Genetically modified NK cells and uses thereof

Abstract

Provided are genetically modified NK cells and uses thereof. The modified NK cells can be used in CAR-NK cell adoptive cell therapy of cancer.

Description

[Technical field]

[0001] Technical Field The present disclosure relates to genetic engineering and immunotherapy, in particular to genetically modified NK cells and their uses in the treatment of disease, such as adoptive cell therapy and / or CAR-NK therapy for cancer. [Background technology]

[0002] background Natural killer (NK) cells comprise 15% of human peripheral blood mononuclear cells (PBMCs) and are cytotoxic lymphocytes that play a key role in defending against tumor and viral infections, and are now known to be a critical component linking the adaptive and innate immune systems.

[0003] NK cell activity is regulated by a series of costimulatory (e.g., NKG2D, CD226) and co-inhibitory surface receptors (e.g., PD-1, TIGIT, CD96, TIM-3, LAG-3, NKG2A) that recognize their corresponding ligands on target cells or antigen-presenting cells. The integration of costimulatory and co-inhibitory signals determines the responsiveness of NK cells.

[0004] TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains), a transmembrane glycoprotein receptor expressed on NK and T cells, is an immune checkpoint molecule that inhibits the activation of T cells and NK cells. It contains an IgV domain, a transmembrane domain, and an immunoreceptor tyrosine-based inhibitory motif (ITIM). CD96 is a member of the same immunoglobulin superfamily and has similar inhibitory activity, but has a lower binding affinity to its ligand CD155 compared to TIGIT. CD155 (mainly) and CD112 act as ligands for TIGIT and CD96 to inhibit T cell- and NK cell-mediated immunity. CD155 (poliovirus receptor, PVR) is poorly expressed in normal human tissues, but highly expressed in various tumor cell lines and primary malignancies. Preclinical and clinical evidence demonstrated that TIGIT blockade with monoclonal antibodies enhances the antitumor and antiviral activities of NK and T cells.

[0005] NKG2A (NK group 2 member A) is an NK cell receptor of the NKG2 family, a type II membrane receptor that forms a heterodimer with CD94. NKG2A dimerizes with CD94 to form inhibitory receptors that recognize HLA-E, which correlate with C-type aggregates. These inhibitory receptors interact with MHC I ligands on target cells, thereby completely inhibiting cell particle polarization and preventing the release of cytotoxic particles. NKG2A contains two ITIMs in its cytoplasmic tail. These ITIMs are phosphorylated after binding to ITIM-containing receptors and promote the recruitment of tyrosine phosphatases such as SH2 domain-containing phosphatase (SHP)-1 and SHP-2. Recruitment of SHP-1 by ITIM-containing receptors appears to inhibit the initiation of signal transduction, as it blocks most downstream signals in INK cells. Tumor cells from hematological and solid tumors showed upregulation of HLA-E expression. In various cancers, poor prognosis is associated with upregulation of HLA-E, and blockade of the CD94 / NKG2A receptor with antibodies could be a therapeutic strategy.

[0006] CISH (cytokine-induced SH2-containing protein), a key negative intracellular immune checkpoint in NK cells, is a member of the Suppressor of Intracellular Cytokine Signaling (SOCS) family and is a key regulator of cytokine and growth factor signaling pathways. Like other members, CISH possesses a central SH2 domain that can interact with phosphotyrosine residues and SOX box motifs that recruit the ubiquitin transferase system and induce proteasomal degradation. CISH is rapidly induced in response to IL-15, but NK cells lacking CISH are more sensitive to IL-15 and are characterized by enhanced proliferation, cytokine production, and cytotoxicity against tumors.

[0007] Genetic modification is expected to redirect the function of various cell types, including T cells, dendritic cells, and NK cells. In particular, much research has been done on genetically redirecting T cells against various tumor antigens. However, difficulties in genetic modification of primary NK cells have led to this field lagging behind T cells to some extent. Some studies have modified NK cells with cytokine transgenes (e.g., IL-2, IL-12, or IL-15 transgenes), thereby enhancing NK cell function by directly providing the cells with the cytokines they require. However, few studies have described the redirection of NK cell specificity by chimeric receptors.

[0008] There remains a need for NK cells that have long-lasting, enhanced anti-tumor activity and reduced side effects during production and use. Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention Disclosed herein are genetically modified NK cells and their use in disease treatment, such as in adoptive cell therapy of cancer.

[0010] As a first aspect, the present specification discloses an isolated genetically modified NK cell that has been modified to impair the functional expression of one or more of TIGIT, NKG2A, and CISH. According to some embodiments, the NK cell may further comprise a chimeric antigen receptor (CAR).

[0011] In some embodiments, an isolated modified NK cell is provided, wherein the NK cell is modified to impair functional expression of at least two of TIGIT, NKG2A, and CISH.

[0012] In some embodiments, an isolated modified NK cell is provided, wherein the NK cell is modified to impair functional expression of one or more of TIGIT, NKG2A, and CISH, and wherein the NK cell further comprises a chimeric antigen receptor (CAR).

[0013] In a second aspect, the present specification discloses a cell population or cell culture comprising the modified NK cells described herein.

[0014] In a third aspect of the present specification, there is disclosed an article of manufacture comprising the modified NK cells, cell populations or cell cultures described herein. In some embodiments, the article of manufacture is a pharmaceutical product, a pharmaceutical composition, or a kit.

[0015] In a fourth aspect, the present specification discloses a method for preparing the modified NK cells described herein.

[0016] In a fifth aspect, the present specification discloses the use of the modified NK cells, cell populations or cell cultures described herein in the preparation of a product for treating a disease.

[0017] As a sixth aspect of the present specification, there is disclosed a method of treating a disease in a subject in need thereof, said method comprising administering an effective amount of an engineered NK cell, cell population, cell culture or product as described herein.

[0018] In a seventh aspect, the present specification discloses a modified NK cell, cell population, cell culture or product as described herein for treating a disease.

[0019] Other objects, features, advantages, and aspects of the present application will become apparent to those skilled in the art from the following description and the appended claims. However, it should be understood that the following description, the appended claims, and specific examples, while showing preferred embodiments of the present application, are given by way of illustrative example. By reading the following, various changes and modifications within the spirit and scope of the disclosed invention will be readily apparent to those skilled in the art. [Brief description of the drawings]

[0020] The novel features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which sets forth illustrative embodiments, in which the principles of the invention are utilized: [Figure 1] FIG. 1 shows the purity of expanded NK cells. [Diagram 2] FIG. 2 shows TIGIT knockout (KO) efficiency by CRISPR / Cas9 in NK cells tested by flow cytometry. [Diagram 3] Figures 3A-3B show the efficiency of NKG2A knockout by CRISPR / Cas9 in NK cells tested by flow cytometry. [Figure 4] FIG. 4 shows the efficiency of CRISPR / Cas9-mediated dual knockout of TIGIT and NKG2A in NK cells tested by flow cytometry. [Diagram 5] FIG. 5 shows CRISPR / Cas9-mediated CISH knockout efficiency in NK cells tested by Western blot. [Figure 6] Figure 6 shows CD155 and HLA-E expression on HT1080 tumor cells. "Negative" refers to the negative control of cells stained with a fluorescently labeled control antibody. [Figure 7] FIG. 7 shows the results of a cytotoxicity test of genetically modified NK cells against HT1080-ZsGreen target cells. [Figure 8] Figures 8A-8B show tumor growth inhibition in A549 tumor-bearing mice after treatment with modified NK. Statistical data was analyzed by two-way ANOVA. *p0.05; **p<0.01. [Figure 9] Figures 9A-9B show the expression of anti-CD19 CAR in CD19 CAR-NK and mCD19 CAR-NK. [Figure 10] Figures 10A-10B show TIGIT knockout efficiency in mCD19CAR-NK tested by flow cytometry. [Figure 11] Figures 11A-11B show CISH knockout efficiency in mCD19CAR-NK tested by Western blot. [Figure 12] Figures 12A-12B show the results of cytotoxicity test of mCD19 CAR-NK cells against Raji-luc target cells. Statistical data is analyzed by two-way ANOVA. *p<0.05; **p<0.01; ****p<0.0001. [Figure 13] Figures 13A-13B show the results of sequential killing assay of mCD19 CAR-NK cells against Raji-luc target cells. Statistical data are analyzed by two-way ANOVA. ***p<0.001. [Figure 14] Figures 14A-14B show the release of IFN-γ cytokines in cytotoxicity assays. Statistical data are analyzed by two-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 15] Figures 15A-15B show the release of IFN-γ cytokines in a sequential killing assay. Statistical data was analyzed by two-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention The following description and examples will describe the embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein, and can be modified. Those skilled in the art should recognize that the present invention has many variations and modifications, and these variations and modifications are included within the scope of the present invention.

[0022] The present disclosure is primarily based on the unexpected finding that impairing the functional expression of TIGIT, NKG2A and / or CISH in NK cells can significantly improve the cytotoxicity of genetically modified NK cells and extend the in vitro or in vivo life span of NK cells. Based on this finding, the present disclosure provides genetically modified NK cells and methods for producing same, where at least one of TIGIT, NKG2A and / or CISH of the NK cells is impaired, and where the NK cells may further comprise or be bound to a chimeric antigen receptor. Further provided herein are cell populations, cell cultures or products comprising the NK cells of the present disclosure, and their uses in the treatment of cancer, autoimmune diseases, infectious diseases, transplant rejection and other age-related diseases.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described in this disclosure, the preferred methods and materials are described.

[0024] As used herein, the terms "a" and "an" are intended to refer to "one or more" (i.e., at least one) of a document grammar object. Unless the context requires otherwise, the singular includes the plural. For example, "an element" means one element or multiple elements.

[0025] "About" means a number, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference number, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0026] Unless otherwise stated, the use of "or" means "and / or."

[0027] As used herein, unless otherwise stated, the terms "comprise", "containing", and "comprising" should be understood to include the stated step or element, or group of steps or elements, but not to exclude any other step or element, or group of steps or elements.

[0028] The phrase "consisting of" is meant to include and limit the content that follows the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that other elements may be present.

[0029] The term "isolated" refers to material that is substantially or essentially free from components that normally accompany it in its natural state. The material may be a cell or a macromolecule, such as a protein or nucleic acid. For example, as used herein, an "isolated cell" refers to a cell that has been purified from the cell in its natural state.

[0030] The term "NK cells" or "natural killer cells" refers to cytotoxic lymphocytes that are important for the innate immune system. NK cells mediate antitumor and antiviral responses and therefore have good clinical potential. The NK cells of the present disclosure may be derived from blood (e.g., autologous or allogeneic PBMC), NK cell lines (e.g., NK-92, NKG, YT, NK-YS, HANK-1, YTS, NKL, etc.), or differentiated stem cells (e.g., iPSC).

[0031] TIGIT, NKG2A and / or CISH As used herein, the term "TIGIT" or "TIGIT gene" refers to a nucleotide molecule that encodes the T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), an immune checkpoint molecule that suppresses the activation of T cells and NK cells.

[0032] A TIGIT gene is a gene encoding a TIGIT polypeptide, such as a TIGIT polypeptide having the sequence set forth in SEQ ID NO: 25, or a TIGIT polypeptide having high identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% identity) to the above-mentioned TIGIT polypeptide or any TIGIT polypeptide known in the art and having the same immune checkpoint function.

[0033] For example, a TIGIT gene may be, but is not limited to, the following: a nucleic acid molecule having the sequence set forth in SEQ ID NO: 26; a TIGIT polypeptide coding sequence having high identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% identity) to the above-mentioned TIGIT genes or any TIGIT gene known in the art, and similarly encoding and expressing a functional TIGIT polypeptide.

[0034] As used herein, the term "NKG2A" or "NKG2A gene" refers to a nucleotide molecule that encodes NK group 2 member A (NKG2A), a type II membrane receptor that heterodimerizes with CD94 and inhibits cell particle polarization by interacting with HLA-E, preventing the release of cytotoxic particles.

[0035] The NKG2A gene is a gene encoding an NKG2A polypeptide such as a TIGIT polypeptide having the sequence set forth in SEQ ID NO:27, or an NKG2A polypeptide having high identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% identity) to the above-mentioned NKG2A polypeptide or any NKG2A polypeptide known in the art and having the same immune checkpoint function.

[0036] For example, the NKG2A gene may be, but is not limited to, the following: a nucleic acid molecule having the sequence set forth in SEQ ID NO:28; an NKG2A polypeptide coding sequence having high identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% identity) to the above NKG2A genes or any NKG2A gene known in the art, and which similarly encodes and expresses a functional NKG2A polypeptide.

[0037] As used herein, the term "CISH" or "CISH gene" refers to a nucleotide molecule that encodes cytokine-induced SH2-containing protein (CISH), a key negative intracellular immune checkpoint in NK cells. CISH (cytokine-induced SH2-containing protein) is a member of the intracellular suppressor of cytokine signaling (SOCS) family and is a key regulator of cytokine and growth factor signaling pathways.

[0038] A CISH gene is a gene that encodes a CISH polypeptide such as a TIGIT polypeptide having the sequence set forth in SEQ ID NO:29, or a CISH polypeptide having high identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% identity) to the above-mentioned CISH polypeptide or any CISH polypeptide known in the art and having the same immune checkpoint function.

[0039] For example, a CISH gene may be, but is not limited to, the following: a nucleic acid molecule having the sequence set forth in SEQ ID NO: 30; a CISH polypeptide coding sequence having high identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% identity) to the above CISH genes or any CISH gene known in the art and which similarly encodes and expresses a functional CISH polypeptide.

[0040] The term "impair / suppress expression" means to suppress, reduce, or eliminate expression of a gene or protein. To suppress or reduce or eliminate expression of a gene (i.e., a gene encoding TIGIT, NKG2A, or CISH), the sequence and / or structure of the gene may be altered such that the gene is not transcribed (for DNA) or translated (for RNA), or is not transcribed or translated to produce a functional protein (e.g., a transcription factor).

[0041] Various methods known in the art for suppressing, reducing or eliminating gene expression are described herein. Some methods can introduce nucleic acid substitutions, additions and / or deletions into wild-type genes. Some methods can also introduce single-strand or double-strand breaks into genes. As described above, to suppress, reduce or eliminate protein expression, the expression of a gene or polynucleotide encoding a protein can be suppressed, reduced or eliminated.

[0042] As used herein, the terms "impair" or "inhibit" refer to a decrease relative to a reference control level by at least 10%, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (i.e., to levels that are absent relative to the reference sample).

[0043] As used herein, the term "inactivation" means preventing expression of a polypeptide product encoded by a gene. Inactivation can occur at any stage or process of gene expression, including but not limited to transcription, translation, protein expression, and can affect any gene or gene product, including but not limited to DNA, RNA (e.g., mRNA), and polypeptides.

[0044] In some embodiments, a gene is suppressed or inactivated by a gene deletion. As used herein, "gene deletion" refers to removing at least a portion of a DNA sequence from or near a gene. In some embodiments, the sequence that is deleted from a gene includes an exon sequence of the gene. In some embodiments, the sequence that is deleted from a gene includes a promoter sequence of the gene. In some embodiments, the sequence that is deleted from a gene includes a flanking sequence of the gene. In some embodiments, a portion of the gene sequence is removed from the gene. In some embodiments, the complete gene sequence is removed from the chromosome. In some embodiments, the host cell comprises a gene deletion as described in any of the embodiments herein. In some embodiments, a gene is suppressed or inactivated by deletion of at least one nucleotide or nucleotide base pair in the gene sequence, resulting in a non-functional gene product. In some embodiments, a gene is inactivated by a gene deletion, where deletion of at least one nucleotide in the gene sequence results in a gene product that no longer has the function or activity of the original gene product, or a dysfunctional gene product.

[0045] In some embodiments, a gene is suppressed or inactivated by gene addition or replacement, where the gene is suppressed or inactivated by adding or replacing at least one nucleotide or nucleotide base pair in the gene sequence. In some embodiments, a gene is inactivated by gene inactivation, where the incorporation or replacement of at least one nucleotide in the gene sequence results in a gene product that no longer has the function or activity of the original gene product, or a dysfunctional gene product. In some embodiments, a gene is inactivated by addition or replacement, where the incorporation or replacement of at least one nucleotide in the gene sequence results in a dysfunctional gene product. In some embodiments, the host cell comprises a gene addition or replacement as described in any embodiment herein.

[0046] Methods and techniques for impairing gene function expression in a host cell include, but are not limited to, clustered, regularly interspaced, short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALEN), zinc-finger nucleases (ZFN), homologous recombination, non-homologous end joining, meganucleases, small interfering RNA (siRNA), and short hairpin RNA (shRNA; also known as short hairpin RNA).

[0047] In some embodiments, TIGIT may be impaired by a CRISPR / Cas9 system comprising a gRNA selected from SEQ ID NOs: 1-6, such as SEQ ID NOs: 3, 2, or 6. In some embodiments, NKG2A may be impaired by a CRISPR / Cas9 system comprising a gRNA selected from SEQ ID NOs: 7-18, such as SEQ ID NOs: 18, 7, or 10. In some embodiments, CISH may be impaired by a CRISPR / Cas9 system comprising a gRNA selected from SEQ ID NOs: 19-24, such as SEQ ID NOs: 21, 19, or 24.

[0048] In some embodiments, dual or triple knockout may be performed with a CRISPR / Cas9 system that includes gRNAs for two or three of TIGIT, NKG2A, and CISH, such as two or more gRNAs selected from the group consisting of gRNAs selected from SEQ ID NOs: 1-6, gRNAs selected from SEQ ID NOs: 7-18, and gRNAs selected from SEQ ID NOs: 19-24. For example, the CRISPR / Cas9 system may include gRNAs of SEQ ID NOs: 3, 18, and / or 21.

[0049] According to the disclosure of the present application, impairment of one or more of TIGIT, NKG2A and / or CISH can increase in vitro and / or in vivo cell expansion, extend in vitro and / or in vivo cell lifespan, improve in vivo cell depletion, increase cytotoxicity of NK cells against target cells, and / or modulate secretion of cytokines, interleukins and / or growth factors from NK cells.

[0050] Chimeric antigen receptors (CARs) The modified NK cells of the present application may further comprise an engineered antigen receptor, such as a chimeric antigen receptor (CAR), including an activating or stimulatory CAR, a costimulatory CAR (see WO2014 / 055668), and / or an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)).

[0051] CARs typically comprise an extracellular antigen (or ligand) binding domain that is associated with one or more intracellular signaling components, in some embodiments, via a linker and / or transmembrane domain. The molecule typically simulates or mimics the signal through a natural antigen receptor, the signal through said receptor in combination with a costimulatory receptor, and / or the signal through a costimulatory receptor alone.

[0052] In some embodiments, the CAR is specifically constructed for a particular antigen (or marker or ligand), e.g., an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen for which an inhibitory response is desired to be induced, e.g., an antigen expressed in a normal or non-diseased cell type. Thus, the CAR typically comprises, in its extracellular portion, one or more antigen-binding molecules that are one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR comprises one or more antigen-binding portions of an antibody molecule, e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0053] In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds to an antigen, such as, for example, a cancer marker or a cell surface antigen of a targeted cell or disease (e.g., a tumor cell or cancer cell), e.g., any target antigen described herein or known in the art.

[0054] In some embodiments, targets of the CAR include, but are not limited to, BCMA, CD19, CD20, CD22, PSMA, ACE2, CD7, CS1, EGFR / EGFRVIII, ErBb2 / HER2, CD3, CD138, and NKG2D.

[0055] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and recombinant host cell production. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment that contains a non-naturally occurring arrangement, e.g., having two or more antibody regions or chains joined by a synthetic linker (e.g., a peptide linker), and / or may not be a fragment produced by enzymatic digestion of a naturally occurring whole antibody. In some aspects, the antibody fragment is an scFv.

[0056] In some embodiments, the CAR comprises an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen (e.g., a complete antigen) expressed on the cell surface. In some embodiments, the CAR comprises an anti-BCMA VHH.

[0057] In some aspects, the antigen-specific binding or recognition component is connected with one or more transmembrane and intracellular signal domains. In some embodiments, the CAR comprises a transmembrane domain fused with the extracellular domain of the CAR. In one embodiment, the transmembrane domain naturally associated with one domain of the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid these domains from binding with the transmembrane domain of the same or different surface membrane protein, thereby minimizing the interaction with other members of the receptor complex.

[0058] In some embodiments, the transmembrane domain is derived from natural or synthetic origin. When the origin is natural, the domain is derived from any membrane-bound or transmembrane protein in some aspects. The transmembrane region includes those derived from (i.e., at least includes) the transmembrane region of the α, β or ζ chain of the T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly includes hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In some embodiments, the CAR includes a CD8 hinge and transmembrane region.

[0059] In some embodiments, a short oligomer or polypeptide linker (e.g., a linker 2-10 amino acids in length, such as a linker that includes a glycine and a serine (e.g., a glycine-serine doublet)) is present to form a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0060] CARs typically include at least one or more intracellular signaling components. In some embodiments, CARs include the intracellular components of the TCR complex, such as the TCR CD3 complex, which mediates T cell activation and cytotoxicity. +The CAR comprises a CD3 ζ (CD3-ζ) or Fc receptor γ and a CD8, CD4, CD25, or CD16 chimeric molecule. In some embodiments, the CAR further ...

[0061] In some embodiments, upon attachment of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of an NK cell.

[0062] In some embodiments, the CAR comprises the signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, ICOS, etc. In some aspects, the same CAR comprises an activating component and a costimulatory component, while in other aspects, the activation domain is provided by one CAR and the costimulatory component is provided by another CAR that recognizes a different antigen.

[0063] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain connected to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric costimulatory domain of CD28 and CD137 (4-1BB, TNFRSF9) connected to a CD3zeta intracellular domain.

[0064] In some embodiments, the CAR comprises two or more costimulatory domains in combination with an activation domain (e.g., a primary activation domain) in the cytoplasmic portion. One example is a receptor that comprises the intracellular components of CD3-zeta, CD28 and 4-1BB.

[0065] In some embodiments, the CAR comprises an anti-BCMA VHH with a human CD8 hinge and transmembrane region, cytoplasmic domain 4-1BB and CD3ζ.

[0066] In some embodiments, the CAR or other antigen receptor further comprises a marker to confirm transduction or engineering of cells to express the receptor, such as a truncated form of the cell surface receptor, such as truncated EGFR (tEGFR).

[0067] In some cases, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some embodiments, first-generation CARs are CARs that provide only CD3 chain induction signals upon antigen binding; in some embodiments, second-generation CARs are CARs that provide such signals and costimulatory signals, for example, CARs that include intracellular signaling domains from costimulatory receptors such as CD28 or CD137; in some embodiments, third-generation CARs are CARs that, in some embodiments, include multiple costimulatory domains from different costimulatory receptors.

[0068] In some embodiments, the CAR or other antigen receptor is an inhibitory CAR (e.g., iCAR) and contains intracellular components that inhibit or suppress responses, such as immune responses, such as ITAM and / or costimulatory responses in cells. Examples of such intracellular signaling components are those found on PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2 / 4 adenosine receptor (including A2AR) immune checkpoint molecules. In some embodiments, the engineered cells contain inhibitory CARs that contain the signaling domains of such inhibitory molecules, or are derived from such inhibitory molecules, and suppress responses of the cells, such as responses induced by activation and / or costimulatory CARs. For example, when the antigen (e.g., CAR) recognized by the activating receptor is also expressed or may be expressed on the surface of normal cells, this CAR is used to reduce the possibility of off-target effects. In some embodiments, an inhibitory receptor, such as an iCAR, that recognizes a marker of normal cell specificity is introduced.

[0069] In some exemplary embodiments, the CAR is designed to include a CD8 signal peptide (e.g., comprising SEQ ID NO:31 or encoded by SEQ ID NO:32), anti-CD19 scFv FMC 63 (e.g., comprising SEQ ID NO:33 or encoded by SEQ ID NO:34), human CD8 hinge and transmembrane region (e.g., comprising SEQ ID NO:35 or encoded by SEQ ID NO:36), cytoplasmic domain 4-1BB (e.g., comprising SEQ ID NO:37 or encoded by SEQ ID NO:38), and / or CD3ζ (e.g., comprising SEQ ID NO:39 or encoded by SEQ ID NO:40).

[0070] Cell populations, cell cultures or products The present specification also provides a cell population, cell culture, or product comprising the modified NK cells disclosed herein.

[0071] In some embodiments of the present disclosure, at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% of the cells of the cell population, cell culture or product are modified NK cells of the present application. In some embodiments, the cell population, cell culture or product does not contain other cells.

[0072] In some embodiments, the cell population, cell culture or product can be used for the treatment of disease, for example as pharmaceutical compositions and preparations. Pharmaceutical compositions and preparations generally optionally include one or more pharma-ceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0073] The term "drug formulation" means that the form of the formulation allows for the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that have unacceptable toxicity to a subject administered the formulation.

[0074] "Pharmaceutically acceptable carrier" means an ingredient in a drug formulation, other than the active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0075] In some embodiments, the choice of carrier is determined in part by the particular cell, binding molecule, and / or antibody and / or administration method. Therefore, there are a variety of suitable formulations. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are typically non-toxic to recipients at the dosages and concentrations employed.

[0076] The formulation or composition may contain one or more active ingredients that can be used for the specific indication, disease or disorder to be treated with the binding molecule or cell, preferably with complementary activities with the binding molecule or cell, and each activity does not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further contains other medicament active agents or pharmaceuticals, such as chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0077] In some embodiments, in the case of genetically engineered cells, the subject is administered about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any of the above values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, or a range defined by any of the above values). 0 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any of the above values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in these ranges, and / or the number of cells per kg of the subject's body weight.

[0078] The pharmaceuticals of the present disclosure can be administered using standard administration techniques, formulations, and / or devices. Formulations and devices, such as syringes and vials, for the storage and administration of the compositions are provided. Administration of cells can be autologous or heterologous. For example, immune response cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood derived immune response cells or their progeny can be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition comprising genetically modified immune response cells), it is usually formulated into an injectable form (solution, suspension, emulsion) per unit dose.

[0079] Formulations include those administered orally, intravenously, intraperitoneally, subcutaneously, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingually, or by suppository. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell population is administered to the subject for circumferential systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0080] Treatment and Use Therapeutic methods and uses of the engineered cells, cell populations, cell cultures, and products of the present disclosure are also provided, which involve administering the cells or compositions comprising the cells to a subject having a disease, condition, or disorder treatable by NK cells.

[0081] As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "therapy") refers to the complete or partial amelioration or reduction of a disease, condition or disorder, or its associated symptoms, side effects or consequences or phenotype. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, relief of direct or indirect pathological consequences of a disease, prevention of metastasis, slowing the rate of disease progression, amelioration or reduction of the disease state, and alleviation or improvement of prognosis.

[0082] As used herein, the term "effective amount" refers to an amount effective, when administered, at a dosage / amount and for a period of time necessary to achieve a desired result, such as a therapeutic result. A "therapeutically effective amount" of an agent (e.g., a drug formulation) refers to an amount effective, at a dosage and for a period of time necessary to achieve a desired therapeutic result, such as treatment of a disease, condition or disorder, and / or a pharmacokinetic or pharmacodynamic effect for the treatment. The therapeutically effective amount can vary depending on factors such as the subject's disease state, age, sex, weight, and cell population to be administered.

[0083] As used herein, a "subject" is a vertebrate, e.g., a mammal, e.g., a human or other animal, typically a human. In some embodiments, the subject suffers from persistent or recurrent disease after treatment with, e.g., chemotherapy, radiation therapy, and / or other therapy, including hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, the subject has not relapsed, but is determined to be at risk of relapse (e.g., at high risk of relapse), and thus is administered a compound or composition prophylactically, such as to reduce the likelihood of relapse or to prevent relapse.

[0084] The disease or disorder includes cancer. Any cancer can be treated using the genetically modified T cells described herein. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a carcinoma or sarcoma. In some embodiments, the cancer is acute lymphocytic leukemia, acute myeloid leukemia, Burkitt's lymphoma, central nervous system lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, chronic myeloproliferative disorder, myeloproliferative dysregulation syndrome, adult acute myeloproliferative disorder, multiple myeloma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the cancer is breast cancer, prostate cancer, testicular cancer, renal cell carcinoma, bladder cancer, liver cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, colorectal cancer, anal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, kidney cancer, head and neck cancer, glioblastoma, mesothelioma, melanoma, chondrosarcoma, or bone or soft tissue sarcoma. In some embodiments, the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bone tumor, brain stem glioma, brain cancer, cerebellar astrocytoma, cerebral astrocytoma, ventricular tumour, medullary cytoma, upper tentorium primitive neuroectodermal tumor, optic pathway and inferior thalamic glioma, or bronchial adenoma.In some embodiments, the cancer is selected from the group consisting of connective tissue proliferative microcytoma, ventricular melanoma, epithelioid hemangioendothelioma (EHE), Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, gastric carcinoid, cardiac cancer, hypopharyngeal cancer, hypothalamic and optic pathway glioma, pediatric intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, lip and oral cancer, lipid sarcoma, non-small cell lung cancer, small cell lung cancer, megalocytic proteinemia, male breast cancer, malignant fibrohistiocytoma of bone, medullary cytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck, oral cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, and the like. fungoides), chronic myxoma, cancer of the nasal cavity and paranasal sinuses, cancer of the nasopharynx, neuroblastoma, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, cancer of the paranasal sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma, makugami primitive neuroectodermal tumor, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, primary central nervous system lymphoma, renal cell carcinoma, retinal blastoma, rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sezary syndrome, nonmyxomatous skin cancer, melanoma, Merkel cell skin cancer, small intestine cancer, squamous cell carcinoma, cervical squamous cell carcinoma, throat cancer, thymoma, thyroid cancer, renal pelvis pelvis) and ureteral transitional cell carcinoma, trophoblastic tumor, gestational age, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenström's macroglobulinemia, or Wilms' tumor.

[0085] Diseases and conditions include autoimmune and inflammatory diseases. Exemplary diseases and conditions include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).

[0086] In addition to cancer, other diseases and conditions include age-related diseases. Exemplary diseases and conditions include atherosclerosis, diabetes, liver fibrosis, and ostarthritis.

[0087] In some embodiments, the methods include adoptive cell therapy for administering to a subject engineered cells of the present disclosure. Such administration can target-promote cellular activation (e.g., NK cell activation) such that disease or condition cells are targetedly destroyed.

[0088] Adoptive cell therapy represents a new model of cancer immunotherapy, but it may be limited by the persistence and functional differences of metastatic T / NK cells. Natural killer (NK) cells are xenotransplantable and may be an off-the-shelf product, making adoptive cell therapy of NK or CAR-NK cells universal.

[0089] For example, we use CRISPR-Cas9 mutagenesis screening method to target TIGIT, NKG2A and / or CISH to reprogram NK cells into long-lived effector cells that accumulate widely in tumors and have better persistence and stronger effector function; and the modified NK cells further containing CAR produce improved anti-tumor effects, thereby providing the modified NK cells promising for adoptive cell therapy of diseases such as tumors.

[0090] The methods and uses provided include adoptive cell therapy methods and uses. In some embodiments, the methods include administering cells or compositions comprising cells to a subject, tissue, or cell, e.g., suffering from, at risk for, or suspected of a disease, condition, or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject with a particular disease or disorder to be treated with adoptive cell therapy, e.g., adoptive NK cell therapy or CAR-NK cell therapy. In some embodiments, the cells or compositions are administered to a subject, e.g., suffering from, or at risk for a disease or disorder. In some aspects, the methods thereby treat (e.g., ameliorate) one or more symptoms of a disease or disorder, e.g., by reducing tumor burden.

[0091] Methods of cell administration for adoptive cell therapy are known and can be used in combination with the provided methods and compositions.In some embodiments, cell therapy, such as adoptive cell therapy, such as adoptive T cell therapy, is carried out by autologous transplantation, where cells are isolated and / or otherwise prepared from the subject who will receive cell therapy or from a sample derived from such a subject.Thus, in some aspects, cells are derived from the subject, such as a patient, who needs treatment, and after isolation and processing of cells, are administered to the same subject.

[0092] In some embodiments, cell therapy, such as adoptive cell therapy, such as adoptive NK or CAR-NK cell therapy, is performed by xenotransplantation, where cells are isolated and / or otherwise prepared from a subject other than the subject (e.g., the first subject) that will or will eventually receive cell therapy. In such embodiments, the cells are then administered to a different subject of the same species, such as the second subject. In some embodiments, the first and second subjects are genetically the same. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0093] Depending on the type and severity of the disease, the dosage of the cells or pharmaceutical composition may include about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg), about 1 μg / kg to 100 mg / kg or more, about 0.05 mg / kg to about 10 mg / kg, 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg. Multiple doses may be administered intermittently, for example, once a week or once every three weeks. Administration may be with an initial higher loading dose followed by one or more lower doses.

[0094] After administration of the cells to a mammal (e.g., a human), the biological activity of the engineered cell population and / or pharmaceutical composition can be measured by any known method. Parameters evaluated include specific binding of the engineered NK cells to an antigen in vivo (e.g., imaging) or ex vivo (e.g., ELISA or flow cytometry). In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as a cytotoxicity assay. In certain embodiments, the biological activity of the cells can also be measured by measuring the expression and / or secretion of certain cytokines. In some aspects, biological activity is measured by assessing clinical outcomes, such as reduction in tumor burden or burden.

[0095] In some embodiments, the cells or pharmaceutical compositions are administered as part of a combination therapy, e.g., administered simultaneously or sequentially in any order with another therapeutic intervention (e.g., another engineered cell or receptor or reagent, e.g., a cytotoxic or therapeutic agent).

[0096] Publications referenced herein and the materials referenced therein are all incorporated herein by reference. All reagents were obtained commercially unless otherwise noted. All parts and percentages are by weight unless otherwise noted. Average results are given unless otherwise noted. Abbreviations used herein are common abbreviations unless otherwise defined. EXAMPLES

[0097] Working Example Example 1 Ex vivo expansion of NK cells using K562 feeder cells K562 cells expressing full-length 4-1BBL and membrane-bound IL-21 (mBIL-21) were constructed using the Sleeping Beauty transfection system (Addgene). Gene sequences encoding mBIL-21 and 4-1BBL were cloned into pSBbi-RB plasmid (Adddgene-60522). pSBbi-RB-mBIL 21-4-1BBL plasmid was transformed into K562 cells by incubating with Sleeping Beauty transposase (SB 100 X, CAT# Adgene-127909) at a ratio of 3:1. The engineered K562 cells were selected under blasticidin pressure for several weeks to confirm the expression of mBIL-21 and 4-1BBL, and then used as NK feeders to fully culture NK cells in vitro.

[0098] Fresh PBMCs from healthy donors were provided by SailyBio (Shanghai, China) and AllCells (Shanghai, China). To stop cell proliferation, K562 feeder cells were pretreated with 50 μg / mL mitomycin C (Sigma-M4287) for 1 h at 37 °C and 5% CO2. PBMCs and inactivated K562 feeder cells were co-cultured at a 1:1 ratio in RPMI-1640 medium containing 10% FBS and 200 U / mL (R&D-202-IL) human IL-2 at 37 °C and 5% CO2. The medium was changed every 2 or 3 days.

[0099] Example 2 Purity measurement of expanded NK cells On day 14, expanded NK cells (1 × 10 5 Cells / well) were incubated with APC-anti-CD3 (Biolegend-300439) and PE-anti-CD56 antibodies (Biolegend-31805) for 1 h at 4° C. After washing with 1% BSA / PBS, cells were further washed and resuspended in 1% BSA-PBS (w / v) and used for flow cytometry and data were analyzed by FlowJo.

[0100] NK cell population (CD3 - CD56+ The purity of the product (characterized by: Table 1 Purity of expanded NK cells from six donors [Table 1] As a result, NK cells were successfully expanded with high purity from fresh PBMCs.

[0101] Example 3 Single or double knockout of TIGIT, NKG2A and / or CISH by CRISPR / Cas9 3.1 sgRNA sequence The small guide RNA (sgRNA) sequences of TIGIT, NKG2A, or CISH were designed on the CRISPOR website (http: / / crispor.tefor.net / ) and synthesized with GenScript (Nanjing, China). The sequences of sgRNAs are shown in Table 2. Table 2. sgRNA sequences for TIGIT, NKG2A, and CISH [Table 2]

[0102] 3.2 Knockout of TIGIT, NKG2A and CISH by CRISPR / Cas9 CRISPR-Cas9 ribonucleoprotein (RNP) complexes were transfected with 4D-Nucleofector TM was delivered to the expanded NK cells via the system (4D-Nuclefector Core Unit, Lonza).

[0103] The medium (RPMI-1640 containing 10% FBS and 200 U / mL human IL-2) was pre-warmed at 37 °C for 30 min in cell culture plates, and NK cells were harvested for nucleofection. RNP (ribonucleoprotein) complexes of Cas9 (100 pmol, Invitrogen-A 36498) and sgRNA (200 pmol) were mixed and incubated at room temperature for 20 min. 1 × 10 cells were added for each reaction at room temperature. 6The amplified NK cells and sgRNA-containing RNP complexes were gently mixed in 100 μL P3 primary nuclear transfection solution (Lonza-V 4 XP-3024), and then the mixture was transferred to the Nucleocuvette vessel. The vessel was inserted into the Lonza 4 D Nuclefector and nuclear transfection was performed using program CM-137. After removing the vessel, pre-warmed medium was immediately added to each cassette well. The medium / cells / RNP mixture was transferred to pre-warmed RPMI-1640 medium in a 6-well plate and incubated at 37 °C, 5% CO2. On day 3, knockout efficiency was then determined by flow cytometry or western blot.

[0104] Dual knockout (DKO) was performed using sgRNA-3 (against TIGIT), sgRNA-18 (against NKG2A), and sgRNA21 (against CISH), which have remarkable effects in single protein knockout.

[0105] 3.3 Measurement of knockout efficiency by flow cytometry We measured the single knockout and dual knockout efficiencies of cell surface proteins TIGIT and NKG2A by flow cytometry.

[0106] NK cells (1×10 5 Cells / well) were incubated with APC-anti-TIGIT antibody (eBioscience-17-900-42) for 1 h at 4° C. After washing with 1% BSA / PBS, cells were washed, resuspended in 1% BSA-PBS (w / v), and used for flow cytometry, and data were analyzed by FlowJo.

[0107] The efficiencies of TIGIT, NKG2A and dual knockout are shown in Figures 2-4 and Tables 3-5, respectively. Table 3. TIGIT knockout efficiency [Table 3] Table 4. NKG2A knockout efficiency [Table 4] Table 5. Efficiency of dual knockout of TIGIT and NKG2A [Table 5] The results indicate that at least some sgRNAs are effective in knocking out TIGIT and / or NKG2A.

[0108] 3.4 Determining knockout efficiency by Western blot The efficiency of intracellular protein CISH alone was determined by Western blot.

[0109] NK cells (1×10 7 Cells) were harvested, washed with cold PBS, and lysed in cell lysis buffer (cell Signaling Technology-9803). Cell lysates containing equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes. After blocking in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with rabbit anti-CISH antibody (Cell Signaling Technology-8731) overnight at 4°C, and then exposed to HRP goat anti-rabbit IgG (Cell Signaling Technology-7070 S) for 2 h at room temperature. Immunoreactive proteins were observed using an enhanced chemiluminescence system (ChemiDocMF, Bio-Rad). The strips were washed with TBST and then incubated with mouse anti-GAPDH antibody (Cell Signaling Technology-97166) overnight at 4°C. After a second antibody incubation (HRP goat anti-mouse IgG (CAT# Bethyl Laboratories-A 90-231 P), the chemiluminescent signal was recaptured.

[0110] The data are shown in Figure 5. The results show that several sgRNAs are effective for CISH knockout.

[0111] Example 4 Cytotoxicity of Modified NK Cells The human fibrosarcoma cell line HT1080 (ECACC-no. 8511505), which expresses endogenous CD155 on the cell surface and upregulates HLA-E expression after IFN-γ induction, was used to evaluate the cytotoxicity of engineered NK cells. Lentivirus expressing ZsGreen was packaged with lentiviral shuttle vectors and packaging plasmids PsPAX.2 (Addgene-1260) and PMD 2.G (Addgene-1259) synthesized at Sangon (Shanghai, China). HT1080 cells transfected with ZsGreen lentivirus in the presence of 0.5 μg / mL IFN-γ were preseeded in 96-well plates for 24 h and allowed to adhere to the flat bottom of the culture plate.

[0112] CD155 and HLA-E expression levels were determined by flow cytometry using PE-anti-CD155 antibody (eBioscience-12-1550-41) and APC-anti-HLA-E antibody (Biolegend-342606). Figure 6 shows high expression levels of CD155 (ligand for TIGIT) and HLA-E (ligand for NKG2A) in HT1080 cells.

[0113] The engineered NK cells were then added to each well at a ratio of 3:10, and the number of live cells was assessed by green fluorescent signals monitored by an IncuCite ZOOM (Essen Bioscience) automated live-cell imaging system.

[0114] The data in Figure 7 showed that when the NK / tumor cell ratio was 3:10, the cytotoxicity of TIGIT, NKG2A and / or CISH knockout NK cells was enhanced, among which TIGIT / NKG2A, TIGIT / CISH and NKG2A / CISH dual knockout showed good anti-tumor activity, and TIGIT+CISH dual knockout showed the best effect among all the tested groups.

[0115] Example 5 In vivo studies of engineered NK Female NSG (Biocytogen, China) mice, aged 6-8 weeks, were placed and handled under specific pathogen-free conditions and provided with autoclaved food and water. All procedures regarding the handling, care, and treatment of animals in the study were performed under the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). On day 0, 4x10 6 A549 cells were injected subcutaneously in 100 mL PBS into the right flank of NSG mice. Tumors were 50–100 mm 3 When the mice reached 1×10 7 NK cells were administered intravenously. Tumor volume and mouse body weight were measured every 3 days. Tumor volume was measured every 3 days using a caliper and calculated using the following equation: V=0.5ab 2 , a and b are the long and short diameters of the tumor, respectively.

[0116] The results are shown in FIG. 8A (tumor growth inhibition) and FIG. 8B (body weight change), respectively.

[0117] The results in Figure 8A show that NK cells with TIGIT / CISH knockout or NKG2A / CISH knockout have superior efficacy in eliminating HT1080 tumor growth in mice, and no significant difference was observed as shown in Figure 8(b), suggesting the safety of NK therapy.

[0118] Example 6 CAR-NK and Engineered CAR-NK Formulations 6.1 Anti-CD19 CAR plasmid and retroviral vector construction The designed CD19-targeted CAR contains the CD8 signal peptide (amino acid sequence of SEQ ID NO:31, coding sequence of SEQ ID NO:32), anti-CD19 scFv FMC63 (amino acid sequence of SEQ ID NO:33, coding sequence of SEQ ID NO:34), human CD8 hinge and transmembrane region (amino acid sequence of SEQ ID NO:35, coding sequence of SEQ ID NO:36), cytoplasmic domain 4-1BB (amino acid sequence of SEQ ID NO:37, coding sequence of SEQ ID NO:38) and CD3ζ (amino acid sequence of SEQ ID NO:39, coding sequence of SEQ ID NO:40). The CAR cDNA construct was synthesized into the multiple cloning site of the retroviral shuttle vector pMSCV SFV.

[0119] On day 0, 1 x 10 7 293T cells (ATCC-CRL-3216) were seeded in 150 mm dishes. The next day, CD19 CAR and vector were co-transfected into 293T cells together with retroviral packaging plasmid, pCMV gag-pol, PMD 2.BaEV (amino acid sequence of SEQ ID NO: 41, coding sequence of SEQ ID NO: 42). On day 2, the medium of transfected 293T cells was replaced with fresh medium. On day 3, retroviral supernatant was collected from transfected 293T cells and filtered through a 0.45 μm polyethersulfone (PES) membrane filter. If necessary, retroviral supernatant was concentrated by ultracentrifugation (Beckman).

[0120] The retroviral titer was measured using the HT1080 (ECACC, no. 85111505) infection method. Briefly, HT1080 cells were seeded overnight at a density of 10,000 / well in 96-well plates. Retroviruses, serially diluted 5-fold in complete DMEM medium, were added to the HT1080 cells and mixed gently. The culture plates were placed in a 37°C incubator for 72 hours. CAR expression in HT1080 cells was characterized by fluorescence using flow cytometry. The retroviral titer was calculated as follows: Retroviral titer (TU / mL) = (number of transformed cells x % fluorescence positive) / (viral volume).

[0121] 6.2 Generation of anti-CD19 CAR-NK cells The expanded NK cells were mixed with retroviral particles at an MOI of 4 and polybrene (Millipore-TR-1003-G, 4 μg / mL), which is known to enhance the efficiency of retroviral transduction of NK cells. The cells were then centrifuged (1500 rpm at 32°C) for 90 min in 6-well plates and then incubated for 18 h in complete RPMI 1640 medium at 37°C. The transduction mixture was then removed by centrifugation and replaced with fresh complete RPMI 1640 medium in the presence of IL-2 (200 U / mL) and IL-15 (140 U / mL).

[0122] 6.3 Generation of engineered CAR-NK cells Four days after transfection, the anti-CD19 CAR-NK was obtained from the anti-CD19 CAR-NK by dual knockout of TIGIT and CISH. The electroporation process of CAR-NK with TIGIT and CISH-RNP complex is described in Example 3.2. Then, mCD19 CAR-NK cells were co-cultured with mitomycin C-treated K562 feeder cells (same treatment as in Example 1) in complete 1640 medium in the presence of IL-2 (200 U / mL) and IL-15 (140 U / mL).

[0123] 6.4 CD19 CAR-NK transduction efficiency measurement The transduction efficiency of CD19 CAR and mCD19 CAR-NK cells was assessed by flow cytometry 8 days after transfection (i.e. after 5 days of co-culture with K562 feeder cells). Briefly, CAR-NK and mCAR NK cells were harvested and incubated with PE-labeled CD19 antigen (Acro Biosystems, CD9-HP2H3, dilution 1:100) for 1 h at 4°C. After washing with 1% BSA / PBS, cells were washed, resuspended in 1% BSA-PBS and used for flow cytometry and data were analyzed by FlowJo.

[0124] The results in Figures 9A and 9B show that anti-CD19 CAR-NK cells are effectively generated with high transduction efficiency.

[0125] 6.5 Measuring knockout efficiency by FACS and Western blot TIGIT and CISH knockout efficiency in mCD19 CAR NK cells was measured by flow cytometry and western blot, respectively. The results shown in Figure 10A, 10B, 11A, and 11B demonstrate the high knockout efficiency of TIGIT and CISH in mCD19 CAR NK cells.

[0126] Example 7 In Vitro Characterization of CAR-NK CAR-NK cells were characterized by tumor cell killing ability and cytokine release 14 days after transfection.

[0127] 7.1 Cytotoxicity measurements To generate luciferase-expressing target cells, lentivirus luciferase was transfected into Raji cells. CD19 CAR-NK and mCD19 CAR-NK cells were co-cultured with 20000 luciferase-expressing Raji (Raji-luc, ATCC-CL86) cells at a ratio of 3:1 and 1:1 for 24 hours. One-Glo luciferase assay reagent (CAT# Promega E6120) was added to each well. After rocking the plate at room temperature for 5 minutes, luminescence was detected using an EnVision reader (PerkinElmer). Supernatants were collected and frozen at -80°C for IFN-γ release. Cytotoxicity was calculated by the following formula: Cytotoxicity = RLU. 試験群 / RLU 対照群 X100%.

[0128] The results in Figures 12A and 12B show that the toxicity of mCD19 CAR-NK cells against cancer cells was significantly increased compared to unmodified CD19 CAR-NK cells, suggesting that the modified CAR-NK cells are more effective in treating cancer.

[0129] 7.2 Sequential kill test The cytotoxicity of mCD19 CAR-NK was further investigated using a sequential killing assay. CD19 CAR-NK and mCD19 CAR-NK cells were co-cultured with 20,000 Raji-luc cells at a ratio of 3:1 and 1:1 for 24 hours, and then the same amount of fresh Raji-luc cells was added. After 24 hours, 100 μL of supernatant was collected and frozen at -80°C for IFN-γ release. One-Glo luciferase assay reagent (CAT#Promega E6120) was added to each well. The plate was rocked at room temperature for 5 minutes, and then luminescence was detected using an EnVision reader (PerkinElmer).

[0130] The results in Figures 13A and 13B show that mCD19 CAR-NK cells had increased cytotoxicity against cancer cells in a sequential killing assay, suggesting that the engineered CAR-NK cells may be more effective in treating cancer.

[0131] 7.3 Cytokine release assay Supernatants collected as described in Examples 7.1 and 7.2 were thawed and subjected to IFN-γ quantification by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs. Recombinant human IFN-γ (cat#PeproTech-300-02) was used as a standard. Plates were precoated with human IFN-γ specific capture antibody (cat#Pierce-M700A). After blocking, 100 μL standard or sample was transferred to each well and incubated for 2 h at ambient temperature. After removing unbound material, biotin-conjugated detection antibody specific for IFN-γ (cat#Pierce-M701B) was added to the wells and incubated for 1 h. Streptavidin-conjugated horseradish peroxidase (HRP) (cat#Invitrogen-SN1004) was then added to the wells and incubated for 30 min at ambient temperature. Color was developed by apportioning 100 μl of TMB matrix, followed by stopping with 100 μl of 2N HCl. Absorbance was read at 450 nM using a microplate spectrophotometer.

[0132] The results in Figures 14A, 14B, 15A, and 15B show that human IFN-γ release of mCD19 CAR-NK cells is much higher than that of CD19 CAR-NK.

[0133] The scope of the present invention is not limited to the embodiments disclosed herein, which are intended as single illustrations of various aspects of the invention, and any functionally equivalent embodiments are within the scope of the present invention. Various modifications of the compositions and methods of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are also intended to fall within the scope of the present invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention.

[0134] Appendix. Sequence information [Table 6]

Claims

1. An isolated modified NK cell that has been modified to impair the functional expression of two or three of TIGIT, NKG2A, and CISH.

2. The modified NK cell described in claim 1, further comprising a chimeric antigen receptor (CAR).

3. 2. The modified NK cell of claim 1, wherein said functional expression is reduced or eliminated by gene knockout, gene mutation, gene deletion, gene silencing, or a combination of any of the foregoing.

4. 2. The modified NK cell of claim 1, wherein said functional expression is reduced or eliminated using a CRISPR system, a TALEN system, a zinc finger nuclease (ZFN) system, a meganuclease system, siRNA, antisense RNA, microRNA, short hairpin RNA, or a combination of any of the foregoing.

5. 2. The modified NK cell of claim 1, wherein the functional expression is reduced or eliminated using a CRISPR system, and the sgRNA used is selected from SEQ ID NOs: 1-6, 7-18, and 19-24.

6. The modified NK cell of claim 1, wherein the impairment of functional expression reduces expression of the target gene in the modified NK cell by at least 50%, 60%, 70%, 80%, 90%, or 95% compared to a corresponding NK cell in which the impairment is not present.

7. 2. The modified NK cell of claim 1, wherein the NK cells are derived from the group consisting of umbilical cord blood, peripheral blood and / or placenta of a vertebrate (e.g., human or rodent cells), and induced pluripotent stem cells (iPSCs), and / or the NK cells are autologous or allogeneic.

8. 3. The modified NK cell of claim 2, wherein the CAR comprises (i) an antigen recognition domain, (ii) an extracellular hinge region, (iii) a transmembrane domain, and (iv) an intracellular signaling domain.

9. the antigen recognition domain is an antibody or antigen-binding fragment thereof that targets an antigen expressed on a target cell but not on a healthy cell, e.g., an antigen recognition domain derived from the variable region of a monoclonal antibody (mAb) bound as a single chain variable fragment (scFv) or the heavy chain variable domain of a heavy chain antibody (VHH); and / or 9. The modified NK cell of claim 8, wherein the antigen recognition domain is a member of a natural ligand / receptor pair, such as a cytokine, an innate immune receptor, a TNF receptor superfamily member, a growth factor, and / or a structural protein.

10. Antigen recognition domains include the following: CD19, CD20, HER2, BCMA and / or EGFR-THR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, reticle, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFRβ, SSEA-4, CD2O, folate receptor α, ERBB2 (HER2 / neu), MUC1, EGFR, NCAM, Protease, PAP, ELF2M, EphrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, G PRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NYK-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR2O, LY6K, 0R51E2, TARP, WT1, NY-ESO-1, LAGEla, MAGE-A1, Legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MADCT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin8, melanAI MART1, Ras mutant, hTERT, sarcoma translocation breakpoint breakpoints), ML-IAP, ERG (TMPRSS2-ETS fusion gene), NA17, PA1X3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut9. The modified NK cell of claim 8, which targets one or more antigens selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCR3MLS and IGLL1, and urokinase-type plasminogen activator receptor (uPAR).

11. The CAR may comprise a CD3ζ endodomain (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:39 or encoded by a nucleotide molecule comprising SEQ ID NO:40), as well as CD27, CD28, 4-1BB (CD137, e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:31 or encoded by a nucleotide molecule comprising SEQ ID NO:32), OX40, CD30, CD4O, PD-1, ICOS, lymphocyte function-associated antigen-1 ( LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp8O (KLRF1), CD16O, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD1O3, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD16O ( 9. The modified NK cell of claim 8, comprising a costimulatory signaling domain selected from: BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly1O8), SLAM (SLAMF1, CD1SO, IPO-3), BLAME (SLAMF8), SELPGG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 and NKG2D.

12. The modified NK cell of claim 8, wherein the CAR is transduced into the NK cell via a vector, such as a lentiviral or retroviral vector (e.g., a gamma-retroviral vector, pMSCV SFFV) and / or via a CRISPR system.

13. The modified NK cell of claim 1 , wherein the modified NK cell is in a cell population, cell culture or product.

14. The modified NK cells, compared to NK cells that do not have impaired functional expression of one or more of TIGIT, NKG2A and CISH, exhibit the following: (a) the functional expression of one or more of TIGIT, NKG2A and CISH is reduced or eliminated; (b) increased in vitro and / or in vivo cell proliferation; (c) the life span of cells in vitro and / or in vivo is extended; (d) in vivo cell depletion is improved; (e) the cytotoxicity of NK cells against target cells is enhanced; and / or (f) NK cells regulate the secretion of cytokines, interleukins and / or growth factors; The modified NK cell of claim 1 , having one or more of the following characteristics:

15. The method comprises the steps of: (i) providing NK cells; (ii) modifying the NK cells to impair functional expression of one or more of TIGIT, NKG2A and CISH; (iii) optionally modifying the NK cells to comprise a chimeric antigen receptor (CAR); and (iv) optionally expanding the modified cells.

2. A method for producing the modified NK cell of claim 1, comprising:

16. 16. The method of claim 15, wherein step (ii) is performed before, simultaneously with, or after step (iii); and / or step (iv) is performed before or after one or more of steps (i)-(iii).

17. Use of the modified NK cells according to any one of claims 1 to 14 in the preparation of a product for the treatment of a disease.

18. The use according to claim 17, wherein the treatment is adoptive cell therapy, preferably CAR-NK adoptive cell therapy.

19. 18. The use according to claim 17, wherein the disease is selected from cancer, autoimmune diseases, infectious diseases, transplant rejection and other age-related diseases.

20. 18. The use according to claim 17, wherein the disease is selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma and leukemia, and / or is a cancer selected from cancers of the blood system, lymphatic system, digestive system, respiratory system, reproductive system, musculoskeletal system and nervous system.

21. 18. The use according to claim 17, wherein the disease is selected from the group consisting of atherosclerosis, diabetes, liver fibrosis and osteoarthritis.

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