acid sequence that contains at least one stem-loop structure and recruits an endonuclease. Any scaffold sequence that recruits a lyase may be used as a genetic element as described herein. Exemplary scaffold sequences are readily apparent to those of skill in the art. For example, Jinek et al., Science (2012) 337 (6096): 8 16-821, Ran et al., Nature Protocols (2013) 8:2281 -2308, PCT Application No. WO2014 / 093694, and PCT Application No. WO2 013 / 176772.

[0130] In some embodiments, the gRNA sequence does not include a scaffold sequence, The sequence is expressed as a separate transcript. In such an embodiment, the gRNA sequence is The scaffold sequence is complementary to a portion of the target nucleic acid sequence and binds (hybridizes) to the scaffold sequence. It further comprises an additional sequence which functions to recruit the endonuclease to the acid.

[0131] In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% %, 99%, or at least 100% complementary (g with the target polynucleotide sequence) U.S. Patent No. 8,697,343, which is incorporated by reference for its teaching of RNA sequence complementarity. (See also Issue 59.) Between the CRISPR guide sequence and the target nucleic acid near the 3' end of the target nucleic acid. It has been demonstrated that mismatches in the nuclease can abolish nuclease cleavage activity (Upadhy ay et al., Genes Genome Genetics (2013) 3(12):223 In some embodiments, the gRNA sequence is located at the 3' end of the target nucleic acid (e.g., at least the last 5, 6, 7, 8, 9, or 10 nucleotides) at the 3' end of the target nucleic acid 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% %, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0132] The target nucleic acid interacts with the endonuclease to target the endonuclease activity to the target nucleic acid. It is flanked at the 3' end by a protospacer adjacent motif (PAM) that may further participate in targeting Generally, PAM sequences adjacent to the target nucleic acid sequence are used to inhibit endonucleases and It depends on the source from which the endonuclease is derived, e.g. from group A hemolytic streptococcus In the Cas9 endonuclease, the PAM sequence is NGG. In the as9 endonuclease, the PAM sequence is NNGRRT. In the Cas9 endonuclease, the PAM sequence is NNNNGATT. In the Cas9 endonuclease from Cox thermophilus, the PAM sequence is NNA. GAA. The Cas9 endonuclease from Treponema denticola The PAM sequence is NAAAAC. In Cpf1 nuclease, the PAM sequence is TTN. do.

[0133] In some embodiments, genetically engineering the cell comprises a Cas endonuclease. In some embodiments, the invention also includes introducing a Cas endonuclease into a cell. The nucleic acid encoding the gRNA and the nucleic acid encoding the gRNA are provided on the same nucleic acid (e.g., a vector). In some embodiments, the nucleic acids encoding the Cas endonuclease and the gRNA are distinct. Alternatively or additionally, the nucleic acid may be provided on a nucleic acid (e.g., a different vector) comprising The AS endonuclease may be provided or introduced into the cell in protein form.

[0134] In some embodiments, the Cas endonuclease is a Cas9 enzyme or a variant thereof. In some embodiments, the Cas9 endonuclease is selected from Group A Streptococcus hemolyticus, Staphylococcus aureus, Neisseria meningitidis, Staphylococcus thermophilus, or Treponema In some embodiments, the Cas endonuclease is derived from Escherichia coli. The nucleotide sequence to be encoded may be codon-optimized for expression in the host cell. In some embodiments, the endonuclease is a Cas9 homolog or ortholog. .

[0135] In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease In some embodiments, the Cas is further modified to alter the activity of the protein. 9 endonucleases are catalytically inactive Cas9s. For example, dCas9 is a catalytically inactive Cas9. It contains mutations at the most active residues (D10 and H840) and has no nuclease activity. Alternatively, or in addition, the Cas9 endonuclease may be mediated by another protein or a portion thereof. In some embodiments, dCas9 can be fused to a repressor region, such as a KRAB region. In some embodiments, such a dCas9 fusion protein is fused to a . are described herein for multiplex gene silencing (e.g., CRISPR interference (CRISPRi)). In some embodiments, dCas9 is used in the constructs described in. is fused to an activator domain such as VPR. dCas9 fusion proteins are used to mediate gene activation (e.g., CRISPR activation) In some embodiments, the constructs described herein are used for dCas9 is a histone demethylase or histone acetyltransferase In some embodiments, the dCas9 is fused to an epigenetic regulatory region such as In some embodiments, the polypeptide is fused to LSD1 or p300, or a portion thereof. dCas9 Fusions Used for CRISPR-Based Epigenetic Regulation In some embodiments, dCas9 or Cas9 is a nuclease domain of Fok1. In some embodiments, a Ca fused to a Fok1 nuclease domain. s9 or dCas9 are used for genome editing. In some embodiments, as9 or dCas9 can be expressed as a fluorescent protein (e.g., GFP, RFP, mCherry, etc.) In some embodiments, the Cas9 / d gene is fused to a fluorescent protein. The Cas9 protein is used to label and / or visualize genomic loci or to transduce Cas genes. It is used to identify cells that express the endonuclease.

[0136] Alternatively, or in addition, the Cas endonuclease is a Cpf1 nuclease. In some embodiments, the host cell is a C. cerevisiae derived from a Prevotella or Francisella species. In some embodiments, the Cpf1 nuclease is expressed. The encoding nuclease sequence may be codon optimized for expression in the host cell.

[0137] In some embodiments, the present disclosure provides methods for the transfection of hematopoietic cells using the CRISPR / Cas9 system. Compositions and methods for inhibiting cell surface lineage-specific antigens, comprising a guide RNA The sequence hybridizes to a nucleotide sequence encoding a cell surface lineage-specific antigen. In some embodiments, the cell surface lineage specific antigen is CD33 and the gRNA is It hybridizes to a portion of the nucleotide sequence encoding the CD33 target (Figure 5). Examples of gRNAs that hybridize to CD33 and are described herein are shown in Table 4. Additional gRNAs may be developed that can be used in the methods.

[0138] Table 4 shows the antibodies that hybridize or are predicted to hybridize to portions of CD33. 1 shows exemplary guide RNA sequences that may be used. [Table 4]

[0139] In some embodiments, HSCs, particularly allogeneic HSCs, can be further engineered and transferred. It may be desirable to reduce the graft-versus-host effect. For example, standard therapy for relapsed AML is hematopoietic stem cell transplantation. However, at least one of the factors limiting the success of HSCT is is a graft-versus-host disease (GVHD) that is associated with the expression of the cell surface molecule CD45. For example, Van Besie, Hematology Am. Soc. tol Educ Program(2013)56, Mawad Curr.Hema See Tol.Malig.Rep.(2013)8(2):132. and CD45RO, an isoform of CD45 (which is expressed in all hematopoietic cells except erythrocytes) In T lymphocytes, CD45RA is expressed on naive cells, whereas CD45RA is expressed on C CD45RO is expressed on memory cells. CD45RA T cells are recruited by HSCT. It is highly likely that the immune system is reactive to host-specific antigens, resulting in GVHD. There remains a need for efficient and safe AML treatments that also reduce the likelihood of graft rejection or GVHD It is believed that CD45-bearing cells are required for survival, but the antigen is CRISPR. CD45 is a type 1 lineage antigen because it can be removed from stem cells using

[0140] In light of the complications that arise due to the progression of GVHD following HSCT, the present disclosure provides Compositions and methods that target 45RA are also provided. Such compositions and methods include It is intended to prevent and / or reduce the occurrence or spread of VHD.

[0141] Therefore, in the case of GVHD, the treatment of the patient involves the following steps: (1) providing the patient with a therapeutically effective administering to the subject a sufficient amount of T cells, wherein the T cells target a CD45RA lineage-specific antigen. and (2) a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that targets the injecting hematopoietic stem cells, wherein the hematopoietic cells express the CD45RA lineage-specific antigen; "It is reducing the reality."

[0142] Additionally, the present disclosure provides a method for the combined inhibition of CD33 and CD45RA lineage specific antigens. The present invention provides compositions and methods for treating such conditions. Such treatment regimens include the following steps: (1) administering to a patient a administering to a subject a therapeutically effective amount of T cells, wherein the T cells express CD33 and C A nucleic acid sequence encoding a chimeric antigen receptor (CAR) targeting the D45RA lineage-specific antigen and (2) infusing the patient with hematopoietic stem cells, either autologous or allogeneic. and reinfusing the hematopoietic cells with the CD33 and CD45RA lineage-specific antigens. The present invention may include reducing the expression of

[0143] In some embodiments, the cell surface lineage specific antigen CD45RA is also expressed by CRISPR. / Cas9 system is used to remove or inhibit in hematopoietic cells. The RNA sequence hybridizes to a portion of the nucleotide sequence encoding CD45RA ( Examples of gRNAs targeting CD45RA are shown in Table 5. Additional gRNAs can be developed that can be resized and used in the methods described herein.

[0144] Table 5 shows antibodies that hybridize to exon 4 or exon 5 of human CD45. 1 shows exemplary guide RNA sequences that are predicted to hybridize. [Table 5]

[0145] Also provided herein are methods for producing cells lacking cell surface lineage-specific antigens, To prepare cells and deliver CRISPR / Cas system components into cells for genome editing. In some embodiments, the vector encoding the lineage-specific cell surface antigen is introduced into Hybridizes or is predicted to hybridize to a portion of the nucleotide sequence. A nucleic acid containing a CRISPR / Cas guide RNA (gRNA) is introduced into the cell. In some embodiments, the gRNA is introduced into the cell in a vector. In some embodiments, a Cas endonuclease is introduced into the cell. Cas endonucleases are expressed in cells as nucleic acids that encode the Cas endonucleases. In some embodiments, the gRNA and Cas endonuclease are introduced into The nucleotide sequences that are loaded are introduced into the cell on the same nucleic acid (e.g., the same vector). In some embodiments, the Cas endonuclease is present in the cell in the form of a protein. In some embodiments, the Cas endonuclease and gRNA are introduced into It is preformed in vitro and introduced into cells as a complex.

[0146] The present disclosure provides engineered vectors capable of encoding one or more components of a CRISPR / Cas9 complex. Further provided are engineered non-naturally occurring vectors and vector systems, wherein the vector comprises ) a (CRISPR)-Cas system guide RNA that hybridizes to a lineage-specific antigen sequence; and (ii) a polynucleotide encoding a Cas9 endonuclease.

[0147] The vectors of the present disclosure can be used to express one or more sequences in mammalian cells using mammalian expression vectors. An example of a mammalian expression vector is pCDM8 (Seed, Nature e(1987)329:840), and pMT2PC (Kaufman et al., EMBO J. (1987) 6:187. When mammalian cells are used, the expression vector A control function is typically provided by one or more control elements. For example, commonly used promoters include polyoma, adenovirus 2, cytomegalovirus Viruses, simian virus 40, and those disclosed herein and known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, , Sambrook et al., MOLECULAR CLONING: A LABORATOR Y MANUAL (Molecular Cloning: A Laboratory Manual). 2nd eds., Col. d Spring Harbor Laboratory,Cold Spring H arbor Laboratory Press,Cold Spring Harbo See, NY, 1989, Chapters 16 and 17.

[0148] The vectors of the disclosure can direct expression of a nucleic acid preferentially in a particular cell type (e.g., For example, tissue-specific regulatory elements are used to express the nucleic acid. The elements include promoters that can be tissue-specific or cell-specific. "Specific," when applied to a promoter, specifies the selective expression of a nucleotide sequence of interest. The same nucleoside can be directed to a single type of tissue (e.g., a seed) and the ... The term "cell type specific" refers to a promoter that is relatively devoid of expression of a promoter sequence. When applied to a cell line, it is possible to direct the selective expression of a nucleotide sequence of interest to a particular cell type, A process that exhibits a relative lack of expression of the same nucleotide sequence of interest in different cell types within the same tissue. The term "cell type specific" when applied to a promoter refers to a promoter that is specific to a single tissue. It also refers to a promoter capable of promoting the selective expression of a nucleotide sequence of interest in a region. The cell type specificity of the motors can be determined by methods well known in the art, e.g., immunohistochemical staining. It can be evaluated using

[0149] Using traditional viral and non-viral based gene transfer methods, CRISPR A nucleic acid encoding Cas9 can be introduced into a mammalian cell or target tissue. using a nucleic acid encoding a component of the CRISPR-Cas system to deliver to cells in culture, or Non-viral delivery systems can be administered to a host organism. Transcripts of vectors described herein), naked nucleic acid, and complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, They have either an episomal or an integrated genome after delivery to a cell. Review of gene therapy procedures.

[0150] Viral vectors can be administered directly to patients (in vivo) or they can be administered in vitro. or can be used to engineer cells in vitro, where the modified cells can be administered to a patient. In one embodiment, the present disclosure relates to, but is not limited to, retroviral vectors for gene transfer. viruses, lentiviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses In addition, the present disclosure utilizes viral-based systems including retroviruses or Vectors capable of integrating into the host genome, such as lentiviruses, are provided. The vector used for expression of the CRISPR-Cas system of the present disclosure is a lentiviral vector. He is a fighter.

[0151] In one embodiment, the present disclosure provides one or more vectors encoding CRISPR-Cas. The method further provides for introducing into a eukaryotic cell a gene encoding a mutated genomic DNA comprising the steps of: Hematopoietic cells, such as hematopoietic stem cells. Examples of stem cells are pluripotent, multipotent and unipotent stem cells. Examples of pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells and induced pluripotent stem cells. In a preferred embodiment, the present disclosure provides a method for producing CRISPR-Cas9. This includes introducing the cells into blood stem cells.

[0152] The vectors of the present disclosure are delivered to eukaryotic cells in a subject. The modification of eukaryotic cells via the system can be carried out in cell culture, where the method involves removing eukaryotic cells from a subject prior to modification. In some embodiments, the method includes isolating the eukaryotic cells and and / or further comprising returning cells derived therefrom to the subject.

[0153] Combination therapy As described herein, an antigen-binding fragment that binds to a cell surface lineage-specific antigen is The agent containing the agonist can be administered to a patient in conjunction with hematopoietic cells lacking cell surface lineage-specific antigens. As used herein, "subject," "individual," and "patient" are used interchangeably. The term is used to refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, , human primates, non-human primates, or murine, bovine, equine, canine, or feline species. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0154] In some embodiments, the agent and / or the hematopoietic cells are pharma- ceutical acceptable. It may be mixed with a carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0155] To carry out the methods described herein, antigen binding proteins that bind to cell surface lineage-specific antigens are used. An effective amount of the agent comprising the fragment and an effective amount of the hematopoietic cells are administered to a subject in need of treatment. As used herein, the term "effective amount" refers to the term The term "therapeutically effective amount" may be used interchangeably and is intended to mean any amount desired for administration to a subject in need thereof. an agent, cell population, or pharmaceutical composition (e.g., an agent and / or a composition comprising hematopoietic cells). In the context of the present disclosure, the term "effective The "amount" refers to an amount that delays the onset of, halts the progression of, or reduces the severity of, the disorder being treated by the disclosed method. A compound, cell population, or pharmaceutical agent sufficient to reduce or alleviate at least one symptom It refers to the amount of a composition. When a combination of active ingredients is administered, an effective amount of the combination may or may not contain amounts of each component that are effective amounts when administered individually. It should be noted that:

[0156] An effective amount will depend on the particular condition being treated, as will be appreciated by those of ordinary skill in the art. Parameters including severity of symptoms, age, physical condition, size, sex and weight of the individual patient , duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and the The amount of the protein that can be effectively used varies depending on factors such as knowledge and opinion. The amount is sufficient to alleviate, relieve, ameliorate, or improve the symptoms of any disease or disorder in a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0157] As described herein, hematopoietic and / or immune cells expressing a chimeric receptor The cells can be autologous to the subject, i.e., the cells are obtained from the subject in need of treatment. , genetically engineered to lack expression of cell surface lineage-specific antigens or expression of chimeric receptor constructs The administration of autologous cells to a subject is similar to the administration of non-autologous cells. Alternatively, the host cells may be administered in an allogeneic manner, resulting in reduced rejection of the host cells compared to administration of the allogeneic lineage-specific cells, i.e., cells are obtained from a first subject and are characterized for expression of cell surface lineage-specific antigens. a first subject genetically engineered to lack expression of a specific or chimeric receptor construct; The allogeneic immune cells are administered to a second subject that is different from the first subject but of the same species. For example, the allogeneic immune cells are administered to a human donor. The transplant may be derived from a donor and administered to a human recipient that is different from the donor.

[0158] In some embodiments, the immune and / or hematopoietic cells are allogeneic and They have also been genetically engineered to reduce graft-versus-host disease. As shown, hematopoietic stem cells were genetically engineered to have reduced expression of CD45RA. This can be done (e.g., using genome editing).

[0159] In some embodiments, an immune cell expressing any of the chimeric receptors described herein is The immune cells are administered to the subject to reduce the number of target cells (e.g., cancer cells) by at least 20%, e.g., 50%. %, 80%, 100%, 2x, 5x, 10x, 20x, 50x, 100x or more It is administered in an amount effective to significantly reduce

[0160] Generally, cells, i.e., immune or hematopoietic cells, are administered to a mammal (e.g., a human). A typical amount may range, for example, from 1 million to 100 billion cells, but this exemplary amount is not intended to be limiting. Amounts below or above the range are also within the scope of the disclosure. For example, a daily dose of cells may be about 1 0 million to about 50 billion cells (e.g., about 5 million, about 25 million, about 500 million, about 1 0 billion, about 5 billion, about 20 billion, about 30 billion, about 40 billion, or any of the above values A range determined by any two of the above), preferably about 10 million to about 100 billion (e.g. For example, about 20 million, about 30 million, about 40 million, about 60 million, about 70 million pieces, about 80 million pieces, about 90 million pieces, about 10 billion pieces, about 25 billion pieces, about 50 billion pieces, about 75 billion, approximately 90 billion, or a range defined by any two of the above values), More preferably, about 100 million to about 50 billion (for example, about 120 million, about 250 million, pieces, about 350 million pieces, about 450 million pieces, about 650 million pieces, about 800 million pieces, about 900 million pieces , approximately 3 billion, approximately 30 billion, approximately 45 billion, or any two of the above values The scope may be determined as follows.

[0161] In one embodiment, the chimeric receptor (e.g., a nucleic acid encoding the chimeric receptor) is The chimeric receptor is introduced into immune cells, and the subject (e.g., a human patient) receives the chimeric receptor-expressing immune cells. Receive an initial dose or dose of an agent (e.g., immune cells expressing a chimeric receptor). One or more subsequent doses should be administered 15, 14, 13, 12, 11, 10, 9, or 100 days after the previous dose. The administration of the agent may be given to the patient at intervals of 8, 7, 6, 5, 4, 3, or 2 days. The dosage may be such that the subject receives administration of the agent, for example, 2, 3, 4 or more times per week. A subject may be administered more than one agent (e.g., a chimeric receptor antagonist) per week. (immune cells expressing the IL-1 receptor), followed by 1 week without agent administration, and finally Additional administration of an agent that enhances immune cell proliferation (e.g., more than once per week of immune cells expressing the chimeric receptor) The chimeric receptor-expressing immune cells will be administered every other day, three times per week. Doses may be administered for 2, 3, 4, 5, 6, 7, 8, or more weeks.

[0162] In the context of this disclosure, insofar as any of the disease conditions listed herein are concerned: The terms "treat," "treatment," and similar terms refer to at least one of the following conditions associated with such conditions: Alleviate or relieve one or more symptoms, or slow or reverse the progression of such symptoms In the context of this disclosure, the term "treat" refers to the onset (i.e., mitigation) of a disease. to halt, delay, or reduce the risk of developing or worsening disease (the period before clinical signs of For example, in the context of cancer, the term "treat" refers to reducing the severity of a patient's tumor. Therapeutic agents are intended to eliminate or reduce tumor burden, or to prevent, delay, or inhibit metastasis. You can taste it.

[0163] In some embodiments, an antigen-binding fragment that binds a cell surface lineage-specific antigen is and a hematopoietic cell population lacking cell surface lineage-specific antigens. In the therapeutic method, the agent is used to target and kill target cells that express cell surface lineage-specific antigens. Hematopoietic cells that lack the antigen are the cell type targeted by the agent. In some embodiments, the patient is treated with the following steps: (1) cellular regeneration; administering to the patient a therapeutically effective amount of an agent that targets a cell surface lineage-specific antigen; and (2) infusing or reinfusing the patient with hematopoietic stem cells, either autologous or allogeneic. wherein the hematopoietic cells have reduced lineage-specific expression of disease-associated antigens. In some embodiments, treatment of a patient may include the following steps: (1) administering a chimeric administering to the patient a therapeutically effective amount of immune cells expressing the receptor, Cells contain nucleic acid sequences encoding chimeric receptors that bind cell surface lineage-specific disease-associated antigens and (2) hematopoietic cells, either autologous or allogeneic (e.g., hematopoietic stem cells). and infusing or reinfusing the hematopoietic cells into the patient, wherein the hematopoietic cells are having expression of a disease-associated antigen.

[0164] Agents containing antigen-binding fragments that bind cell surface lineage-specific antigens and cell surface The efficacy of therapeutic methods using hematopoietic cell populations lacking lineage-specific antigens is known in the art. The patient may be assessed by any method apparent to a trained medical professional. The efficacy of the treatment may be measured by measuring the survival of the subject or the cancer burden in the subject or in a tissue or sample thereof. In some embodiments, the efficacy of a treatment can be assessed by measuring specific populations of cells or In some embodiments, the therapeutic effect is assessed by quantifying the number of cells belonging to the lineage. The efficacy of the treatment was assessed by quantifying the number of cells presenting cell surface lineage-specific antigens. do.

[0165] In some embodiments, the antigen-binding fragment binds to a cell surface lineage-specific antigen. The agent and the hematopoietic cell population are administered simultaneously.

[0166] In some embodiments, an antigen-binding fragment that binds a cell surface lineage-specific antigen is The agent (e.g., an immune cell expressing a chimeric receptor described herein) is In some embodiments, the antibody binds a cell surface lineage-specific antigen. Agents that contain antigen-binding fragments that bind to the chimeric receptors described herein (e.g., The immune cells expressing the hematopoietic cells are , 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 1 It may be administered 2 weeks, 3 months, 4 months, 5 months, 6 months or earlier.

[0167] In some embodiments, the hematopoietic cells are provided with an antigen-binding domain that binds a cell surface lineage-specific antigen. An agent comprising a fragment (e.g., an immune cell expressing a chimeric receptor described herein) In some embodiments, the hematopoietic cell population is administered prior to administration of cell surface lineage-specific At least about 1, 2 days after administration of an agent comprising an antigen-binding fragment that binds to a target antigen , 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, Administered at 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months or earlier do.

[0168] In some embodiments, agents targeting cell surface lineage-specific antigens and hematopoietic cells In some embodiments, the cell populations are administered substantially simultaneously. An agent targeting the hematopoietic cell population is administered, the patient is evaluated for a period of time, and then In some embodiments, the hematopoietic cell population is administered and the patient is evaluated for a period of time. followed by administration of an agent that targets the cell surface lineage-specific antigen.

[0169] Multiple administrations (e.g., doses) of agents and / or hematopoietic cell populations are also within the scope of the present disclosure. In some embodiments, the agent and / or the hematopoietic cell population is administered to the subject once. In some embodiments, the agent and / or the hematopoietic cell population are administered more than once. administered to the subject at least twice (e.g., at least 2, 3, 4, 5, or more times) In some embodiments, the agent and / or the hematopoietic cell population are periodically administered, e.g. The drug is administered to the subject, for example, every six months.

[0170] In some embodiments, the subject is a human subject with a hematopoietic malignancy. As used herein, hematopoietic malignancies are those that involve the development of hematopoietic cells (e.g., precursor and stem cells). Hematopoietic malignancies refer to malignant abnormalities involving blood cells. Examples of hematopoietic malignancies include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. Leukemia is an acute bone marrow necrosis factor (ANC). Myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia or chronic These include myeloblastic leukemia, myeloblastic leukemia, and chronic lymphocytic leukemia.

[0171] In some embodiments, the leukemia is acute myeloid leukemia (AML). AML is a They have progressively acquired critical genetic changes that disrupt key differentiation and proliferation control pathways. Characterized as a heterogeneous, clonal, neoplastic disease originating from transformed cells (Dohner et al., NEJM, (2015) 373:1136). CD33 glycoprotein is expressed in the majority of myeloid leukemia cells, as well as in normal bone marrow and monocytic precursors, and in AML It is believed to be an attractive target for therapeutics (Laszlo et al., Blood Rev. 2014) 28(4):143-53). Based on anti-CD33 monoclonal antibody Clinical trials using the treatment have shown promise in treating a subset of AML patients when used in combination with standard chemotherapy. Although these effects have been associated with safety and efficacy concerns, .

[0172] Other attempts aimed at targeting AML cells have been directed at selectively inhibiting CD4+ in AML. This involves the generation of T cells expressing chimeric antigen receptors (CARs) that target B. uckley et al., Curr. Hematol. Malig. Rep. (2):65(20 15 However, data are limited as to how effective this approach is in treating patients. It is unclear how effective this will be (whether all targeted cells will be eliminated). Because myeloid lineage cells are essential for life, depleting a subject from myeloid lineage cells may adversely affect the survival of the patient. This disclosure is directed, at least in part, to the prevention and treatment of AML. The aim is to solve such problems.

[0173] Alternatively or additionally, the methods described herein can be used to treat, but are not limited to, lung cancer, Ear, nose, and throat cancer, colon cancer, melanoma, pancreatic cancer, breast cancer, prostate cancer, breast cancer, ovarian cancer, Basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer , digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, pharyngeal cancer, liver cancer, fibroma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), Ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, kidney cancer, and cancer of the respiratory system , sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, cancer of the urinary system, and other cancers and The present invention may be used to treat non-hematopoietic cancers, including sarcomas and non-hematopoietic cancers.

[0174] Carcinomas are cancers of epithelial origin. Carcinomas contemplated for treatment by the methods of the present disclosure include, but are not limited to, However, acinar carcinoma, lobular carcinoma, follicular adenocarcinoma (adenocystic carcinoma, adenomyoepithelioma, cribriform carcinoma and circular carcinoma) (also called cystoma), adenomatous carcinoma, adenocarcinoma, adrenal cortical carcinoma, alveolar carcinoma, alveolar cell carcinoma (pulmonary Bronchiolaryngeal carcinoma, also called alveolar cell tumor and pulmonary adenomatosis), basal cell carcinoma cell carcinoma), basal cell carcinoma (carcinoma basocel) lulare) (also called basaloma, or basilloma, and pilomatrix carcinoma), Basal cell carcinoma, basal squamous cell carcinoma, breast carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic Carcinoma, cerebriform carcinoma, cholangiocellular carcinoma (also called cholangiocarcinoma and cholangiocarcinoma), choriocarcinoma , colloid carcinoma, comedocarcinoma, body carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, columnar carcinoma, columnar cell Carcinoma, ductal carcinoma, compact carcinoma, embryonal carcinoma, brain-like carcinoma, supraocular carcinoma, epidermoid carcinoma, epithelial adenoma Noid carcinoma, exophytic carcinoma, ulcerative carcinoma, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, giant cell Carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma (liver carcinoma, malignant Hepatocarcinoma and hepatoma), Hürthle cell carcinoma, hyaline carcinoma, adrenal-like carcinoma, Infantile embryonal carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ, Krumpecher carcinoma, Kurtz Key cell carcinoma, lenticular carcinoma, lenticular Carcinoma (carcinoma lenticulare), lipoadenoma, lymphoepithelial carcinoma, Mastitis carcinoma, medullary carcinoma (carcinoma medullare), medullary carcinoma (me dullary carcinoma), carcinoma melano des), melanotic carcinoma, mucinous carcinoma, mucinous carcinoma secretory carcinoma, carcinoma mucocellulare, mucoepidermal Carcinoma, mucinous carcinoma (carcinoma mucosum), mucinous carcinoma (mucous c arcinoma), myxomatoid carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma , ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of the kidney (renal (also called pancreatic adenocarcinoma and hypernephrotic tumor), storage cell carcinoma, sarcomatoid carcinoma, Schnabel-Hall carcinoma, Idar carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma Squamous cell carcinoma, globular cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma squamous cell carcinoma), squamous cell carcinoma a) Carcinoma of the cord, telangiectatic carcinoma um), carcinoma telangiectodes, metastasis row cell carcinoma, nodular carcinoma (carcinoma tuberosum), nodular carcinoma (t These include verrucous carcinoma, verrucous carcinoma, and choriocarcinoma. The disclosed method is for detecting a tumor in the breast, cervix, ovary, prostate, lung, colon or rectum, pancreas, stomach, or other organs. Alternatively, it may be used to treat a subject with cancer of the kidney.

[0175] Sarcomas are mesenchymal neoplasms arising in bone and soft tissue. Different types of sarcomas are recognized: These include liposarcoma (such as myxoid and pleomorphic liposarcoma), leiomyosarcoma, and striated muscle Sarcoma, malignant peripheral nerve sheath tumor (also called malignant nerve sheath tumor, neurofibrosarcoma, or neurogenic sarcoma) Ewing's tumor (Ewing's sarcoma of bone, extraosseous (i.e., non-bone) Ewing's sarcoma sarcoma, synovial sarcoma, endothelial sarcoma, angiosarcoma, and primitive neuroectodermal tumors (PNETs). Lymphangiosarcoma, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, desmoid tumor (also called aggressive fibromatosis) ), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma , malignant mesenchymal tumor, alveolar soft part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small round cell tumor, Gastric stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (osteogenic sarcoma) Also known as sarcomas - skeletal and extraskeletal, as well as chondrosarcomas.

[0176] In some embodiments, the cancer being treated may be a refractory cancer. As used herein, these are cancers that are resistant to prescribed standard treatments. The disease may initially appear to respond to treatment (and then relapse), or may become completely unresponsive to treatment. The general standard of care depends on the type of cancer and how advanced it is in the subject. It depends on the chemotherapy, or surgery, or radiation, or a combination of these. Those skilled in the art are aware of such standard treatments. Subjects treated according to the indications may therefore already have received another treatment for these cancers. Alternatively, the cancer may be intractable (e.g., analysis of cancer cells or or given the subject's medical history), the subject may not have received other previous treatments. Examples of refractory cancers include, but are not limited to, leukemia, melanoma, renal cell carcinoma, colon cancer, and liver cancer. (liver cancer), pancreatic cancer, non-Hodgkin's lymphoma and lung cancer.

[0177] Any of the immune cells expressing the chimeric receptors described herein may be used as pharmaceutical compositions. The compound may be administered in a pharma- ceutical acceptable carrier or excipient.

[0178] The phrase "pharmaceutical acceptable salt" as used in connection with the compositions and / or cells of the present disclosure "Possible" means that the compound is physiologically tolerable and incompatible when administered to a mammal (e.g., human). Refers to molecular entities or other ingredients of a composition that generally do not undergo beneficial reactions. As used herein, the term "pharmaceutical acceptable" refers to a compound that is soluble in a mammal, more preferably a mammalian animal. Preferably, the method is approved by a federal or state government regulatory agency for use in humans. or be listed in the United States Pharmacopeia or other generally recognized pharmacopoeia "Acceptable" means that the carrier is compatible with the active ingredients of the composition (e.g., nucleic acids, vectors, cells, and is compatible with the host cell (such as a cellular or therapeutic antibody) and does not adversely affect the subject to which the composition is administered. None of the pharmaceutical compositions and / or cells used in the methods are intended to be used in any therapeutically effective manner. A pharma- ceutically acceptable carrier, excipient, or stabilizer may be added to the lyophilized formulation or aqueous solution. It may be included in the dosage form.

[0179] Pharmaceutically acceptable carriers, including buffers, are well known in the art and include, for example, phosphate, Citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins Proteins; Amino acids; Hydrophobic polymers; Monosaccharides; Disaccharides; Other carbohydrates metal complexes; and / or non-ionic surfactants. on:The Science and Practice of Pharmacy( The Science and Practice of Pharmacy, 20th Ed. (2000) Lippincott Wills See iams and Wilkins, Ed. K. E. Hoover.

[0180] Kits for therapeutic use cell surface lineage-specific antigens in combination with hematopoietic cell populations lacking cell surface lineage-specific antigens. Kits for the use of targeting agents are also within the scope of the present disclosure. Any agent that contains an antigen-binding fragment that binds a cell surface lineage-specific antigen (e.g., (e.g., an immune cell expressing a chimeric receptor as described herein) and a medicamentously acceptable a first pharmaceutical composition comprising a therapeutically effective carrier and a hematopoietic cell (e.g., and a second pharmaceutical composition comprising a population of human hematopoietic stem cells and a pharma- ceutical acceptable carrier. The container may include one or more of the above containers.

[0181] In some embodiments, the kit comprises a The included instructions may include instructions for administering the intended activity in a subject. The kit may include instructions for administering the first and second pharmaceutical compositions to a subject to achieve the above objective. Selecting subjects for treatment based on identifying whether they are in need of the treatment In some embodiments, the instructions further include instructions for use in treating The method includes administering the first and second pharmaceutical compositions to a subject.

[0182] The agents that target cell surface lineage-specific antigens described herein and the first and second The instructions for use of the second pharmaceutical composition shall include dosages, dosing schedules, and instructions for use of the second pharmaceutical composition for the intended treatment. Containers typically contain information about the dosage form, dosage form, and route of administration. The kits of the present disclosure may be packaged in a single unit dose package (multi-dose package) or sub-unit dose package. The notice is typically a statement on a label or packaging insert. The packaging insert indicates that the pharmaceutical composition treats, delays the onset of, or reduces a disease or disorder in a subject. indicates that the term is used to enhance and / or mitigate

[0183] The kits provided herein are in suitable packaging. Packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. For use with certain devices such as inhalers, nasal administration devices, or injection containers. Packaging for the purpose of providing a sterile access port is also contemplated. may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle. The container may also have a sterile access port. The component is a chimeric receptor mutant as described herein.

[0184] The kit may optionally provide additional components such as buffers and instruction information. In some embodiments, the container includes a label or packaging insert on or in association with the container. The present disclosure provides an article of manufacture containing the contents of the above kit.

[0185] general technology The practice of the present disclosure involves, unless otherwise indicated, molecular biology (recombinant techniques) which are within the skill of the art. The laboratory will use standard techniques in biology, microbiology, cell biology, biochemistry, and immunology. Such techniques are well described in the literature and are discussed in Molecular Cloning: A Laboratory Manual (Molecular Cloning: A Laboratory Manual) econd edition(Sambrook et al., 1989)Cold Spring Harbor Press, Oligonucleotide Synthesis ( Oligopeptide Synthesis) (MJ Gait, ed. 1984), Methods in Molecular Biology (Methods in Molecular Biology), Humana Pr ess, Cell Biology: A Laboratory Notebook Cellular Biology: Laboratory Notes (J.E.Cellis, ed., 1989) Acad. emic Press, Animal Cell Culture (R I. Freshney, ed. 1987), Introduction to Cell and Tissue Culture (Introduction to Cell and Tissue Culture) (JPMat her and P.E. Roberts, 1998) Plenum Press, Ce. ll and Tissue Culture: Laboratory Procedure res (Cell and tissue culture: laboratory procedures) (A. Doyle, J.B. Griffiths) hs,and DGNewell,eds.1993-8)J.Wiley and Sons, Methods in Enzymology (A Academic Press, Inc.), Handbook of Experiments Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.), Gene Transfer Vect. Vectors for Mammalian Cells (JM Miller and MPCalos, eds., 1987) , Current Protocols in Molecular Biology( Current Protocols in Molecular Biology (FM Ausubel et al., eds. 1987) , PCR:The Polymerase Chain Reaction (Mullis et al., eds. 1994), Current Proc. Latest protocols in immunology (J. E.Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Simple Protocols in Molecular Biology) (Wil ey and Sons,1999), Immunobiology (CA . Janeway and P. Travers, 1997), Antibodies ( Antibodies)(P.Finch,1997), Antibodies: a practice approach (Antibodies: Practical methods) (D. Catty., ed., IRL Pres s,1988-1989), Monoclonal antibodies:a pra A practical approach to monoclonal antibodies (P. Sheph erd and C. Dean, eds., Oxford University Pr. ess, 2000), Using antibodies: a laboratory The Use of Antibodies: A Laboratory Manual (E. Harlow and D. La ne(Cold Spring Harbor Laboratory Press,1 999), The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach(D NA Cloning: Practical Methods), Volumes I and II (DNGlo ver ed.1985), Nucleic Acid Hybridization( Nucleic Acid Hybridization) (BD Hames & SJ Higgins e ds.(1985), Transcription and Translation( Transcription and Translation) (BD Hames & SJ Higgins, eds. (19 84)), Animal Cell Culture (RIFre shney, ed. (1986)), Immobilized Cells and E. Immobilized Cells and Enzymes (IRL Press, (1986)), and B.Perbal,A practical Guide To Molecular Cloning (A Practical Guide to Molecular Cloning) (1984) FMAusube Examples include l et al. (eds.).

[0186] Without further elaboration, it is believed that one of ordinary skill in the art can, based on the foregoing, utilize the present disclosure to its fullest extent. Accordingly, the specific embodiments described below are to be construed as merely illustrative. These and other aspects of the present disclosure are not intended to be limiting in any way whatsoever. All publications used are incorporated by reference for the purpose or subject matter referred to herein. So it is incorporated.

[0187] Example 1: In vitro depletion of CD33 in human leukemia cell lines To test the ability of the CRSPR-Cas9 system to target CD33 in vitro, Human leukemia cell line K-562 was treated with Neon® (Termo Fisher Scientific) Using a chromatin profiling system, Cas9-GFP (PX458, S. pyogenes) and GN Co-transfect with a guide RNA containing an N PAM sequence (Figure 4), where the guide The RNA was designed to target the hCD33 genomic sequence. After 48 hours, cells expressing Cas9 were identified and isolated using GFP FACS sorting. The cells were then incubated for 96 hours and analyzed by flow cytometry for CD33 Expression was examined (Figure 5). Flow cytometry plots using anti-CD33 antibody showed that C K-5 before (top graph) and after (bottom graph) delivery of as9 vector and guide RNA As shown in Figure 5, after transfection, cells 98% of the cells lacked CD33 expression.

[0188] This example shows the efficiency of CD33 expression using the CRISPR-Cas9 system in human leukemia cells. Demonstrate effective removal.

[0189] Example 2: In vitro depletion of CD45 in human leukemia cell lines We targeted CD45RA in vitro using the CRISPR-Cas9 system. Briefly, TIB-67 reticulum cell sarcoma mouse macrophage-like cells were cultured with Neon® Using reagents (Termo Fisher Scientific), Cas9-GFP was (PX458, S. pyogenes) and CRIS targeting hCD45RA genomic sequences The transfection was performed with PR sgRNA (containing the “NGG” PAM sequence). 48 hours after transfection, cells expressing the CRISPR-Cas9 system were transfected with GFP F The cells were then incubated for 96 hours and then cultured at 4°C for 10 min. CD45RA expression was examined (Figure 6). Flow cytometry using anti-CD45RA antibody Plots are shown before (top graph) and after (bottom graph) delivery of Cas9 vector and guide RNA. Rough) CD45RA expression is shown.

[0190] Similar to Example 1, where CD33 expression was successfully reduced in leukemia cells, the results of this example were consistent with CR. 1 shows efficient targeting of CD45RA using the ISPR-Cas9 system.

[0191] Example 3: Cell surface lineage-specific targeting of CD33 in acute myeloid leukemia (AML) This example involves targeting the CD33 antigen in AML. The specific steps of this example are shown below. This is outlined in Section 6. [Table 6]

[0192] I. CD33-targeted chimeric antigen receptor (CAR) T cell therapy A. Generation of anti-CD33 CAR constructs The chimeric antigen receptors that target CD33 described herein are arranged in the 5' to 3' order The pHIV-Zsgreen lentiviral backbone (www.addgene.org / 18121 / ), peptide signal, CD33scFv, hinge, transmembrane of CD28 molecule domain, the intracellular domain of CD28, and the signal transduction domain of the TCR-ζ molecule.

[0193] First, a peptide signal, anti-CD33 light chain (SEQ ID NO:1), flexib The linker and anti-CD33 heavy chain (SEQ ID NO:2) were transformed with pHIV-Zsgree The product is cloned into the EcoRI site of n, optionally with a Kozak sequence.

[0194] It has the basic structure of light chain-linker-heavy chain-hinge-CD28 / ICOS-CD3ζ An exemplary nucleic acid structure of a chimeric receptor that binds CD33 is shown below. Part 1: Light chain-linker heavy chain (SEQ ID NO:16): Kozak initiation site thickened The peptide signal L1 is in italics. The anti-CD33 light and heavy chains are in bold. The double-stranded ribosomes are shown in italics and separated by a linker. [ka] Part 2: Hinge-CD28 / ICOS-CD3ζ NotI restriction site is upsized The restriction site is shown in bold. The translation stop site is shown in bold. The BamHI restriction site is underlined. [ka] [ka] [ka]

[0195] In the next step, the hinge region, the CD28 region (SEQ ID NO:3), and the T The cytoplasmic component of CR-ζ was transformed into pHIV-Zsgreen (which already contained the peptide signal and C D33scFv) into the NotI and BmHI sites of The CD28 domain can be replaced with the ICOS domain (SEQ ID NO:4).

[0196] In addition to the CD28 and ICOS domains, the CD28 and ICOS intracellular signaling domains A fusion region was engineered containing a fragment of the nucleotide sequence (SEQ ID NO:5) to express additional In this configuration, the chimeric receptor is an antigen-binding domain. fragment, anti-CD33 light chain variable region, linker, anti-CD33 heavy chain variable region, CD28 / ICOS hybrid domain (containing the transmembrane domain of CD28) and the sigma of the TCR-ζ molecule It contains the signal transmission region.

[0197] Exemplary amino acid sequences of building blocks that can be used to generate chimeric receptors are provided herein. The CD28 domain (SEQ ID NO:6), the ICOS domain (SEQ ID NO:7), the CD28 / ICOS hybrid region (SEQ ID NO:8), and and TCR-ζ, etc., are presented herein. Alternatively, chimeric receptors can be similarly produced. (Section B).

[0198] B. Alternative Methods for the Generation of Anti-CD33 CAR Constructs A schematic of an exemplary chimeric receptor is shown in Figure 7, panels A-D. The chimeric receptor is a receptor that binds extracellular C D8 hinge region, transmembrane and cytoplasmic signaling domains, and the CD3ζ signal chain The antibody is generated using an extracellular humanized scFv that recognizes the CD33 antigen linked to the IgG antibody (Figure 7, Panel B). DNA encoding the anti-CD33 chimeric receptor was successfully used with a humanized scFv. and (Essand et al., J Intern Med. (2013) 27 3(2):166). Alternatives are CD28 or CD28 / OX1 or CD28 / 4- CAR T cells containing OX-1 or 41-BB instead of the 1-BB hybrid (Figure 7, panels C and D).

[0199] To prepare the anti-CD33 scFv sequence, the heavy and The coding regions of the main and light chains (SEQ ID NOs: 1 and 2) were amplified using specific primers. The DNA is amplified using the DNA fragment and cloned into the pHIV-Zsgreen vector for expression in cells. To evaluate the binding strength of scFv (single-chain variable fragment) to the target antigen, To this end, the vector (p Transform E. coli Top10F with HIV-Zsgreen to prepare the plasmid. The resulting expression vector encoding the cFv antibody was transfected into Hek293T cells. After culturing the transfected cells for 5 days, the supernatant was collected and The antibody is then purified.

[0200] The resulting antibodies can be synthesized using framework substitutions according to protocols known in the art. For example, the protocol provided by BioAtla (San Diego) can be used to humanize the The present invention refers to a fragment library that encodes synthetic CDRs derived from a template antibody. The human framework is restricted to germline sequences from functionally expressed antibodies. Ligation to fragments encoding human framework regions from the pool (bioat la.com / applications / express-humanization / ).

[0201] Affinity maturation may be performed to improve antigen binding affinity. This can be done using phage display techniques, etc. The method can be carried out using common techniques known in the art (Schier R., J. Mol. Biol (1996), 263:551). The mutants were then used to determine their biological activity. (e.g., binding affinity) using, for example, Biacore analysis. To identify hypervariable region residues that may be good candidates for modification, alanine skimming may be performed. Random mutagenesis can be performed to identify hypervariable region residues significantly involved in antigen binding. To improve the affinity of antibodies, combinatorial libraries as described above can be used (Rajp al., PNAS (2005) 102(24):8466). has developed a platform for rapid and efficient affinity maturation of antibodies, May be used for body optimization purposes (bioatla.com / applicat ions / functional-maturation / ).

[0202] C. Assembly of the CAR construct Next, anti-CD33 scFv was coupled to the extracellular CD8 hinge region, the transmembrane and cytoplasmic CD28 synthase. Briefly, anti-C Primers specific to the D33scFv sequence are used to amplify the scFv as described above. A plasmid carrying the complete human CD8 coding sequence (pUN1-CD8) (www.inv ivogen.com / puno-cd8a) to identify the CD8 hinge and transmembrane domains. The CD3ζ fragment was amplified from the complete human CD3 Invivogen plasmid pORF9-hCD247a (hCD247a) carrying the 3ζ coding sequence ttp: / / www.invivogen.com / PDF / pORF9-hCD247 a_10E26v06.pdf) and finally, CD28 (amino acids 153-2 20, which corresponds to the transmembrane and signal transduction domain of CD28) from activated T cells. Amplify from cDNA generated using RNA collected by the Lysol method. Anti-CD3 The fragment containing 3-scFv-CD8-hinge+transmembrane-CD28-CD3ζ was Assemble the fragments using sequence overlap extension (SOE) PCR. It is then cloned into the pELPS lentiviral vector. pELPS is the third generation It is a derivative of the lentiviral vector pRRL-SIN-CMV-eGFP-WPRE. , in which the CMV promoter was replaced with the EF-1α promoter and the HIV central polyp A phosphoryl tract was inserted 5' of the promoter (Milone et al., Mol Ther. (2009)(8):1453, Porter et al., NEJM(2011)(8):725 ). All constructs are verified by sequencing.

[0203] Alternatively, ICOS, CD27, 41BB, or OX-40 instead of the CD28 region Creating a CAR containing a signaling domain and introducing it into T cells to eradicate CD33-positive cells The ability of the receptor to bind to the IL-1 receptor will be tested (Figure 7, Panel C). The creation of "third generation" chimeric receptors is also contemplated. (Figure 7, panel D), which is CD3z-CD28-41BB or CD3z-CD28 -Combining multiple signaling domains such as OX40 to further enhance efficacy (Sad elain et al., Cancer Discov. (2013) 4:388).

[0204] D. Anti-CD33CAR T cell preparation Primary human CD8 + T cells were isolated by immunomagnetic separation (Miltenyi Biotec). T cells are isolated from the peripheral blood of patients by incubating in complete medium (10% heat-inactivated FBS, 100 U 100 μg / mL penicillin, 100 μg / mL streptomycin sulfate, 10 mM HEPES (RPMI 1640 supplemented with 100 mM NaCl) and cultured as previously described (Levine et al. , J. Immunol. (1997) 159(12):5921) Anti-CD3 and anti-CD Stimulation is performed with 28 mAb-coated beads (Invitogen).

[0205] Packaging cell lines are used to produce viral vectors that can then be used to transduce target cells. To produce lentiviral particles containing the anti-CD33 chimeric receptor, The CAR generated in step (1) was transfected into immortalized normal fetal kidney 293T packaging cells. The cells were incubated in 10% FBS, 100 U / mL penicillin, The cells were cultured in high glucose DMEM containing 100 μg / mL streptomycin. 48-72 hours after transfection, the supernatant was collected and the recombinant lentivirus was then transfected into FB Enrich in DMEM without S. Primary CD8 + The T cells were then cultured in the presence of polybrene at approximately Transduce cells at a multiplicity of infection (MOI) of 5–10. tems) was added every other day (50 IU / mL). T cells were cultured for approximately 14 days after stimulation. The transduction efficiency of human primary T cells was evaluated using the ZsGreen reporter gene (Clontech). cch (Mountain View, Calif.) expression.

[0206] E. Infusion of CAR T cells into patients Prior to in vivo infusion of anti-CD33 CAR T cells into patients, the cells were soaked in phosphate-buffered saline. The cells were washed with water and concentrated. Haemonetics Corporation, Braintree, Massachusetts Cell processors such as the Sigma-Aldrich Cell Processing Center (St. Louis, MO) are used for washing and concentration steps prior to formulation. The definitive T cells expressing anti-CD33 chimeric receptors were cultured in immune-mediated immunization medium supplemented with human serum albumin. Finally, patients receive 1-100 mL of T cells over 1-3 days. 0×10 7 Injected at 100 mg / kg (Maude et al., NEJM (2014) 371 (16) The number of T cells expressing anti-CD33 chimeric receptors injected will depend on the condition of the cancer patient. The outcome depends on many factors, including the type of disease, age of the patient, and previous treatments.

[0207] In addition, the present invention provides a method for the development of a chimeric receptor targeting CD33 in AML patients. Alternatively, immune cells expressing chimeric receptors that target CD45RA may be considered. Two different methods: 1) Immune cells expressing anti-CD33 chimeric receptors and anti-CD47RA The aim of this study was to generate immune cells expressing the chimeric receptor separately and to introduce both types of immune cells into patients. or 2) by injecting the cells separately, or 3) by targeting both CD33 and CD45RA simultaneously. This can be achieved by generating immune cells that can 2):151(2014)).

[0208] II.CD34 + CD33 - Autologous hematopoietic stem cell transplantation (HSCT) using cells A process for isolating stem cells from patients, conditioning regimens, and injecting the stem cells into patients. Protocols vary widely depending on the patient's age, symptoms, medical history, and the center where treatment is delivered. It is understood that the protocol described below operates in a similar manner. Therefore, the protocol described below is merely an example and is not intended to be construed as a substitute for the prior art. Regular optimization is performed by the vendor.

[0209] A. Using peripheral blood stem cell (PBSC) mobilization followed by adoptive transfer of anti-CD33 CAR T cells Isolation of hematopoietic stem cells AML patients were treated with granulocyte colony-stimulating factor (G-CSF) at a dose of 10 mg / kg per day. Stimulate by intravenous administration. CD34 + Cell positive selection was performed using immunomagnetic beads and immunomagnetic Performed using enrichment device. CD34 + 2 x 10 of cells 6 The minimum quantity per kg of body weight is It is expected that the cells will be collected using a Fenwall CS 3000+ cell separator. (Park et al., Bone Marrow Transplantation (200 3)32:889).

[0210] B. Patient Preparative Regimen The conditioning regimen for autologous peripheral blood stem cell transplantation (PBSCT) consisted of etoposide (VP- 16) + cyclophosphamide (CY) + total body irradiation (TBI). Simply stated, the regimen is 1.8 g / m2 over 26 hours as a single dose. 2 Intravenous Etoposide (VP-16) in a constant infusion (civ), followed by a 2-hour infusion for 3 days Cyclophosphamide (CY) at 60 mg / kg body weight intravenously per day, followed by the next 3 days The study consisted of total body irradiation (TBI) at 300 cGy per day.

[0211] To calculate dose, use ideal body weight or actual body weight, whichever is less. As described above, the condition of the cancer patient, the age of the patient, previous treatment, and the type of facility where the procedure is performed Factors such as type of chemotherapy and other factors are all taken into consideration when determining the exact conditioning regimen.

[0212] C. Plasmid construction of CRISPR-Cas9 system targeting CD33 Add lentiCRISPRv2 containing insert Cas9 and puromycin resistance ene (plasmid #52961) (Sanjana et al., Nat Methods s(2014)(8):783). Single-stranded guide RNA (sgRNA) CD33 guide sequence For cloning, lentivirus CRISPRv2 was cloned using FastDigest BsmBI and cleaved and dephosphorylated with FastAP (Fermentas) at 37°C for 2 hours. The gRNA targeting CD33 was optimized online at crispr.mit.edu. Alternatively, the gRNA has the sequence shown in Figure 12 (SEQ ID NO: 1). Q ID NO:11). The CD33 gRNA oligonucleotide was The polynucleotides were obtained from ed DNA Technologies (IDT) and The DNA was phosphorylated with ATPase (Fermentas) at 37°C for 30 min and incubated at 95°C for 5 min. Anneal by heating and cooling to 25°C at 1.5°C / min. Use T7 ligase. The oligonucleotides are annealed using The resulting fragment is ligated into gel-purified vector (Qiagen) at 25° C. for 5 minutes. The plasmid was then purified using an endotoxin-free midi-prep kit (Qiagen). (Sanjana et al., Nat Methods (2014)(8) :783).

[0213] Alternatively, the two vector systems can be transfected according to a previously reported protocol (Mandal et al., 2013). ll Stem Cell (2014) 15(5):643) (gR (when NA and Cas are expressed from separate vectors). Here, Mandal et al. Using a viral vector-expressed CRISPR-Cas system to transfect human hematopoietic stem cells Efficient removal of the gene was achieved.

[0214] Briefly, a human codon-optimized Cas9 gene containing a C-terminal SV40 nuclear localization signal is The gene is cloned into a CAG expression plasmid carrying 2A-GFP. To instruct Cas9 to cleave the sequence, a guide RNA (gRNA (SEQ ID NO: 3) was used. DNO:11) was isolated from a plasmid containing the human U6 polymerase III promoter. The gRNA sequence oligonucleotides are expressed in the Integrated DNA The primers were obtained from IDT Technologies and annealed to contain the BbsI restriction site. The human U6 promoter requires a "G" base to initiate transcription. The requirement for PAM (protospacer adjacent motif) The target is GN 20 It contains the nucleotide sequence GG.

[0215] In addition to injecting patients with CD33-depleted hematopoietic stem cells, patients were subsequently The protocol will proceed with the injection of RA-depleted HSCs. Alternatively, the inventors will Using the PR-Cas9 system, CD34 + CD33 - CD45RA - Cells were generated and CD3 3 and CD45RA genes simultaneously. Exemplary guide RNA sequences for this purpose are shown in Tables 4 and 5.

[0216] D.CD34 + CD33 - CD34 for generating cells + Transfection of HSC cells Cushion Freshly isolated peripheral blood-derived CD34 + Cells (from step 4) are cultured in cell culture grade Stem Cell Factor (SCF) 300ng / mL, FLT3-L 300 ng / mL, thrombopoietin (TPO) 100ng / mL, and IL-3 60ng 1 × 10 cells in serum-free CellGro SCGM medium in the presence of 0.1% DMSO / mL 6 pieces / m After 24 hours of prestimulation, CD34 + HSCs were transduced with Cas9 and CD33 gR Amaxa Human CD34 cells were transfected with lentivirus-containing CRISPRv2. Transfection was performed using the Nucleofector kit (U-008) (#VPA-1003). (Mandal et al., Cell Stem Cell (2014) 1 5(5):643). 24–48 hours after transfection, CD34 + CD33 - Cells are selected with 1.2 μg / mL puromycin. After puromycin selection, CD 34 + CD33 - Cells are maintained in puromycin-free medium for 2-3 days.

[0217] E. CD34 in patients + CD33 - Re-injection of cells CD34 transfected in vitro with CRISPR-Cas9-CD33 + Thin cells (CD34+ CD33 - cells) immediately using a standard blood administration set without a filter. The blood is then reinjected through a Hickman catheter using a 100% ethanol solution (Hacein-Bey Abina et al., JAMA (2015) 313(15):1550).

[0218] In general, patients undergoing the treatment protocols outlined above will experience recurrence of circulating blasts and cytopenias. Additionally, the potential mechanisms of AML in specific patients are being monitored. Depending on the type, achievement of treatment may be determined by the reappearance of informative molecular or cytogenetic markers, Alternatively, monitoring may be performed by examining informative flow cytometry patterns. For example, BCR-ABL signaling in Philadelphia chromosome-positive AML The reappearance of the gene was confirmed by probes for BCR (on chromosome 22) and ABL (on chromosome 9). It is detected using fluorescent in situ hybridization (FISH).

[0219] To evaluate the success of CRISPR-Cas9-mediated CD33 depletion, peripheral blood CD3 4 + Cells are isolated from the patient (post-transplant) and CD33 expression is analyzed, e.g., by flow cytometry. The results are evaluated using immunohistochemistry, Western blotting, or immunohistochemistry.

[0220] As described herein, the HSCT reported in this case was either autologous or allogeneic. Either approach may be used, both of which are suitable and may be incorporated into the methods described in this disclosure. Cut.

[0221] III. Optional Step: Continued Treatment of the Patient with CD33 Antibody Attached to a Toxin A. Treatment of patients with the CD33 immunotoxin gemtuzumab ozogamicin (GO) Patients were treated with 9 mg / m2 as a 2-hour intravenous infusion in two doses, 2 weeks apart. 2 Anti-CD33 antibody Treat with gemtuzumab ozogamicin (GO) (Larson et al., Cancer (2016) 05), 104(7):1442-52). GO inhibits the cell proliferation inhibitor calicheamicin. It consists of a conjugated humanized monoclonal antibody against CD33 (Figure 8).

[0222] Alternatively, the anti-CD33 antibody may be a cytotoxin that binds diphtheria toxin, Pseudomonas aeruginosa exotoxin A (PE), or lysine. Toxins such as ribosome adenosine triphosphate (RTA) can be conjugated to different toxins and made (Wayne et al., Bl. ood(2014)123(16):2470). Similarly, anti-CD45RA antibodies can be used to treat may be conjugated and included in a treatment regimen.

[0223] Example 4: T cell and NK cell lines expressing anti-CD33 chimeric receptors express CD33 Induce cell death in target cells Binding of chimeric receptors to CD33 Chimeric receptors that bind CD33 (e.g., CART1, CART2, CART3) Using conventional recombinant DNA technology, the pHIV-Zsgreen vector (Addg The vector containing the chimeric receptor was inserted into the GFP-derived vector (Cat. No. 63311, Cambridge, Massachusetts). The lentiviral particles are then used to generate lentiviral particles that can be transfected into different cell types, such as T cell lines (e.g. It is used to transduce NK cell lines (e.g., NK92 cells) and NK cell lines (e.g., 293T cells). The expression of the chimeric receptor was examined by Western blotting (Figure 9, Panel A) and flow cytometry. The results were detected by immunocytometry (Figure 9, Panel B).

[0224] Cells expressing the chimeric receptor are selected by fluorescence-activated cell sorting (FACS). We then assayed their ability to bind CD33. Lysates of 293T cells expressing CD33 or CD33-allophycocyanin (APC) were analyzed by immunofluorescence staining. The samples were subjected to protein electrophoresis and Stain with a protein stain (Figure 10, Panel A) or transfer to a membrane and stain with anti-CD3ζ In both cases, the chimeric receptor and its It binds between its target CD33.

[0225] K562 cells expressing the chimeric receptor were also analyzed by flow cytometry using CD33 as a probe. Binding to CD33 was assessed by immunofluorescence in situ hybridization (Figure 10, Panel C). The number of cells positive for the expression of receptors (CART1, CART2, or CART3) and CD33 binding was increased compared to cells containing the empty vector control, indicating that the chimeric receptor It was shown to bind to D33.

[0226] Cytotoxicity induced by cells expressing chimeric receptors NK-92 cells expressing the chimeric receptor were cultured on target cells presenting CD33 on their cell surface ( For example, K562 cells are a human chronic myeloid leukemia line that is CD33+. To perform the cytotoxicity assay, the effector In vivo, the target cells (immune cells such as NK-92 cells) were treated with lentiviruses encoding chimeric receptors. Several days after infection, cells expressing the chimeric receptor were cultured in FA Using CS analysis, we identified a fluorescent marker ( For example, GFP+ or Red+ was selected. The selected cells expressing the IgG antibody were grown for one week. 14 days after infection, the target cells (target cell surface Cells expressing the lineage-specific antigen CD33 were incubated with carboxyfluorescein succinimide. Staining with CFSE and target cells expressing the chimeric receptor A cytotoxicity assay was performed that included counting both the number of cells that were positive for different ratios of target cells. and cells expressing the chimeric receptor were co-incubated for 4.5 h in round-bottom 96-well plates. ion, followed by addition of 7-aminoactinomycin D (7-AAD) to remove non-viable cells. Flow cytometry was performed to enumerate viable and nonviable target cell populations. As shown in panels A and B of FIG. 11, the chimeric receptors CART1, CART2, or NK92 cells expressing either CART3 or CART3 were able to upregulate the target K562 at each cell ratio evaluated. It induced a significant amount of cell death in the cells.

[0227] We determined that cell death of K562 cells was dependent on specific targeting of the chimeric receptor to CD33. To identify these genes, K562 cells were genetically engineered to lack CD33 using the CRISPR / Cas system. Briefly, a human codon-optimized Cas9 endonuclease and A gRNA targeting a portion of the IgC region of CD33 was expressed in K562 cells and CD33 We obtained a population of K562 cells that were deficient in the chimeric receptor. The cells were expanded and cocultured with NK92 cells expressing the chimeric receptor. Incubation and cytotoxicity assays were performed as described above. As shown, pooled CD33-deficient K562 cells express NK92 chimeric receptors. Co-incubation with cells showed a slight reduction in cell death. Single clones of CD33-deficient K562 cells were isolated and expanded, and cytotoxicity assays were performed. When used to treat pulmonary circulation, an even more significant reduction in cytotoxicity was observed (Figure 12, Panel B). ).

[0228] Expression of chimeric receptors in primary T cells Primary T cell populations were isolated from PMBCs obtained from donors using CD4+, CD8+, or CD 4+ / CD8+ cells were isolated using FACS to select for positivity, resulting in a highly pure population. (Figure 13, Panels A and B). Primary T cell populations (CD4+, CD8+, or CD4+ / CD8+ cells) were transfected with chimeric receptors (e.g., CART1 and CART8) Transduce primary T cells expressing the resulting chimeric receptor with a lentiviral vector containing Cytotoxicity assays were performed using the cells as described above. Co-incubation of +T cell population with K562 cells (1000 target K562 cells) did not cause cytotoxicity in K562 cells (Figure 14, panel A). Either CD8+ or CD4+ / CD8+ cells expressing the Mela receptor, and 100 In the cytotoxicity assay using 0 target K562 cells, CD8+ or CD4+ / CD 8+ cells were able to induce cell death in a low percentage of K562 cells (Figure 14, Panel B).

[0229] Genetic Engineering of Human Hematopoietic Stem Cells Several gRNAs were designed to hybridize to the IgC region of CD33 ( For example, see Table 4, SEQ ID NO: 11 or 28 to 31. It was co-expressed with Cas9 endonuclease in K562 cells. was evaluated by flow cytometry (Figure 15). Crispr3 (SEQ ID NO: 28) and Crispr5 (SEQ ID NO:29), expressing a CD33-targeting CRISPR / Cas system compared to control cells expressing 3 A significant decrease in CD33 was observed in cells expressing CD33.

[0230] CD33-deficient hematopoietic stem cells were subjected to various cellular functions including proliferation, erythropoietic differentiation, and colony formation. Briefly, we also evaluated the characteristics of CD33-deficient hematopoietic stem cells and control The erythrocytes were induced to differentiate by exposing them to hemin, resulting in the formation of erythroid precursors. The marker CD71 was assessed at different time points by flow cytometry (Figure 1 6, Panels A and B). CD33-deficient hematopoietic stem cells undergo erythropoietic differentiation and The cytometry profile appeared similar to that of control cells (CD33+). The cells were also subjected to MTT assay to measure the metabolic activity of CD33-deficient hematopoietic stem cells. As shown in Figure 16, panel C, CD33-deficient hematopoietic stem cells behaved comparably to controls. Finally, the ability of the cells to proliferate and form cell colonies was assessed using a microscopic colony formation assay. Again, CD33-deficient hematopoietic stem cells showed similar proliferation rates to control cells. These results suggest that the CD33 CRISP signaling pathway is involved in the formation of colonies. R / Cas ablation did not significantly affect the ability of cells to proliferate, differentiate, or form colonies. Indicates that there is no

[0231] Other embodiments All of the features disclosed herein may be used in any combination. Each feature disclosed is intended to be non-exclusive and is not to be construed as limiting the present invention, as merely illustrative, and not restrictive. Thus, unless expressly stated otherwise, each feature disclosed may be interchangeable. are merely examples of a comprehensive set of equivalent or similar features.

[0232] From the above description, one skilled in the art can easily ascertain the essential features of the present disclosure. Various modifications and improvements of the present disclosure may be made without departing from the spirit and scope thereof. The present invention is applicable to various uses and conditions. Therefore, other embodiments are within the scope of the appended claims. be.

[0233] Equivalent Several inventive embodiments have been described and illustrated herein, and those skilled in the art will appreciate that Performing the functions described herein and / or achieving the results and / or one or more benefits. Numerous other means and structures for achieving this goal are readily conceivable, and such modifications and Each of the improvements and / or modifications is considered to be within the scope of the embodiments of the invention described herein. More generally, a person skilled in the art would understand all of the parameters described herein. The dimensions, materials, and configurations are meant to be exemplary and actual parameters, dimensions, materials, and configurations may vary. The materials and / or configurations may vary depending on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will readily recognize that, using no more than routine experimentation, We recognize or have identified many equivalents to the specific inventive embodiments described herein. Accordingly, the above described embodiments are presented by way of example only and are not intended to be limiting unless otherwise specified by the appended claims and claims. Within this range of equivalents, embodiments of the present invention may be practiced other than as specifically described and claimed. Inventive embodiments of the present disclosure may incorporate each individual feature described herein. The present invention relates to a method, system, article, material, kit, and / or method. Any two or more of the features, systems, articles, materials, kits, and / or methods A combination is any such feature, system, article, material, kit, and / or method. If they are not mutually inconsistent, they are within the scope of the invention of this disclosure.

[0234] All definitions provided and used herein are based on dictionary definitions of the terms provided. Covering definitions and / or ordinary meanings in documents incorporated by reference should be understood as managing.

[0235] All references, patents, and patent applications disclosed herein are hereby incorporated by reference. are incorporated by reference in relation to the subject matter covered, and in some cases, the entire document is included. obtain.

[0236] The indefinite article "a" or "an" as used in this specification and claims is expressly intended to be used interchangeably. Unless otherwise indicated, "at least one" should be understood to mean "at least one."

[0237] The phrase "and / or" as used in this specification and claims thus constrains "Either or both" of the combined components, i.e., in some cases, " and " shall be understood to mean components that exist separately in other cases. The elements indicated by " / or" are of the same form, i.e., The other components should be considered as "one or more" of the components specifically identified. The phrase "and / or" refers to any of the elements of the may be present optionally other than the components specifically identified by ". Thus, non-limiting examples include As such, the meaning of "A and / or B" together with open-ended terms such as "including" is When used in the present invention, in one embodiment, only A (optionally including components other than B) is used, and in another embodiment, In one embodiment, it means only B (optionally including components other than A), and in another embodiment, can mean both A and B (optionally including other components), etc.

[0238] As used in this specification and the claims, "or" means "and / or" as defined above. " should be understood to have the same meaning as ". For example, separating items in a list where "or" or "and / or" is to be construed as inclusive, i.e. i.e., the inclusion of at least one, but not all, of the members of a number or list; Includes additional unlisted items. Only one item is clearly indicated to the contrary: "One of "only" or "exactly one of," or "consisting of," etc., when used in this claim. , implies the inclusion of exactly one member of a number or list of members. As used in the specification, "or" means "either," "one of," "only one of," or When preceded by an exclusive term such as "exactly one of," it indicates exclusive alternatives (i.e., The term "basic" should be interpreted only as indicating "one or the other, but not both." As used in this claim, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law. has.

[0239] As used herein and in the claims, the phrase "at least one" means one or more In a reference to a list of components, any of the components in the list of components means at least one component selected from one or more, but not in the list of components It does not necessarily include at least one of each and every component specifically listed. , does not exclude any combination of the elements of the list of elements. "At least one" means any element other than those specifically identified in the list of elements. It is possible that components may be present optionally, and those components specifically identified are Therefore, as a non-limiting example, “A and and at least one of A and B" (or, equivalently, "at least one of A or B"; or Synonymous with "at least one of A and / or B" is, in one embodiment, the presence of B. None (optionally including components other than B), and optionally including more than one In another embodiment, without the presence of A (optionally including components other than A), optionally one In yet another embodiment, B is at least one including more than At least one A, including at least one B, including at least one and optionally other components), etc.

[0240] Unless expressly stated to the contrary, any part in this specification that includes more than one step or action. In any method claimed herein, the order of the method steps or actions is not necessarily sequential. It should also be understood that no limitations to the order of the method steps or actions may be employed.

Claims

1. A method of treating a hematopoietic malignancy comprising administering to a subject in need thereof (i) an effective amount of an agent that targets a lineage-specific cell surface antigen, wherein said the agent comprises an antigen-binding fragment that binds said lineage-specific cell surface antigen. Effective quantity of quality, and (ii) a hematopoietic cell population that lacks said lineage-specific cell surface antigen; The method of claim 1, further comprising administering

2. The agent comprises an antigen-binding fragment that binds the lineage-specific cell surface antigen. The method of claim 1, wherein the immune cell expresses a chimeric receptor.

3. 10. The method of claim 1, wherein the immune cells, the hematopoietic cells, or both are allogeneic or autologous. is the method described in 2.

4. The method according to any one of claims 1 to 3, wherein the hematopoietic cells are hematopoietic stem cells.

5. The method according to any one of claims 1 to 4, wherein the lineage-specific cell surface antigen is a type 2 lineage-specific cell surface antigen. The method according to any one of claims 1 to 5.

6. The method of claim 5, wherein the type 2 lineage-specific cell surface antigen is CD33.

7. The lineage-specific antigen-binding fragment in the chimeric receptor is a human protein. A single chain antibody fragment (scFv) that specifically binds a foreign cell surface antigen.

7. The method according to any one of 1 to 6.

8. The method of claim 7, wherein the scFv binds to human CD33.

9. The scFv has the same complementarity determining regions (CDRs) as those in SEQ ID NO:

12. and a heavy chain variable region having the same CDRs as in SEQ ID NO:

13. The method of claim 8, comprising a light chain variable region.

10. The scFv comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 12; The antibody of claim 9, comprising a light chain variable region having the amino acid sequence of SEQ ID NO:

13. How to.

11. The method according to any one of claims 1 to 4, wherein the lineage-specific cell surface antigen is a type 1 lineage-specific cell surface antigen. The method according to any one of claims 1 to 5.

12. The method of claim 11, wherein the type 1 lineage-specific cell surface antigen is CD19.

13. The method according to any one of claims 1 to 12, wherein the immune cells are T cells.

14. The chimeric receptor (a) the hinge region; (b) transmembrane region; (c) at least one costimulatory domain; (d) a cytoplasmic signaling region, or (e) combinations thereof; The method of any one of claims 1 to 13, further comprising:

15. The chimeric receptor is CD27, CD28, 4-1BB, OX40, CD30, CD4 0, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR, HVEM, and combinations thereof and at least one costimulatory signaling domain derived from a costimulatory receptor selected from The method of claim 14 .

16. The at least one costimulatory signaling region is 4-1BB, CD28, or ICO The method of claim 15, wherein the strain is derived from S.

17. The at least one costimulatory signaling region comprises a signaling region of CD28 and 17. The method of claim 16, comprising the signal transduction region of ICOS.

18. said at least one costimulatory signaling region being derived from CD28 and said chimeric receptor The receptor further comprises a second costimulatory signaling region derived from 4-1BB or ICOS.

18. The method according to item 16 or 17.

19. The chimeric receptor comprises a cytoplasmic signaling region derived from CD3ζ.

19. The method according to any one of claims 18 to 18.

20. 2. The chimeric receptor of claim 1 , wherein the chimeric receptor comprises a hinge region that is derived from CD8α or CD28α.

20. The method according to any one of 1 to 19.

21. The chimeric receptor comprises a transmembrane domain derived from CD8, CD28, or ICOS. The method according to any one of claims 1 to 20.

22. The chimeric receptor comprises, from the N-terminus to the C-terminus: (i) an scFv that binds to a lineage-specific cell surface antigen, a hinge region derived from CD8α, CD8 a transmembrane domain derived from CD3, a costimulatory domain derived from 4-1BB, and a cytoplasmic signal domain derived from CD3ζ Transmission area, (ii) an scFv that binds to a lineage-specific cell surface antigen, a hinge region derived from CD8α, The transmembrane domain from CD28, the costimulatory domain from CD28, and the cytoplasmic signaling domain from CD3ζ transmission area, or (iii) an scFv that binds to a lineage-specific cell surface antigen, a hinge region derived from CD8α, D28-derived transmembrane domain, CD28-derived first costimulatory domain, 4-1BB-derived second costimulatory domain region, and a cytoplasmic signaling region derived from CD3ζ, The method according to any one of claims 1 to 21, comprising:

23. The hematopoietic stem cells are ＋ / CD33 － The method according to any one of claims 4 to 22, Method of posting.

24. 23. The hematopoietic stem cells are derived from bone marrow cells or peripheral blood mononuclear cells (PBMCs). The method described above.

25. The subject has Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. The method according to any one of claims 1 to 24, comprising:

26. The subject has acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or 26. The method of claim 25, wherein the patient has leukemia that is chronic lymphoblastic leukemia.

27. A nucleic acid comprising a nucleotide sequence encoding a chimeric receptor, wherein said chimeric receptor The receptor comprises an antigen-binding fragment that binds CD33, a transmembrane domain, and a cytoplasmic signal transduction domain. and wherein the antigen-binding fragment has a sequence similar to that in SEQ ID NO:

12. A heavy chain variable region having the same CDRs and the same CDRs as in SEQ ID NO:

13. A nucleic acid comprising a light chain variable region having R.

28. 28. The nucleic acid of claim 27, wherein the chimeric receptor further comprises at least one costimulatory domain. acid.

29. The at least one costimulatory signaling region is selected from the group consisting of CD27, CD28, 4-1BB, O X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA -1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR, HVEM, and combinations thereof. Nucleic acid.

30. The at least one costimulatory signaling region is 4-1BB, CD28, or ICO 30. The nucleic acid of claim 29, which is derived from S.

31. The at least one costimulatory signaling region is a signaling region of CD28 and The nucleic acid of claim 30 comprising a COS signal transduction region.

32. said at least one costimulatory signaling region being derived from CD28 and said chimeric receptor The receptor further comprises a second costimulatory signaling region derived from 4-1BB or ICOS.

32. The nucleic acid according to item 30 or 31.

33. Any of claims 27 to 32, wherein the cytoplasmic signaling region is derived from CD3ζ.

3. The nucleic acid according to claim 1 .

34. Any of claims 27 to 33, wherein the hinge region is derived from CD8α or CD28α. The nucleic acid according to claim 1.

35. The transmembrane domain of any one of claims 27 to 34 is derived from CD8, CD28, or ICOS. The nucleic acid according to any one of claims 1 to 4.

36. The chimeric receptor comprises, from the N-terminus to the C-terminus: (i) the antigen-binding fragment, the hinge region derived from CD8α, and the transmembrane region derived from CD8 , a costimulatory domain derived from 4-1BB, and a cytoplasmic signaling domain derived from CD3ζ, (ii) the antigen-binding fragment, the hinge region derived from CD8α, and the transmembrane region derived from CD28 domain, a costimulatory domain derived from CD28, and a cytoplasmic signaling domain derived from CD3ζ, teeth (iii) the antigen-binding fragment, the hinge region derived from CD8α, the transmembrane region derived from CD28 a first costimulatory domain derived from CD28, a second costimulatory domain derived from 4-1BB, and Cytoplasmic signaling domain derived from 3ζ The nucleic acid according to any one of claims 27 to 35, comprising:

37. The antigen-binding fragment has a heavy chain having the amino acid sequence of SEQ ID NO:

12. and a light chain variable region having the amino acid sequence of SEQ ID NO:

13. The nucleic acid according to any one of claims 27 to 36.

38. A vector comprising the nucleic acid of any one of claims 27 to 37.

39. A chimeric receptor encoded by the nucleic acid of any one of claims 27 to 38.

40. 40. An immune cell expressing the chimeric receptor of claim 39.

41. The immune cell of claim 40, which is a T cell.

42. 42. The method of claim 40 or 41, wherein the immune cells are obtained from a patient with a hematopoietic malignancy. Immunoglobulin G (IMG)-dependent endothelial cell death (EMD) assay.

43. A genetically engineered hematopoietic cell, comprising a nucleotide sequence that is present on the hematopoietic cell prior to genetic engineering. Genetically engineered hematopoietic cells that lack lineage-specific cell surface antigens.

44. All or part of the endogenous gene encoding the lineage-specific cell surface antigen is deleted.

44. The genetically engineered hematopoietic cell of claim 43.

45. All or a portion of the endogenous gene encoding the lineage-specific cell surface antigen is genomicly edited. The genetically engineered hematopoietic cell of claim 44, wherein the cell is deleted using a gene expression vector.

46. The genome editing is carried out using zinc finger nucleases (ZFNs), transcription activator-like enzymes (TAEs), and The claimed invention includes a talenase (TALEN), a CRISPR-Cas system, or 46. ​​The genetically engineered hematopoietic cell of claim 45.

47. 47. The method according to claim 43, wherein the lineage-specific cell surface antigen is CD33 or CD19. A genetically engineered hematopoietic cell according to any one of claims 1 to 4.

48. The lineage-specific cell surface antigen is CD33 and the immunoglobulin constant (I 47. The genetically engineered hematopoietic cell of claim 46, wherein a portion of the GPCR region is deleted.

49. The genetic engineering method according to any one of claims 43 to 48, wherein the hematopoietic cells are hematopoietic stem cells. Bioprocessed hematopoietic cells.

50. CD34 ＋ / CD33 － 50. The genetically engineered hematopoietic cell of claim 49. Cell.

51. Any of claims 43 to 50, derived from bone marrow cells or peripheral blood mononuclear cells (PBMC). A genetically engineered hematopoietic cell according to claim 1.

52. 1. A method for producing a cell lacking a lineage-specific cell surface antigen, comprising: Providing cells; In the cells (i) hybridizing to a portion of the nucleotide sequence encoding the lineage-specific cell surface antigen; A nucleic acid comprising a nucleotide sequence of a CRISPR-Cas system guide RNA (gRNA) that encodes , and (ii) a Cas endonuclease; To introduce A method comprising:

53. 53. The method of claim 52, wherein the Cas endonuclease is Cas9 or Cpf1. method.

54. 52. The method of claim 52, wherein (i) and (ii) are encoded on the same nucleic acid or on different nucleic acids. Or the method described in 53.

55. (i) and (ii) are introduced into said cell as a preformed ribonucleoprotein complex. The method according to any one of claims 52 to 54,

56. The portion of the nucleotide sequence to which the gRNA hybridizes is from 18 to 22 amino acids. The method according to any one of claims 52 to 55, which consists of nucleotides.

57. 57. The method of claim 52, wherein the lineage-specific cell surface antigen is CD33 or CD19. The method according to any one of claims 1 to 5.

58. The method of any one of claims 52 to 57, wherein the cell is a hematopoietic cell.

59. 59. The method of claim 58, wherein the hematopoietic cells are hematopoietic stem cells.

60. The hematopoietic stem cells are ＋ 60. The method of claim 59, wherein:

61. The gRNA is a nucleotide sequence of any one of SEQ ID NOs: 11 and 28 to 31. The method of any one of claims 52 to 60, comprising a nucleotide sequence.

62. (i) a combination of an immune cell according to any one of claims 40 to 42 and a lineage-specific cell surface antigen; Agents comprising antigen-binding fragments that target and bind said lineage-specific cell surface antigens , and (ii) a structure lacking a lineage-specific cell surface antigen as defined in any one of claims 43 to 51; Blood cell populations, Including the kit.

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